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  • 251.
    Reddy Vanga, Sudarsana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sävmarker, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Ng, Leelee
    Monash Univ, Biomed Discovery Inst, Dept Physiol, Clayton, Vic 3800, Australia.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Chai, Siew Yeen
    Monash Univ, Biomed Discovery Inst, Dept Physiol, Clayton, Vic 3800, Australia.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Aryl Sulfonamides2018In: ACS OMEGA, ISSN 2470-1343, Vol. 3, no 4, p. 4509-4521Article in journal (Refereed)
    Abstract [en]

    The insulin-regulated aminopeptidase (IRAP) is a membrane-bound zinc metallopeptidase with many important regulatory functions. It has been demonstrated that inhibition of IRAP by angiotensin IV (Ang IV) and other peptides, as well as more druglike inhibitors, improves cognition in several rodent models. We recently reported a series of aryl sulfonamides as small-molecule IRAP inhibitors and a promising scaffold for pharmacological intervention. We have now expanded with a number of derivatives, report their stability in liver microsomes, and characterize the activity of the whole series in a new assay performed on recombinant human IRAP. Several compounds, such as the new fluorinated derivative 29, present submicromolar affinity and high metabolic stability. Starting from the two binding modes previously proposed for the sulfonamide scaffold, we systematically performed molecular dynamics simulations and binding affinity estimation with the linear interaction energy method for the full compound series. The significant agreement with experimental affinities suggests one of the binding modes, which was further confirmed by the excellent correlation for binding affinity differences between the selected pair of compounds obtained by rigorous free energy perturbation calculations. The new experimental data and the computationally derived structure-activity relationship of the sulfonamide series provide valuable information for further lead optimization of novel IRAP inhibitors.

  • 252.
    Rodriguez, David
    et al.
    Stockholm Univ, Sci Life Lab, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.;Stockholm Univ, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Chakraborty, Saibal
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Warnick, Eugene
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Crane, Steven
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Gao, Zhan-Guo
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    O'Connor, Robert
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Jacobson, Kenneth A.
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Structure-Based Screening of Uncharted Chemical Space for Atypical Adenosine Receptor Agonists2016In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 11, no 10, p. 2763-2772Article in journal (Refereed)
    Abstract [en]

    Small molecule screening libraries cover only a small fraction of the astronomical number of possible drug-like compounds, limiting the success of ligand discovery efforts. Computational screening of virtual libraries representing unexplored chemical space could potentially bridge this gap. Drug development for adenosine receptors (ARs) as targets for inflammation and cardiovascular diseases has been hampered by the paucity of agonist scaffolds. To identify novel AR agonists, a virtual library of synthetically tractable nucleosides with alternative bases was generated and structure-based virtual screening guided selection of compounds for synthesis. Pharmacological assays were carried out at three AR subtypes for 13 ribosides. Nine compounds displayed significant activity at the ARs, and several of these represented atypical agonist scaffolds. The discovered ligands also provided insights into receptor activation and revealed unknown interactions of endogenous and clinical compounds with the ARs. The results demonstrate that virtual compound databases provide access to bioactive matter from regions of chemical space that are sparsely populated in commercial libraries, an approach transferrable to numerous drug targets.

  • 253.
    Rokka, Johanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Faresjö, Rebecca
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Olberg, Dag
    Oslo University.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvanen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Journal of Labelled Compounds and Pharmaceuticals: Neurosciences session2019In: ISRS 2019, Wiley Online Library , 2019, Vol. 62, p. S78-S79Conference paper (Refereed)
  • 254.
    Rosenström, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Design and Synthesis of AT2 Receptor Selective Angiotensin II Analogues Encompassing β- and γ-Turn Mimetics2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Important information on the bioactive conformation of biologically active peptides may be obtained by studies of rigid peptides or well-defined secondary structure mimetics incorporated into pseudopeptides. The structural requirements for the interaction of angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) with its AT1 and AT2 receptors were the subject of this study.

    The main objectives of this work were to synthesize secondary structure mimetics and incorporate these into Ang II. Ang II has been suggested to adopt a turn conformation around Tyr4 when interacting with its AT1 receptor. Therefore, two γ- and one β-turn mimetic scaffolds based on the benzodiazepine structure were synthesized and decorated with side chains. The scaffolds replaced the turn region around Tyr4. Most of the pseudopeptides obtained after incorporation into Ang II exhibited high AT2/AT1 selectivity and nanomolar affinity to the AT2 receptor. One pseudopeptide encompassing a β-turn mimetic also displayed AT1 receptor affinity.

    We hypothesized that the position of the guanidino group of the arginine residue and the N-terminal end, in relation to the tyrosine side chain, was critical for AT2 receptor affinity. Conformational evaluation of the pseudopeptides revealed that in all the compounds with AT2 receptor affinity the arginine side chain and the N-terminal end could reach common regions, not accessible to the inactive compound. It is proposed that Ang II has a more extended bioactive conformation when binding to the AT2 receptor than when binding to the AT1 receptor.

    Furthermore, in a Gly scan of Ang II only replacement of the arginine residue reduced the affinity for the AT2 receptor considerably. Some N-terminal modified Ang II analogues were also synthesized and it was concluded that truncated Ang II analogues interact with the AT2 receptor differently than Ang II.

    Three of the synthesized pseudopeptides were evaluated in AT2 receptor functional assays and were found to act as agonists.

    List of papers
    1. A Selective AT2 Receptor Ligand with a γ-Turn-Like Mimetic replacing the Amino Acid Residues 4-5 of Angiotensin II
    Open this publication in new window or tab >>A Selective AT2 Receptor Ligand with a γ-Turn-Like Mimetic replacing the Amino Acid Residues 4-5 of Angiotensin II
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    2004 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 47, no 4, p. 859-870Article in journal (Refereed) Published
    Abstract [en]

    Three angiotensin II (Ang II) analogues encompassing a benzodiazepine-based γ-turn-like scaffold have been synthesized. Evaluation of the compounds in a radioligand binding assay showed that they had no affinity to the rat liver AT1 receptor. However, one of the compounds displayed considerable affinity to the pig uterus AT2 receptor (Ki = 3.0 nM) while the other two lacked affinity to this receptor. It was hypothesized that the reason for the inactivity of one of these analogues to the AT2 receptor was that the guanidino group of the Arg2 residue and/or the N-terminal end of the pseudopeptide could not interact optimally with the receptor. To investigate this hypothesis, a conformational analysis was performed and a comparison was carried out with the monocyclic methylenedithioether analogue cyclo(S−CH2−S)[Cys3,5]Ang II which is known to bind with high affinity to the AT2 receptor (Ki = 0.62 nM). This comparison showed that, in the compounds with high AT2 receptor affinity, the guanidino group of the Arg2 residue and the N-terminal end could access common regions of space that were not accessible to the inactive compound. To examine the importance of the guanidino group for binding, the Arg side chain was removed by substituting Arg2 for Ala2 in the analogue having the high affinity. This analogue lacked affinity to AT2 receptors, which supports the role of the guanidino group in receptor binding.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92323 (URN)10.1021/jm030921v (DOI)
    Available from: 2004-11-10 Created: 2004-11-10 Last updated: 2017-12-14Bibliographically approved
    2. Synthesis and AT2 receptor-binding properties of angiotensin II analogues
    Open this publication in new window or tab >>Synthesis and AT2 receptor-binding properties of angiotensin II analogues
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    2004 (English)In: Journal of Peptide Research, ISSN 1397-002X, E-ISSN 1399-3011, Vol. 64, no 5, p. 194-201Article in journal (Refereed) Published
    Abstract [en]

    The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2–8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 μm). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.

    Keywords
    angiotensin II, angiotensin III, AT1 binding, AT2 binding, Gly scan, peptide synthesis, structure–activity relationship
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-92324 (URN)10.1111/j.1399-3011.2004.00184.x (DOI)
    Available from: 2004-11-10 Created: 2004-11-10 Last updated: 2017-12-14Bibliographically approved
    3. New selective AT2 receptor ligands encompassing a γ-turn mimetic replacing the amino acid residues 4-5 of angiotensin II act as agonists
    Open this publication in new window or tab >>New selective AT2 receptor ligands encompassing a γ-turn mimetic replacing the amino acid residues 4-5 of angiotensin II act as agonists
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    2005 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 48, no 12, p. 4009-4024Article in journal (Refereed) Published
    Abstract [en]

    New benzodiazepine-based γ-turn mimetics with one or two amino acid side chains were synthesized. The γ-turn mimetics were incorporated into angiotensin II (Ang II) replacing the Val3-Tyr4-Ile5 or Tyr4-Ile5 peptide segments. All of the resulting pseudopeptides displayed high AT2/AT1 receptor selectivity and exhibited AT2 receptor affinity in the low nanomolar range. Molecular modeling was used to investigate whether the compounds binding to the AT2 receptor could position important structural elements in common areas. A previously described benzodiazepine-based γ-turn mimetic with high affinity for the AT2 receptor was also included in the modeling. It was found that the molecules, although being structurally quite different, could adopt the same binding mode/interaction pattern in agreement with the model hypothesis. The pseudopeptides selected for agonist studies were shown to act as AT2 receptor agonists being able to induce outgrowth of neurite cells, stimulate p42/p44mapk, and suppress proliferation of PC12 cells.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-95767 (URN)10.1021/jm0491492 (DOI)
    Available from: 2007-04-17 Created: 2007-04-17 Last updated: 2017-12-14Bibliographically approved
    4. Design, synthesis, and incorporation of a beta-turn mimetic in angiotensin II forming novel pseudopeptides with affinity for AT(1) and AT(2) receptors
    Open this publication in new window or tab >>Design, synthesis, and incorporation of a beta-turn mimetic in angiotensin II forming novel pseudopeptides with affinity for AT(1) and AT(2) receptors
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    2006 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 49, no 20, p. 6133-6137Article in journal (Refereed) Published
    Abstract [en]

    A benzodiazepine-based beta-turn mimetic has been designed, synthesized, and incorporated into angiotensin II. Comparison of the mimetic with beta-turns in crystallized proteins showed that it most closely resembles a type II beta-turn. The compounds exhibited high to moderate binding affinity for the AT(2) receptor, and one also displayed high affinity for the AT(1) receptor. Molecular modeling showed that the high-affinity compounds could be incorporated into a previously derived model of AT(2) receptor ligands.

    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-95769 (URN)10.1021/jm051222g (DOI)000240826200029 ()17004728 (PubMedID)
    Available from: 2007-04-17 Created: 2007-04-17 Last updated: 2018-01-13Bibliographically approved
  • 255.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Affibody Molecules for HER3-targeted Theranostics of Malignant Tumours2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The HER3 receptor plays a strong role in disease progression and resistance to therapies in several cancer types. Due to its endogenous expression and low overexpression in malignant tumours, it is a particularly challenging target. The primary aim of this thesis project was to develop, evaluate and characterize affibody molecules for theranostic applications in HER3-expressing malignant tumours.

    Paper I investigated the in vivo targeting properties and therapeutic efficacy of a bivalent affibody construct fused with an albumin binding domain, ZHER3-ABD-ZHER3. This construct could slow down the growth of HER3-expressing tumour xenografts without causing health problems or side effects in mice.

    Paper II compared the in vitro and in vivo properties of two HER3-targeting affibody molecules (Z08698 and Z08699) to select an imaging probe for HER3 diagnostics. While the two constructs had similar properties, Z08698 demonstrated better blood clearance and better radioactivity retention in tumours.

    Paper III and IV present the development of a HER3 imaging probe for PET using gallium and cobalt isotopes. We demonstrated that imaging of HER3 expression could be obtained as soon as 3 h pi using gallium-68. Additionally, we demonstrated that affibody molecules labelled with a neutral cobalt-NOTA complex had a lower radioactivity uptake in the liver than molecules radiolabelled with a positive gallium-NOTA complex. Imaging contrast increased over time. As the dose of the injected protein increased, the activity uptake in normal organs decreased, whereas the tumour uptake remained the same, which improved the imaging contrast and allowed discrimination between xenografts with high and low HER3 expression. This modification did not influence tumour activity uptake.

    Paper V presents the HER3-targeting affibody molecule trimer as a tool to block hepatic uptake in order to increase the imaging contrast in the liver. The trimer demonstrated its ability to bind to endogenous receptors in the liver, which decreased the hepatic uptake of the radiolabelled monomer. This phenomenon enabled the monomer to pass the liver barrier, which increased tumour radioactivity uptake and improved imaging contrast.

    List of papers
    1. In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct
    Open this publication in new window or tab >>In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct
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    2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43118Article in journal (Refereed) Published
    Abstract [en]

    Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with In-111 via a DOTA chelator. The residence time of In-111-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. In-111-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, In-111-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct In-111-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of In-111-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-318958 (URN)10.1038/srep43118 (DOI)000394748000001 ()28230065 (PubMedID)
    Funder
    Swedish Cancer Society, CAN2013-586, CAN 2016/463, CAN2014-474, CAN2015/350Swedish Research Council, Swedish Research Council 621-2012-5236, 2015-02509, 2015-02353VINNOVA, 2016-04060
    Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-09-20Bibliographically approved
    2. Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors
    Open this publication in new window or tab >>Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors
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    2015 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 34, no 2, p. 1042-1048Article in journal (Refereed) Published
    Abstract [en]

    Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium-111 (In-111) and assessed in vitro and in vivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with In-111 provided stable conjugates. In vitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT-474 breast carcinoma cells. In mice bearing BT-474 xenografts, the tumor uptake of the two conjugates was receptor-specific. Direct in vivo comparison of In-111-HEHEHE-Z08698-NOTA and In-111-HEHEHE-Z08699-NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for In-111-HEHEHE-Z08698-NOTA [12 +/- 3 vs. 8 +/- 1,4 h post injection (p.i)] due to more efficient blood clearance. In-111-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

    Keywords
    NOTA, indium-111, affibody molecules, HER3, molecular imaging
    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-260279 (URN)10.3892/or.2015.4046 (DOI)000357965600060 ()26059265 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Research Council
    Available from: 2015-08-21 Created: 2015-08-18 Last updated: 2018-09-20Bibliographically approved
    3. Affibody-mediated PET imaging of HER3 expression in malignant tumours
    Open this publication in new window or tab >>Affibody-mediated PET imaging of HER3 expression in malignant tumours
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    2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15226Article in journal (Refereed) Published
    Abstract [en]

    Human epidermal growth factor receptor 3 (HER3) is involved in the progression of various cancers and in resistance to therapies targeting the HER family. In vivo imaging of HER3 expression would enable patient stratification for anti-HER3 immunotherapy. Key challenges with HER3-targeting are the relatively low expression in HER3-positive tumours and HER3 expression in normal tissues. The use of positron-emission tomography (PET) provides advantages of high resolution, sensitivity and quantification accuracy compared to SPECT. Affibody molecules, imaging probes based on a non-immunoglobulin scaffold, provide high imaging contrast shortly after injection. The aim of this study was to evaluate feasibility of PET imaging of HER3 expression using Ga-68-labeled affibody molecules. The anti-HER3 affibody molecule HEHEHE-Z08698-NOTA was successfully labelled with Ga-68 with high yield, purity and stability. The agent bound specifically to HER3-expressing cancer cells in vitro and in vivo. At 3 h pi, uptake of Ga-68-HEHEHE-Z08698-NOTA was significantly higher in xenografts with high HER3 expression (BT474, BxPC-3) than in xenografts with low HER3 expression (A431). In xenografts with high expression, tumour-to-blood ratios were >20, tumour-to-muscle >15, and tumour-to-bone >7. HER3-positive xenografts were visualised using microPET 3 h pi. In conclusion, PET imaging of HER3 expression is feasible using Ga-68-HEHEHE-Z08698-NOTA shortly after administration.

    National Category
    Medical Image Processing Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-266695 (URN)10.1038/srep15226 (DOI)000362985400001 ()26477646 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Research Council
    Available from: 2015-11-12 Created: 2015-11-10 Last updated: 2018-09-20Bibliographically approved
    4. Evaluation of radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression
    Open this publication in new window or tab >>Evaluation of radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression
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    2017 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 51, no 6, p. 1765-1774Article in journal (Refereed) Published
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-targeted therapies. Several HER3‑targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-to-blood ratios significantly increased between 3 and 24 h p.i. (p<0.05). At 24 h p.i., tumour-to-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3‑expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.

    Keywords
    HER3, affibody, PET imaging, Cobalt-55/57, NOTA-chelator
    National Category
    Radiology, Nuclear Medicine and Medical Imaging
    Identifiers
    urn:nbn:se:uu:diva-343404 (URN)10.3892/ijo.2017.4152 (DOI)000416685600014 ()
    Funder
    Knut and Alice Wallenberg FoundationSwedish Cancer Society, CAN2014/474Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Swedish Research Council, 2012-05236Swedish Cancer Society, CAN2015/350Swedish Cancer Society, CAN2016/463VINNOVA, 2016-04060
    Available from: 2018-02-27 Created: 2018-02-27 Last updated: 2018-09-20Bibliographically approved
    5. Improved contrast of affibody-mediated imaging of HER3 expression through co-injection of affibody trimer for in vivo blocking of hepatic uptake
    Open this publication in new window or tab >>Improved contrast of affibody-mediated imaging of HER3 expression through co-injection of affibody trimer for in vivo blocking of hepatic uptake
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    HER3, affibody molecule, molecular imaging, imaging contrast
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-360288 (URN)
    Available from: 2018-09-12 Created: 2018-09-12 Last updated: 2018-09-20
  • 256. Rosjo, Helge
    et al.
    Opstad, Per-Kristian
    Hoff, Jon Erik
    Godang, Kristin
    Christensen, Geir
    Stridsberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemical endocrinology.
    Omland, Torbjorn
    Effect of short- and long-term physical activities on circulating granin protein levels2013In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 185, p. 14-19Article in journal (Refereed)
    Abstract [en]

    Background: The classic chromogranin-secretogranin (granin) proteins are produced in the myocardium and throughout the neuroendocrine system, but while chromogranin (Cg) A and B levels are high in the adrenal medulla, secretogranin (Sg) II production is higher in the pituitary gland. Whether these differences may influence the response to physical activity is not known. Methods: We measured circulating granin proteins during (1) a short-term maximal bicycle exercise stress test and (2) a 7 day military ranger course of continuous physical activity and sleep and energy deprivation. Results: In 9 healthy subjects performing the exercise stress test (7 male, age 45 +/- 5 y [mean +/- SEM], duration 10.13 +/- 1.14 min), CgB levels increased from before to immediately after the test: 1.20 +/- 0.12 vs. 1.45 +/- 0.09 nmol/L, p = 0.013. Metabolic equivalents, representing an index of performed work, were closely associated with the change (Delta) in CgB levels during stress testing and explained 74% of the variability in (Delta)CgB levels (p = 0.004). CgA and SgII levels were not increased after exercise stress testing. In the second cohort of 8 male subjects (age 25 +/- 1 y) participating in the ranger course, CgB levels increased from day 1 and wire significantly elevated on days 5 and 7. CgA also increased gradually with levels significantly elevated on day 7, while SgII was markedly increased on day 5 whereas levels on days 3 and 7 were unchanged compared to baseline levels. Conclusion: We demonstrate a heterogeneous response to short- and long-term physical activities among circulating granin proteins with the most potent effect on CgB levels.

  • 257.
    Roslin, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Dahl, Kenneth
    Massachusetts Gen Hosp, Div Nucl Med & Mol Imaging, Boston, MA 02114 USA; Harvard Med Sch, Dept Radiol, Boston, MA USA.
    Nordeman, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Reaction of 11C‐benzoyl chlorides with metalloid reagents: 11C‐labeling of benzyl alcohols, benzaldehydes, and phenylketones from [11C]CO2018In: Journal of Labelled Compounds and Radiopharmaceuticals, ISSN 0362-4803, Vol. 61, no 5, p. 447-454Article in journal (Refereed)
    Abstract [en]

    In this article, we describe the carbon‐11 (11C, t1/2 = 20.4 minutes) labeling of benzyl alcohols, benzaldehydes, and ketones using an efficient 2-€step synthesis in which 11C-€carbon monoxide is used in an initial palladium-€mediated reaction to produce 11C-€benzoyl chloride as a key intermediate. In the second step, the obtained 11C-€benzoyl chloride is further treated with a metalloid reagent to furnish the final 11C-€labeled product. Benzyl alcohols were obtained in moderated to high non‐isolated radiochemical yields (RCY, 35%-90%) with lithium aluminum hydride or lithium aluminum deuteride as metalloid reagent. Changing the metalloid reagent to either tributyltin hydride or sodium borohydride, allowed for the reliable syntheses of 11C-€benzaldehydes in RCYs ranging from 58% to 95%. Finally, sodium tetraphenylborate were utilized to obtain 11C-€phenyl ketones in high RCYs (77%-95%). The developed method provides a new and efficient route to 3 different classes of compounds starting from aryl iodides or aryl bromides.

  • 258.
    Rosén, Josefin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Lövgren, Anders
    Uppsala University.
    Kogej, Thierry
    DECS Global Compound Sciences, AstraZeneca R&D, Mölndal.
    Muresan, Sorel
    DECS Global Compound Sciences, AstraZeneca R&D, Mölndal.
    Gottfries, Johan
    Pharmnovo Inc., Göteborg.
    Backlund, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    ChemGPS-NPweb: chemical space navigation online2009In: Journal of Computer-Aided Molecular Design, ISSN 0920-654X, E-ISSN 1573-4951, Vol. 23, no 4, p. 253-259Article in journal (Refereed)
    Abstract [en]

    Internet has become a central source for information, tools, and services facilitating the work for medicinal chemists and drug discoverers worldwide. In this paper we introduce a web-based public tool, ChemGPS-NPWeb (http://chemgps.bmc.uu.se), for comprehensive chemical space navigation and exploration in terms of global mapping onto a consistent, eight dimensional map over structure derived physico-chemical characteristics. ChemGPS-NPWeb can assist in compound selection and prioritization; property description and interpretation; cluster analysis and neighbourhood mapping; as well as comparison and characterization of large compound datasets. By using ChemGPS-NPWeb, researchers can analyze and compare chemical libraries in a consistent manner. In this study it is demonstrated how ChemGPS-NPWeb can assist in interpreting results from two large datasets tested for activity in biological assays for pyruvate kinase and Bcl-2 family related protein interactions, respectively. Furthermore, a more than 30-year-old suggestion of “chemical similarity” between the natural pigments betalains and muscaflavins is tested.

  • 259.
    Rotili, Dante
    et al.
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Samuele, Alberta
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Tarantino, Domenico
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Ragno, Rino
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Musmuca, Ira
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Ballante, Flavio
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Botta, Giorgia
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Morera, Ludovica
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Pierini, Marco
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Cirilli, Roberto
    Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
    Nawrozkij, Maxim B
    Volgograd State Technical University, prospekt Lenina, 28, 400131 Volgograd, Russia.
    Gonzalez, Emmanuel
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Clotet, Bonaventura
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Artico, Marino
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Esté, José A
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Maga, Giovanni
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Mai, Antonello
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    2-(Alkyl/aryl)amino-6-benzylpyrimidin-4(3H)-ones as inhibitors of wild-type and mutant HIV-1: enantioselectivity studies.2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 7, p. 3558-62Article in journal (Refereed)
    Abstract [en]

    The single enantiomers of two pyrimidine-based HIV-1 non-nucleoside reverse transcriptase inhibitors, 1 (MC1501) and 2 (MC2082), were tested in both cellular and enzyme assays. In general, the R forms were more potent than their S counterparts and racemates and (R)-2 was more efficient than (R)-1 and the reference compounds, with some exceptions. Interestingly, (R)-2 displayed a faster binding to K103N RT with respect to WT RT, while (R)-1 showed the opposite behavior.

  • 260.
    Rotili, Dante
    et al.
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Tarantino, Domenico
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Nawrozkij, Maxim B
    Volgograd State Technical University, pr. Lenina, 28, 400131 Volgograd, Russia.
    Babushkin, Alexandre S
    Volgograd State Technical University, pr. Lenina, 28, 400131 Volgograd, Russia.
    Botta, Giorgia
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Marrocco, Biagina
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Cirilli, Roberto
    Dipartimento del Farmaco, Istituto Superiore di Sanita ̀ ,, Viale Regina Elena 299, 00161 Rome, Italy.
    Menta, Sergio
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Badia, Roger
    IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auto ̀ noma de Barcelona, 08916 Badalona, Spain.
    Crespan, Emmanuele
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Ballante, Flavio
    Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy; Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, School of Medicine, 700 South Euclid Avenue, St. Louis, Missouri 00185, United States.
    Ragno, Rino
    Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Esté, José A
    IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auto ̀ noma de Barcelona, 08916 Badalona, Spain.
    Maga, Giovanni
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Mai, Antonello
    Dipartimento di Chimica e Tecnologie del Farmaco, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy; Istituto Pasteur  Fondazione Cenci Bolognetti, Universita ̀ degli Studi di Roma “ La Sapienza ” , P.le A. Moro 5, 00185 Roma, Italy.
    Exploring the role of 2-chloro-6-fluoro substitution in 2-alkylthio-6-benzyl-5-alkylpyrimidin-4(3H)-ones: effects in HIV-1-infected cells and in HIV-1 reverse transcriptase enzymes.2014In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, no 12, p. 5212-25Article in journal (Refereed)
    Abstract [en]

    A comparison of the effects of the 6-(2-chloro-6-fluorobenzyl)-2-(alkylthio)pyrimidin-4(3H)-ones (2-Cl-6-F-S-DABOs) 7-12 and the related 6-(2,6-difluorobenzyl) counterparts 13-15 in HIV-1 infected cells and in the HIV-1 reverse transcriptase (RT) assays is here described. The new 2-Cl-6-F-S-DABOs showed up to picomolar activity against wt HIV-1. Against clinically relevant HIV-1 mutants and in enzyme assays, the simultaneous C5(methyl)/C6(methyl/ethyl) substitution in the 2-Cl-6-F- and 2,6-F2-benzyl series furnished compounds with the highest, wide-spectrum inhibitory activity against HIV-1. Three representative 2-Cl-6-F-S-DABOs carrying two (9c, 10c) or one (10a) stereogenic centers were resolved into their individual stereoisomers and showed a significant diastereo- and enantioselectivity in HIV-1 inhibition, the highest antiviral activity well correlating with the R absolute configuration to the stereogenic center of the C6-benzylic position in both cellular and enzymatic tests. Application of previously reported COMBINEr protocol on 9c and 10c confirmed the influence of the stereogenic centers on their binding modes in the HIV-1 RT.

  • 261.
    Rudling, Axel
    et al.
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Gustafsson, Robert
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Almlof, Ingrid
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Homan, Evert
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Scobie, Martin
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Berglund, Ulrika Warpman
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Helleday, Thomas
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Stenmark, Pal
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space2017In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 19, p. 8160-8169Article in journal (Refereed)
    Abstract [en]

    Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 mu M. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.

  • 262. Rundlöf, Torgny
    et al.
    Mathiasson, Marie
    Bekiroglu, Somer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hakkarainen, Birgit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bowden, Tim
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Survey and qualification of internal standards for quantification by 1H NMR spectroscopy2010In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 52, no 5, p. 645-651Article in journal (Refereed)
    Abstract [en]

    In quantitative NMR (qNMR) selection of an appropriate internal standard proves to be crucial. In this study, 25 candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts, purity, solubility, and ease of use. The 1H chemical shift (δ) values, assignments, multiplicities and number of protons (for each signal), appropriateness (as to be used as internal standards) in four different deuterated solvents (D2O, DMSO-d6, CD3OD, CDCl3) were studied. Taking into account the properties of these 25 internal standards, the most versatile eight compounds (2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid and fumaric acid) were qualified using both differential scanning calorimetry (DSC) and NMR spectroscopy employing highly pure acetanilide as the reference standard. The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards. Finally, the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

  • 263.
    Rydevik, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the in vitro production of drug metabolites using fungi of the Cunninghamella species. The metabolites were characterized with mainly liquid chromatography-mass spectrometry using ion-trap and quadrupole-time-of-flight instruments. A fungal in vitro model has several advantages e.g., it is easily up-scaled and ethical problems associated with animal-based models are avoided.

    The metabolism of bupivacaine and the selective androgen receptor modulators (SARMs) S1, S4 and S24 by the fungi Cunninghamella elegans and Cunninghamella blakesleeana was investigated. The detected metabolites were compared with those formed in vitro and in vivo by human and horse and most phase I metabolites formed by mammals were also formed by the fungi. The higher levels of bupivacaine metabolites in the fungal samples allowed an extensive mass spectrometric structural characterization which shows that the fungi are relevant metabolic models.

    Glucuronides are important drug metabolites but they are difficult to synthesize. The discovery that the fungus Cunninghamella elegans formed large amounts of glucosides led to the idea that they could be used to form glucuronides. A new concept was developed where a fungal incubate containing a SARM S1 glucoside was mixed with the free radical tetramethylpiperidinyl-1-oxy (TEMPO), sodium bromide and sodium hypochlorite which produced a glucuronide. Isolation and characterization by nuclear magnetic resonance spectroscopy proved that the new method could produce glucuronides for use as reference material.

    An investigation of reactive metabolite formation of the drugs paracetamol, mefenamic acid and diclofenac by the fungus Cunninghamella elegans was performed. It was demonstrated for the first time that the fungus could produce glutathione, glutathione ethyl-ester, cysteine and N-acetylcysteine conjugates that are indicative of a preceding formation of reactive intermediates. A comparison with conjugates formed by human liver microsomes showed that both models formed identical metabolites.

    The presented investigations prove that Cunninghamella fungi are relevant drug metabolism models. They show that the fungi to a large extent forms the same metabolites as mammals and that they can produce metabolites for use as reference material in, e.g. doping control. It was also demonstrated that the fungal model can be used in the important assessment of drug toxicity.

    List of papers
    1. Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
    Open this publication in new window or tab >>Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
    2012 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, p. 1338-1346Article in journal (Refereed) Published
    Abstract [en]

    RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial.

    METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d9. Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MSn and hydrogen/deuterium exchange.

    RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana.

    CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

    National Category
    Medicinal Chemistry Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-173545 (URN)10.1002/rcm.6225 (DOI)000303597100009 ()
    Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2018-01-12Bibliographically approved
    2. The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
    Open this publication in new window or tab >>The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
    Show others...
    2013 (English)In: Xenobiotica, ISSN 0049-8254, E-ISSN 1366-5928, Vol. 43, no 5, p. 409-420Article in journal (Refereed) Published
    Abstract [en]

    1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites.

    2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation.

    4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

    National Category
    Pharmaceutical Sciences Medicinal Chemistry Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-196318 (URN)10.3109/00498254.2012.729102 (DOI)000316952100002 ()
    Note

    MEDLINE AN 2013135005(Journal; Article; (JOURNAL ARTICLE))

    Available from: 2013-03-07 Created: 2013-03-07 Last updated: 2018-01-11Bibliographically approved
    3. Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
    Open this publication in new window or tab >>Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
    2013 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 84, p. 278-284Article in journal (Refereed) Published
    Abstract [en]

    A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost.

    Keywords
    High resolution mass spectrometry, UPLC, Glucuronides, SARM, TEMPO
    National Category
    Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-205709 (URN)10.1016/j.jpba.2013.06.012 (DOI)000322752800040 ()23867089 (PubMedID)
    Available from: 2013-08-22 Created: 2013-08-22 Last updated: 2017-12-06Bibliographically approved
    4. Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
    Open this publication in new window or tab >>Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
    Show others...
    2014 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 98, p. 36-39Article in journal (Refereed) Published
    Abstract [en]

    In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of beta-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM Si was incubated with the fungus C elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid beta-conjugate of hydroxylated SARM Si bearing the glucuronide moiety on carbon C-10.

    National Category
    Medicinal Chemistry Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-220769 (URN)10.1016/j.jpba.2014.05.001 (DOI)000339859800005 ()
    Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2018-01-11Bibliographically approved
    5. A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
    Open this publication in new window or tab >>A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medicinal Chemistry Pharmaceutical Sciences Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-220770 (URN)
    Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2018-01-11
  • 264.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Mikael, Hedeland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry2012In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, p. 1338-1346Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial.

    METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d9. Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MSn and hydrogen/deuterium exchange.

    RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana.

    CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

  • 265.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hellqvist, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometryManuscript (preprint) (Other academic)
  • 266.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lagojda, Andreas
    Bayer CropScience AG.
    Thevis, Mario
    Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Cologne.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation2014In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 98, p. 36-39Article in journal (Refereed)
    Abstract [en]

    In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of beta-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM Si was incubated with the fungus C elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid beta-conjugate of hydroxylated SARM Si bearing the glucuronide moiety on carbon C-10.

  • 267.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Thevis, Mario
    Krug, Oliver
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)2013In: Xenobiotica, ISSN 0049-8254, E-ISSN 1366-5928, Vol. 43, no 5, p. 409-420Article in journal (Refereed)
    Abstract [en]

    1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites.

    2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation.

    4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

  • 268.
    Rydfjord, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Sävmarker, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Konda, Vivek
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Skillinghaug, Bobo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Continuous flow chemistry with a non-resonant microwave applicator2014In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 248Article in journal (Other academic)
  • 269.
    Rönn, Robert
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. ORGFARM.
    Sabnis, Yogesh A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. ORGFARM.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. ORGFARM.
    Johansson, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. ORGFARM.
    Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS32006In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 14, no 2, p. 544-559Article in journal (Refereed)
  • 270.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Low sub-minimal inhibitory concentrations of antibiotics generate new types of resistance2019In: Sustainable Chemistry and Pharmacy, ISSN 2352-5541, Vol. 11, p. 46-48Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance is a major threat to modern medicine. Routes of transmission of resistant bacteria are complex and include spread between humans, between humans and animals, between animals and to humans and animals via the environment. Recent findings have shown that resistant bacteria can be selectively enriched even at antibiotic concentrations several hundred-fold lower than previously expected, such as those found in sewage water. In addition, these low concentrations can select for high level resistant bacteria with very low fitness cost in contrast to resistant bacteria selected at high concentrations such as during antibiotic treatment of patients. This calls for action to determine what concentrations and combinations of antibiotics that can be considered safe in waste water and ensure proper measures to reduce the antropogenic contamination with antibiotics.

  • 271.
    Sawant, Rajiv T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Stevens, Marc Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Acetic acid-promoted cascade N-acyliminium ion/aza-Prins cyclization: stereoselective synthesis of functionalized fused tricyclic piperidines2017In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 53, no 13, p. 2110-2113Article in journal (Refereed)
    Abstract [en]

    A novel acetic acid-promoted metal-free cascade N-acyliminium ion/aza-Prins cyclization of o-formyl carbamates and homoallylamines is reported. This one-pot protocol provides efficient and rapid access to masked cis-hydroxyhexahydropyrido[1,2-c] quinazolin-6-ones with concomitant generation of two stereogenic centers, four C-C/C-O/C-N bonds and two new rings in good yield and excellent diastereoselectivity.

  • 272.
    Sawant, Rajiv T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Stevens, Marc Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala Univ, Dept Med Chem, Uppsala Biomed Ctr, POB 574, SE-75123 Uppsala, Sweden;Beactica AB, Uppsala Business Pk,Virdings Alle 2, S-75450 Uppsala, Sweden.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala Univ, Dept Med Chem, Uppsala Biomed Ctr, POB 574, SE-75123 Uppsala, Sweden.
    Microwave-Assisted aza-Friedel-Crafts Arylation of N-Acyliminium Ions: Expedient Access to 4 -Aryl 3,4-Dihydroquinazolinones2018In: ACS OMEGA, ISSN 2470-1343, Vol. 3, no 10, p. 14258-14265Article in journal (Refereed)
    Abstract [en]

    A one-pot microwave-assisted aza-Friedel-Crafts arylation of N-acyliminium ions, generated in situ from o-formyl carbamates and different amines, is reported. This metal-free protocol provides rapid access to diverse 4-aryl 3,4-dihydroquinazolinones in excellent yield without any aqueous workup. A solvent-directed process for the selective aza-Friedel-Crafts arylation of electron-rich aryl/heteroaryl/butenyl-tethered N-acyliminium ions is also described.

  • 273.
    Sawant, Rajiv T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Stevens, Marc Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Sköld, Christian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Microwave-Assisted Branching Cascades: A Route to Diverse 3,4-Dihydroquinazolinone-Embedded Polyheterocyclic Scaffolds2016In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 18, no 20, p. 5392-5395Article in journal (Refereed)
    Abstract [en]

    A novel metal-free microwave-assisted branching cascades strategy for the efficient synthesis of 3,4-dihydro-quinazolinone-embedded polyheterocyclic scaffolds is reported. Starting from in situ generated key N-acyliminium ion precursors, 12 distinct and skeletally diverse polycyclic frameworks were accessed in a single step/pot via adjustment of the nucleophile(s) and reaction conditions. Postcascade functionalization of these compounds was also demonstrated, proving the utility of this method in accessing structurally diverse chemical entities.

  • 274.
    Schaal, Wesley
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Computational Studies of HIV-1 Protease Inhibitors2002Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human Immunodeficiency Virus (HIV) is the causative agent of the pandemic disease Acquired Immune Deficiency Syndrome (AIDS). HIV acts to disrupt the immune system which makes the body susceptible to opportunistic infections. Untreated, AIDS is generally fatal. Twenty years of research by countless scientists around the world has led to the discovery and exploitation of several targets in the replication cycle of HIV. Many lives have been saved, prolonged and improved as a result of this massive effort. One particularly successful target has been the inhibition of HIV protease. In combination with the inhibition of HIV reverse transcriptase, protease inhibitors have helped to reduce viral loads and partially restore the immune system. Unfortunately, viral mutations leading to drug resistance and harmful side-effects of the current medicines have identified the need for new drugs to combat HIV.

    This study presents computational efforts to understand the interaction of inhibitors to HIV protease. The first part of this study has used molecular modelling and Comparative Molecular Field Analysis (CoMFA) to help explain the structure-active relationship of a novel series of protease inhibitors. The inhibitors are sulfamide derivatives structurally similar to the cyclic urea candidate drug mozenavir (DMP-450). The central ring of the sulfamides twists to adopt a nonsymmetrical binding mode distinct from that of the cyclic ureas. The energetics of this twist has been studied with ab initio calculations to develop improved empirical force field parameters for use in molecular modelling.

    The second part of this study has focused on an analysis of the association and dissociation kinetics of a broad collection of HIV protease inhibitors. Quantitative models have been derived using CoMFA which relate the dissociation rate back to the chemical structures. Efforts have also been made to improve the models by systematically varying the parameters used to generate them.

  • 275.
    Schowen, Katharine Barbara
    et al.
    Univ Kansas, Dept Chem, Lawrence, KS 66045 USA..
    Schowen, Richard L.
    Univ Kansas, Dept Chem, Lawrence, KS 66045 USA.;Univ Kansas, Dept Mol Biosci, Lawrence, KS 66047 USA.;Univ Kansas, Dept Pharmaceut Chem, Lawrence, KS 66047 USA..
    Borchardt, Susan E.
    Borchardt, Paul M.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Audus, Kenneth L.
    Univ Kansas, Sch Pharm, Lawrence, KS 66047 USA..
    Augustijns, Patrick
    Katholieke Univ Leuven, Drug Delivery & Disposit, Dept Pharmaceut & Pharmacol Sci, B-3000 Leuven, Belgium..
    Nicolazzo, Joseph A.
    Monash Univ, Monash Inst Pharmaceut Sci, Drug Delivery Disposit & Dynam, Parkville, Vic 3052, Australia..
    Raub, Thomas J.
    Eli Lilly & Co, Lilly Res Labs, Drug Disposit, Indianapolis, IN 46285 USA..
    Schoneich, Christian
    Univ Kansas, Dept Pharmaceut Chem, Lawrence, KS 66047 USA..
    Siahaan, Teruna J.
    Univ Kansas, Dept Pharmaceut Chem, Lawrence, KS 66047 USA..
    Takakura, Yoshi
    Kyoto Univ, Dept Biopharmaceut & Drug Metab, Grad Sch Pharmaceut Sci, Kyoto 6068501, Japan..
    Thakker, Dhiren R.
    Univ N Carolina, Div Pharmacotherapy & Expt Therapeut, Eshelman Sch Pharm, Chapel Hill, NC 27599 USA..
    Wolfe, Michael S.
    Harvard Med Sch, Brigham & Womens Hosp, Ann Romney Ctr Neurol Dis, Boston, MA 02115 USA..
    A Tribute to Ronald T. Borchardt-Teacher, Mentor, Scientist, Colleague, Leader, Friend, and Family Man2016In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 105, no 2, p. 370-385Article in journal (Other academic)
  • 276.
    Sebastiano, Matteo Rossi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Doak, Bradley C.
    Monash Univ, MIPS, Dept Med Chem, Victoria, Australia.
    Backlund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Poongavanam, Vasanthanathan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Over, Björn
    AstraZeneca R&D Gothenburg, Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, Mölndal, Sweden.
    Ermondi, Giuseppe
    Univ Turin, Dept Mol Biotechnol & Hlth Sci, Turin, Italy.
    Caron, Giulia
    Univ Turin, Dept Mol Biotechnol & Hlth Sci, Turin, Italy.
    Matsson, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Impact of Dynamically Exposed Polarity on Permeability and Solubility of Chameleonic Drugs Beyond the Rule of 52018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 9, p. 4189-4202Article in journal (Refereed)
    Abstract [en]

    Conformational flexibility has been proposed to significantly affect drug properties outside rule-of-5 (Ro5) chemical space. Here, we investigated the influence of dynamically exposed polarity on cell permeability and aqueous solubility for a structurally diverse set of drugs and clinical candidates far beyond the Ro5, all of which populated multiple distinct conformations as revealed by X-ray crystallography. Efflux-inhibited (passive) Caco-2 cell permeability correlated strongly with the compounds’ minimum solvent-accessible 3D polar surface areas (PSA), whereas aqueous solubility depended less on the specific 3D conformation. Inspection of the crystal structures highlighted flexibly linked aromatic side chains and dynamically forming intramolecular hydrogen bonds as particularly effective in providing “chameleonic” properties that allow compounds to display both high cell permeability and aqueous solubility. These structural features, in combination with permeability predictions based on the correlation to solvent-accessible 3D PSA, should inspire drug design in the challenging chemical space far beyond the Ro5.

  • 277. Sehgelmeble, Fernando
    et al.
    Jansson, Juliette
    Ray, Colin
    Rosqvist, Susanne
    Gustavsson, Susanne
    Nilsson, Linda I
    Minidis, Alexander
    Holenz, Jörg
    Rotticci, Didier
    Lundkvist, Johan
    Arvidsson, Per I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Sulfonimidamides as Sulfonamides Bioisosteres: Rational Evaluation through Synthetic, in Vitro, and in Vivo Studies with γ-Secretase Inhibitors2012In: ChemMedChem, ISSN 1860-7179, E-ISSN 1860-7187, Vol. 7, no 3, p. 396-399Article in journal (Refereed)
    Abstract [en]

    The proof of the pudding: A proof-of-concept study using γ-secretase inhibitors as a model has shown that sulfonimidamides act as bioisosteres for sulfonamides. Detailed in vitro and in vivo profiling reveal that the sulfonimidamide motif imparts desirable properties such as decreased lipophilicity and plasma protein binding, accompanied by increased solubility. Our data support a wider use of this unique functional group in the design of new pharmacologically active agents.

  • 278.
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS.

    In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines.

    In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

    List of papers
    1. Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
    Open this publication in new window or tab >>Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
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    2015 (English)In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 14, no 6, p. 1504-1516Article in journal (Refereed) Published
    Abstract [en]

    Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning-using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials.

    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-260735 (URN)10.1158/1535-7163.MCT-14-0792 (DOI)000358054300025 ()25911689 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Research CouncilSwedish Childhood Cancer Foundation
    Available from: 2015-08-24 Created: 2015-08-24 Last updated: 2017-12-04Bibliographically approved
    2. Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
    Open this publication in new window or tab >>Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
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    2016 (English)In: CELL CHEMICAL BIOLOGY, ISSN 2451-9448, Vol. 23, no 11, p. 1428-1438Article in journal (Refereed) Published
    Abstract [en]

    Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

    National Category
    Cell and Molecular Biology Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-311191 (URN)10.1016/j.chembiol.2016.09.013 (DOI)000388373200015 ()27984028 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Foundation for Strategic Research
    Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2019-04-29Bibliographically approved
    3. Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
    Open this publication in new window or tab >>Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Cancer and Oncology Medicinal Chemistry
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-320596 (URN)
    Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2018-01-13
    4. Targeting tumor cells based on PDE3A expression
    Open this publication in new window or tab >>Targeting tumor cells based on PDE3A expression
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Medicinal Chemistry Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-320597 (URN)
    Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2018-01-13
  • 279.
    Senkowski, Wojciech
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nazir, Madiha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cellsManuscript (preprint) (Other academic)
  • 280. Shalaly, Nancy Dekki
    et al.
    Aneiros, Eduardo
    Blank, Michael
    Mueller, Johan
    Nyman, Eva
    Blind, Michael
    Dabrowski, Michael A
    Andersson, Christin V
    Sandberg, Kristian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers2015In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, no 9, p. 1112-1123Article in journal (Refereed)
    Abstract [en]

    According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl(-) channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1β. This aptamer potentiated whole-cell Cl(-) currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

  • 281.
    Shwter, Abdrabuh N.
    et al.
    Univ Malaya, Dept Biomed Sci, Fac Med, Kuala Lumpur 50603, Malaysia..
    Abdullah, Nor Azizan
    Univ Malaya, Dept Pharmacol, Fac Med, Kuala Lumpur 50603, Malaysia..
    Alshawsh, Mohammed A.
    Univ Malaya, Dept Pharmacol, Fac Med, Kuala Lumpur 50603, Malaysia..
    El-Seedi, Hesham R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Univ Malaya, Dept Chem, Fac Sci, Kuala Lumpur 50603, Malaysia..
    Al-Henhena, Nawal A.
    Univ Malaya, Dept Biomed Sci, Fac Med, Kuala Lumpur 50603, Malaysia..
    Khalifa, Shaden A. M.
    Karolinska Univ Hosp, Dept Expt Hematol, SE-14186 Stockholm, Sweden..
    Abdulla, Mahmood A.
    Univ Malaya, Dept Biomed Sci, Fac Med, Kuala Lumpur 50603, Malaysia..
    Chemopreventive effect of Phaleria macrocarpa on colorectal cancer aberrant crypt foci in vivo2016In: Journal of Ethnopharmacology, ISSN 0378-8741, E-ISSN 1872-7573, Vol. 193, p. 195-206Article in journal (Refereed)
    Abstract [en]

    Ethnopharmacological relevance: Natural products are important ingredients for pharmaceutical applications specifically new entities for treating cancer and other diseases. Phaleria macrocarpa is native of Indonesia and considered as a prolific source of bioactive substances useful for chemoprevention. Aim of the study: To investigate the chemopreventive properties of Phaleria macrocarpa on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. Methods: The biological activities of the ethanol extract of P. macrocarpa fruits were evaluated both in vitro and in vivo. First the extract was investigated for its in vitro antioxidant activity by the total phenolic content and ferric reducing antioxidant power assay. Then the chemopreventive effect of P. macrocarpa was performed on AOM-induced aberrant crypt foci as colorectal carcinoma model in rats. Result: the crude ethanolic extract of P. macrocarpa has high antioxidant activity and modulated the oxidative stress as proved by the up-regulation of glutathione-s-transferase and superoxide dismutase. Immunohistochemical staining of the treated sections showed overexpression of PCNA and Bax, reduced crypt sizes and numbers, indicating the characteristic feature of apoptotic cancer cells. PCNA is a landmark of cell damage and turn-over and can be associated with clinical cancer mutation. The most potent doses were 250 mg/kg and 500 mg/kg as compared to 35 mg/kg 5-fluorouracil. Conclusion: In this sense, the potential modulation of the colorectal pathophysiological pathway by P. macrocarpa natural compounds mostly flavonoids offer a great possibility for the discovery of new leads towards the colorectal cancer.

  • 282. Sibley, Graham Edward Morris
    et al.
    Malmstroem, Lars Jonas
    Larsson, Johanna Maria.
    2-amino-1,3,4-thiadiazine and 2-amino-1,3,4-oxadiazine based antifungal agents2017Patent (Other (popular science, discussion, etc.))
  • 283.
    Silvestri, Laura
    et al.
    Rome Center for Molecular Design Dipartimento di Chimica e Tecnologie del Farmaco, Facolta ̀ di Farmacia e Medicina.
    Ballante, Flavio
    Rome Center for Molecular Design Dipartimento di Chimica e Tecnologie del Farmaco, Facolta ̀ di Farmacia e Medicina.
    Mai, Antonello
    Istituto Pasteur - Fondazione Cenci Bolognetti Dipartimento di Chimica e Tecnologie del Farmaco, Facolta ̀ di Farmacia e Medicina.
    Marshall, Garland R
    Rome Center for Molecular Design Dipartimento di Chimica e Tecnologie del Farmaco, Facolta ̀ di Farmacia e Medicina; Visiting Professor from the Department of Biochemistry and Molecular Biophysics, Wash ington University School of Medicine, St. Louis, Missouri 63110, United States.
    Ragno, Rino
    Rome Center for Molecular Design Dipartimento di Chimica e Tecnologie del Farmaco, Facolta ̀ di Farmacia e Medicina.
    Histone deacetylase inhibitors: structure-based modeling and isoform-selectivity prediction.2012In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 52, no 8, p. 2215-35Article in journal (Refereed)
    Abstract [en]

    An enhanced version of comparative binding energy (COMBINE) analysis, named COMBINEr, based on both ligand-based and structure-based alignments has been used to build several 3-D QSAR models for the eleven human zinc-based histone deacetylases (HDACs). When faced with an abundance of data from diverse structure-activity sources, choosing the best paradigm for an integrative analysis is difficult. A common example from studies on enzyme-inhibitors is the abundance of crystal structures characterized by diverse ligands complexed with different enzyme isoforms. A novel comprehensive tool for data mining on such inhomogeneous set of structure-activity data was developed based on the original approach of Ortiz, Gago, and Wade, and applied to predict HDAC inhibitors' isoform selectivity. The COMBINEr approach (apart from the AMBER programs) has been developed to use only software freely available to academics.

  • 284. Simmen, K. A.
    et al.
    De Kock, H. A.
    Raboisson,, P. J.
    Hu, L.
    Tahri, A.
    Surleraux,, D. L. N. G.
    Nilsson, K. M.
    Samuelsson, B. B.
    Rosenquist, Å. A. K.
    Ivanov, V.
    Pelcman, M.
    Belfrage,, Anna Karin G. L.
    Johansson, P. M.
    Vendeville,, S. M. H.
    Macrocylic inhibitors of hepatitis C virus2012Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    Inhibitors of HCV replication of formula (I)

    and the N-oxides, salts, and stereoisomers, wherein

    • each dashed line represents an optional double bond;
    • X is N, CH and where X bears a double bond it is C;
    • R1 is —OR7, —NH—SO2R8;
    • R2 is hydrogen, and where X is C or CH, R2 may also be C1-6alkyl;
    • R3 is hydrogen, C1-6alkyl, C1-6alkoxyC1-6alkyl, C3-7cycloalkyl;
    • R4 is aryl or Het; n is 3, 4, 5, or 6;
    • R5 is halo, C1-6alkyl, hydroxy, C1-6alkoxy, phenyl, or Het;
    • R6 is C1-6alkoxy, or dimethylamino;
    • R7 is hydrogen; aryl; Het; C3-7cycloalkyl optionally substituted with C1-6alkyl; or C1-6alkyl optionally substituted with C3-7cycloalkyl, aryl or with Het;
    • R8 is aryl; Het; C3-7cycloalkyl optionally substituted with C1-6alkyl; or C1-6alkyl optionally substituted with C3-7cycloalkyl, aryl or with Het;
    • aryl is phenyl optionally substituted with one, two or three substituents;
    • Het is a 5 or 6 membered saturated, partially unsaturated or completely unsaturated heterocyclic ring containing 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and being optionally substituted with one, two or three substituents;
    • pharmaceutical compositions containing compounds (I) and processes for preparing compounds (I). Bioavailable combinations of the inhibitors of HCV of formula (I) with ritonavir are also provided.
  • 285.
    Sjöberg, Hans
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Release and transport of drugs in some complex and interacting systems2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Drug formulations come in many forms, some intended for direct and immediate action and others aiming at a controlled manner of drug delivery. Viewed in a more general way drug transport often involves charged units, it takes place in a medium which in many respects must be considered complex - to some degree even reactive. Drug transport may also be competitive because of thermo- and hydrodynamic interactions, These three aspects of drug transport in general is discussed and exemplified in specific studies: 1) A conductivity study as a fundamental investigation for iontophoretic drug delivery; 2) a methodological development and diffusivity investigation of a steroidal hormone transport in silica containing silicone; 3) an investigation of competitive diffusion transport of small amphiphilic molecules in a polyelectrolyte containing semi-solid system.

    Iontophoresis - drug administration by electric current - normally requires charged drug molecules, hence their mobility and state of dissociation are essential parameters. The medium in which the drug is situated regulates the electrolytic drug content (e.g. dissociation equilibrium) and thus the iontophoretic efficiency. The conductivity of the local anesthetic, IidocaineHCl was investigated by high precision conductometry in aqueous and octanol solutions. From conductivity data and applied advanced electrolyte theory drug mobility parameters as well as, dissociation and association constants were obtained. The drug ion mobility was found, to be a factor 50 lower in octanol. The equilibrium constants obtained show that increasing drug concentration favors ion formation in water while favoring ion pair association in octanol. Hence, such a drug behaves very differently in different media, with obvious clinical implications.

    The dynamic diffusion in the complex system silicone/silica/estradiol was studied by means of a new equipment design which has considerably improved the possibility to perform precise quantitative dynamic diffusion measurements in such systems, The method avoids all disturbing interfaces between different media and allows for a precise spatial resolution of concentration of the diffusing drug. Data showed the method to be dependable and accurate. The effect on estradiol diffusivity due to varied silica content in the silicone is found to be paramount, increasing [SiO2] from 17 to 30% decreased the diffusivity by 68%. Some mechanisms are discussed.

    The diffusion of amphiphiles within and the release from agarose hydrogels containing the polyelectrolyte carrageenan was investigated. All drugs exhibit same apparent diffusivity in pure agarose gel. The diffusivity decreased when carrageenan was introduced in the gel. The order of the decrease follows the order of increased hydrophobic character of the drug. Competing diffusion was studied for amitriptyline and chlorpromazine, the former released significantly faster than chlorpromazine when simultaneously released from a hydrogel slab. Data indicate the possibility to adjust the simultaneous release rates individually for the two drugs by means of polyion charge density.

  • 286.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Andersson, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sundgren-Andersson, Anna K.
    AstraZeneca R&D, Global Med Dev, SE-43183 Molndal, Sweden..
    Halldin, Magnus M.
    Karolinska Inst, AlzeCure Fdn, Sci Pk, SE-14157 Huddinge, Sweden..
    Stalberg, Olle
    Karlstad Univ, Dept Engn & Chem Sci, SE-65188 Karlstad, Sweden..
    Assessment of Free Drug Concentration in Cyclodextrin Formulations Is Essential to Determine Drug Potency in Functional In Vitro Assays2016In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 105, no 9, p. 2913-2920Article in journal (Refereed)
    Abstract [en]

    Cyclodextrins (CD) have the ability to form inclusion complexes with drugs and can be used as excipients to enhance solubility of poorly soluble drugs. To make accurate estimations of the potency of the drug, knowledge of the free drug concentration is important. The aim of this study was to evaluate the applicability of calculated free drug concentrations toward response measurements in a transient receptor potential vanilloid receptor-1 cell-based in vitro assay. This included accounting for potential competitive CD binding of 2 transient receptor potential vanilloid receptor-1 active entities: 1 antagonist, and 1 agonist (capsaicin). Solubility of the CD-drug complexes was measured, and the ligand to substrate affinity in CD formulations was determined according to the phase-solubility technique. The total concentration of antagonist, agonist, CD, and the binding constants between ligands and CD were used to calculate the free concentration of CD ligands. For capsaicin and 2 of the 3 investigated model drugs, the calculated free drug concentration was consistent with the experimental in vitro data while it was overestimated for one of the compounds. In conclusion, the suggested approach can be used to calculate free drug concentration and competitive binding in CD formulations for the application of cell-based drug functionality assays.

  • 287.
    Skogh, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Development of Substance P 1–7 Related Peptides and Peptidomimetics: Targeting Neuropathic Pain2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The neuropeptide substance P 1–7 (SP1–7, H-Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-OH) and its amidated analogue SP1–7 amide, have displayed intriguing effects in experimental models for neuropathic pain acting on a specific, yet unknown SP1–7 target. The aim of this thesis was to design and synthesise SP1–7 related peptides and peptidomimetics, to be used as research tools to study the SP1–7 system, and to serve as drug leads in the neuropathic pain area.

    The in vivo structure activity relationship (SAR) of the SP1–7 amide was elucidated using Ala-substituted, N-terminally truncated and N-methylated variants. By evaluation of the anti-allodynic effect in spared nerve injury (SNI) mice and the pharmacokinetic properties it is suggested that Phe7 acts as a message residue and Arg1 as an address residue, both important for the overall anti-allodynic activity. In contrast, Lys3 could be substituted by alanine, and the Pro2-Lys3 and Pro4-Gln5 bond could be N-methylated with retained anti-allodynic effect. The Pro2-Lys3 bond was found most sensitive towards proteolysis and indeed, N-methylation of this bond delivered peptides completely inert in plasma. Conversely, prolonged plasma stability did not improve the overall in vivo activity for these peptides. Instead, the SP1–7 amide remained the most potent peptide in vivo, despite fast degradation in plasma.    

    Besides peptide synthesis, the synthetic work included development of a Pd-catalysed aminocarbonylation protocol using an amino acid nucleophile, which was used for the synthesis of an imidazole-based peptidomimetic. This peptidomimetic was equipotent to the SP1–7 amide, and more potent than the drug gabapentin, in regard to its anti-allodynic effect in SNI mice, and it is a promising drug lead for further development. The Pd-catalysed aminocarbonylation protocol was refined further, in regards to reaction scope and requirements for solid-phase peptide synthesis and has proven useful for N-capping, isotopic labelling, and intramolecular cyclisation of peptides.

    In summary, the work presented herein resulted in an in vivo SAR for SP1–7 related peptides, a novel small molecule SP1–7 peptidomimetic, and methods expanding the toolbox for synthesising modified peptides and peptidomimetics – a field in drug discovery that presently gaining increasing attention.

    List of papers
    1. Importance of N-and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration
    Open this publication in new window or tab >>Importance of N-and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration
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    2017 (English)In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 106, p. 345-351Article in journal (Refereed) Published
    Abstract [en]

    The heptapeptide SP1-7 (1, Arg(1)-Pro(2)-Lys(3)-Pro(4)-Gln(5)-Gln(6)-Phe(7)) is the major bioactive metabolite formed after proteolytic processing of the neuropeptide substance P (SP, Arg(1)-Pro(2)-Lys(3)-Pro(4)-GIn(5)-Gln(6)-Phe(7)-Phe(8)-Gly(9)-Leu(10)-Meti(11)-NH2). The heptapeptide 1 frequently exhibits opposite effects to those induced by SP, such as exerting antinociception, or attenuating thermal hyperalgesia and mechanical allodynia. The heptapeptide SP1-7 amide (2, Arg(1)-Pro(2)-Lys(3)-Pro(4)-Gln(5)-Gln(6)-Phe(7)-NH2 ) is often more efficacious than 1 in experimental pain models. We have now assessed the anti-allodynic outcome after systemic administration of 2 and a series of Ala substituted and truncated analogues of 2, in the spared nerve injury (SNI) mice model and the results obtained were correlated with in vitro plasma stability and permeability measurements. It is herein demonstrated that an intact Arg(1) in SP1-7 amide analogues is fundamental for retaining a potent in vivo effect, while Lys(3) of 2 is less important. A displacement with Ala(1) or truncation rendered the peptide analogues either inactive or with a significantly attenuated in vivo activity. Thus, the pentapeptide SP3-7 amide (7, t(1/2) = 11.1 min) proven to be the major metabolite of 2, demonstrated an in vivo effect itself although considerably less significant than 2 in the SNI model. Intraperitoneal administration of 2 in a low dose furnished the most powerful anti-allodynic effect in the SNI model of all the analogous evaluated, despite a fast proteolysis of 2 in plasma (t(1/2) = 6.4 min). It is concluded that not only the C-terminal residue, that we previously demonstrated, but also the N-terminal with its basic side chain, are important for achieving effective pain relief. This information is of value for the further design process aimed at identifying more drug-like SP1-7 amide related peptidomimetics with pronounced antiallodynic effects.

    Place, publisher, year, edition, pages
    Elsevier, 2017
    Keywords
    Neuropathic pain, Spared nerve injury (SNI), SP1-7, Neuropeptides, Plasma stability, Structure-activity relationship, Message-address concept
    National Category
    Pharmacology and Toxicology
    Identifiers
    urn:nbn:se:uu:diva-334033 (URN)10.1016/j.ejps.2017.06.004 (DOI)000406988600036 ()28587787 (PubMedID)
    Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-02-28Bibliographically approved
    2. Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration
    Open this publication in new window or tab >>Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration
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    2017 (English)In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 109, p. 533-540, article id S0928-0987(17)30497-9Article in journal (Refereed) Published
    Abstract [en]

    Substance P 1-7 (SP1-7, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1-7 amide 1 (Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) was previously shown to be superior to the endogenous SP1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1-7 amide (3) and the [MeGln5]SP1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg1 and C-terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP1-7 amide (1), the [MeLys3]SP1-7 amide (3) amide and the [MeGln5]SP1-7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain.

    Keywords
    Anti-allodynia, N-methylation, SP(1–7) amide, Solid phase peptide synthesis (SPPS), Spared nerve injury (SNI), Substance P (SP)
    National Category
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-335651 (URN)10.1016/j.ejps.2017.09.007 (DOI)000413325000055 ()28887235 (PubMedID)
    Funder
    Swedish Research Council, 9459The Swedish Brain FoundationBerzelii Centre EXSELENT
    Available from: 2017-12-07 Created: 2017-12-07 Last updated: 2018-02-28
    3. Aminocarbonylation of 4-Iodo-1H-imidazoles with an Amino Acid Amide Nucleophile: Synthesis of Constrained H-Phe-Phe-NH2 Analogues
    Open this publication in new window or tab >>Aminocarbonylation of 4-Iodo-1H-imidazoles with an Amino Acid Amide Nucleophile: Synthesis of Constrained H-Phe-Phe-NH2 Analogues
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    2013 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 78, no 23, p. 12251-12256Article in journal (Refereed) Published
    Abstract [en]

    A simple and an expedient process to prepare 5-aryl-1benzyl-1H-imidazole-4-carboxamides by the aminocarbonylation of 5aryl-4-iodo-1H-imidazoles using ex situ generation of CO from Mo(CO)(6) with an amino acid amide nucleophile is reported. Furthermore, a microwave-assisted protocol for the direct C-5 arylation of 1-benzyl-1H-imidazole and a regioselective C-4 iodination method to acquire starting material for our aminocarbonylation are presented. The method can be used to prepare imidazole based peptidomimetics, herein exemplified by the synthesis of constrained H-Phe-Phe-NH2 analogues.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-214017 (URN)10.1021/jo4020613 (DOI)000328231600069 ()
    Available from: 2014-01-08 Created: 2014-01-07 Last updated: 2018-02-28Bibliographically approved
    4. An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice
    Open this publication in new window or tab >>An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice
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    2018 (English)In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 28, no 14, p. 2446-2450Article in journal (Refereed) Published
    Abstract [en]

    The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.

    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-343682 (URN)10.1016/j.bmcl.2018.06.009 (DOI)000438467200020 ()29929882 (PubMedID)
    Funder
    Swedish Research Council, 9459
    Note

    Title in dissertation reference list: An Imidazole-Based H-Phe-Phe-NH2 Peptidomimetic with Anti-Allodynic Effect in Spared Nerve Injury Mice and without Neurotoxic Liability

    Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2018-09-24Bibliographically approved
    5. Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide Synthesis: A Method for Capping, Cyclization, and Isotope Labeling
    Open this publication in new window or tab >>Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide Synthesis: A Method for Capping, Cyclization, and Isotope Labeling
    2017 (English)In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 19, no 11, p. 2873-2876Article in journal (Refereed) Published
    Abstract [en]

    A new synthetic approach for introducing N-capping, groups onto peptides attached to a solid support, Combining aminocarbonylation under mild conditions Wing a palladacycle precatalyst and, solid-phase peptide synthesis is reported. The use of la silacarboxylic acid as an in situ CO-releasing molecule allowed the reaction to be performed single vial. The method also enables versatile substitution of side chains, side-chain to side-chain cyclizations, and selective aryl labeling of modified peptides.

    Place, publisher, year, edition, pages
    AMER CHEMICAL SOC, 2017
    National Category
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-327450 (URN)10.1021/acs.orglett.7b01068 (DOI)000402850900025 ()28498670 (PubMedID)
    Funder
    Danish National Research Foundation, DNRF118
    Available from: 2017-08-25 Created: 2017-08-25 Last updated: 2018-02-28Bibliographically approved
  • 288.
    Skogh, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Lesniak, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sköld, Christian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Karlgren, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Gaugaz, Fabienne Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Diwakarla, Shanti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jonsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fransson, Rebecca
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Johansson, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice2018In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 28, no 14, p. 2446-2450Article in journal (Refereed)
    Abstract [en]

    The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.

  • 289.
    Slazak, Blazej
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Polish Acad Sci, W Szafer Inst Bot, 46 Lubicz St, PL-31512 Krakow, Poland..
    Kapusta, Malgorzata
    Univ Gdansk, Fac Biol, Dept Plant Cytol & Embryol, 59 Wita Stwosza St, PL-80308 Gdansk, Poland..
    Malik, Sohaib
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bohdanowicz, Jerzy
    Univ Gdansk, Fac Biol, Dept Plant Cytol & Embryol, 59 Wita Stwosza St, PL-80308 Gdansk, Poland..
    Kuta, Elzbieta
    Jagiellonian Univ, Inst Bot, Dept Plant Cytol & Embryol, 9 Gronostajowa St, PL-30387 Krakow, Poland..
    Malec, Przemyslaw
    Jagiellonian Univ, Fac Biochem Biophys & Biotechnol, 7 Gronostajowa St, PL-30387 Krakow, Poland..
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense2016In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 244, no 5, p. 1029-1040Article in journal (Refereed)
    Abstract [en]

    Main conclusionThe distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

  • 290.
    Smeets, Cleo J. L. M.
    et al.
    Univ Groningen, Univ Med Ctr Groningen, Dept Genet, Groningen, Netherlands..
    Zmorzynska, Justyna
    Univ Groningen, Univ Med Ctr Groningen, Dept Genet, Groningen, Netherlands..
    Melo, Manuel N.
    Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Ctr Life Sci, Groningen, Netherlands..
    Stargardt, Anita
    Acad Med Ctr, Dept Cell Biol & Histol, Amsterdam, Netherlands..
    Dooley, Colette
    Torrey Pines Inst Mol Studies, Port St Lucie, FL USA..
    Bakalkin, Georgy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    McLaughlin, Jay
    Univ Florida, Dept Pharmacodynam, Gainesville, FL 32610 USA..
    Sinke, Richard J.
    Univ Groningen, Univ Med Ctr Groningen, Dept Genet, Groningen, Netherlands..
    Marrink, Siewert-Jan
    Reits, Eric
    Acad Med Ctr, Dept Cell Biol & Histol, Amsterdam, Netherlands..
    Verbeek, Dineke S.
    Univ Groningen, Univ Med Ctr Groningen, Dept Genet, Groningen, Netherlands..
    Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA232016In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 25, no 13, p. 2728-2737Article in journal (Refereed)
    Abstract [en]

    Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides alpha-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-D-aspartate-mediated non-opioidmechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal alpha-helix associated with decreased kappa-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA-receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.

  • 291. Smith, M.
    et al.
    Martin, J.
    Smith, D.
    Maag, H.
    Naiera, I.
    Jiang, W. R.
    Klumpp, K.
    Leveque, V.
    Cammack, N.
    Johansson, N. G.
    Kalayanov, G.
    Belfrage, A. K.
    Benkestock, K.
    Farnell, K.
    Hiscock, S.
    Lindborg, B.
    Maltseva, T.
    Morisson, V.
    Pinho, P.
    Sund, C.
    Tozer, M.
    Winquist, A.
    Zhou, X. X.
    MEDI 236-Synthesis and anti-HCV activity of 4'-substituted nucleosides2006In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 232Article in journal (Refereed)
  • 292.
    Sooriyaarachchi, Sanjeewani
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chofor, Rene
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Risseeuw, Martijn D. P.
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pouyez, Jenny
    Univ Namur, Dept Chem, Rue Bruxelles 61, B-5000 Namur, Belgium..
    Dowd, Cynthia S.
    George Washington Univ, Dept Chem, Washington, DC 20052 USA..
    Maes, Louis
    Univ Antwerp, LMPH, Univ Pl 1, B-2610 Antwerp, Belgium..
    Wouters, Johan
    Univ Namur, Dept Chem, Rue Bruxelles 61, B-5000 Namur, Belgium..
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Van Calenbergh, Serge
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with -Arylpropyl Analogues of Fosmidomycin2016In: ChemMedChem, ISSN 1860-7179, E-ISSN 1860-7187, Vol. 11, no 18, p. 2024-2036Article in journal (Refereed)
    Abstract [en]

    Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the -position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.

  • 293.
    Stevens, Marc Y.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Chow, Shiao Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Estrada, Sergio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Asplund, Veronika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Åberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Synthesis of C-11-labeled Sulfonyl Carbamates through a Multicomponent Reaction Employing Sulfonyl Azides, Alcohols, and [C-11]CO2016In: ChemistryOpen, ISSN 2191-1363, Vol. 5, no 6, p. 566-573Article in journal (Refereed)
    Abstract [en]

    We describe the development of a new methodology focusing on C-11-labeling of sulfonyl carbamates in a multicomponent reaction comprised of a sulfonyl azide, an alkyl alcohol, and [C-11] CO. A number of C-11-labeled sulfonyl carbamates were synthesized and isolated, and the developed methodology was then applied in the preparation of a biologically active molecule. The target compound was obtained in 24 +/- 10% isolated radiochemical yield and was evaluated for binding properties in a tumor cell assay; in vivo biodistribution and imaging studies were also performed. This represents the first successful radiolabeling of a non-peptide angiotensin II receptor subtype 2 agonist, C21, currently in clinical trials for the treatment of idiopathic pulmonary fibrosis.

  • 294.
    Stevens, Marc Y.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Sawant, Rajiv T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Synthesis of arylsulfonyl azides via diazotransfer using an imidazole-1-sulfonyl azide salt: Scope and N-15 NMR labeling experiments2014In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 248Article in journal (Other academic)
  • 295. Stroem, Peter
    et al.
    Malmquist, Jonas
    Hansson, Jonas
    Yngve, Ulrika
    Claesson, Alf.
    Tritium labeling of a class of spirooxazaindolamines possessing analgesic properties.2004In: Synth. Appl. Isot. Labelled Compd., Proc. Int. Symp., 8th, 2004, p. 285-288Conference paper (Refereed)
    Abstract [en]

    In order to study the mol. action of a class of spirooxazaindolamines with promising analgesic activity, tritiated material was prepd. Efficient synthesis of 3H-labeled material allowed essential information on the mol. mechanism for this class of compds. to be obtained. Different approaches for the introduction of the 3H-label on the spirooxazaindole skeleton via 3H-methyliodide were evaluated involving lithium-mediated coupling, zinc-mediated coupling and Stille coupling. Tritiation using 3H2 (g) with a Pd-catalyst was also tested. [on SciFinder(R)]

  • 296.
    Strömstedt, Adam A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Felth, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bohlin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bioassays in Natural Product Research: Strategies and Methods in the Search for Anti-inflammatory and Antimicrobial Activity2014In: Phytochemical Analysis, ISSN 0958-0344, E-ISSN 1099-1565, Vol. 25, no 1, p. 13-28Article, review/survey (Refereed)
    Abstract [en]

    Introduction: Identifying bioactive molecules from complex biomasses requires careful selection and execution of relevant bioassays in the various stages of the discovery process of potential leads and targets.

    Objective: The aim of this review is to share our long-term experience in bioassay-guided isolation, and mechanistic studies, of bioactive compounds from different organisms in nature with emphasis on anti-inflammatory and antimicrobial activity.

    Methods: In the search for anti-inflammatory activity, in vivo and in vitro model combinations with enzymes and cells involved in the inflammatory process have been used, such as cyclooxygenases, human neutrophils and human cancer cell lines. Methods concerning adsorption and perforation of bacteria, fungi, human cells and model membranes, have been developed and optimised, with emphasis on antimicrobial peptides and their interaction with the membrane target, in particular their ability to distinguish host from pathogen.

    Results: A long-term research has provided experience of selection and combination of bioassay models, which has led to an increased understanding of ethnopharmacological and ecological observations, together with in-depth knowledge of mode of action of isolated compounds.

    Conclusion: A more multidisciplinary approach and a higher degree of fundamental research in development of bioassays are often necessary to identify and to fully understand the mode of action of bioactive molecules with novel structure-activity relationships from natural sources. 

    Selection and execution of relevant bioassays are critical in the various stages of the discovery process of potential drug leads and targets from natural sources. The aim of this review is to share our long-term experience in bioassay-guided isolation of bioactive compounds from different organisms in nature with emphasis on anti-inflammatory and antimicrobial activity. We conclude that an increased multidisciplinary approach and a higher degree of fundamental research in development of bioassays are essential to discover complex structure-activity relationships.

  • 297.
    Strömstedt, Adam A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Kristiansen, Per Eugen
    Univ Oslo, Dept Mol Biosci, Box 1041, N-0316 Oslo, Norway.
    Gunasekera, Sunithi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Grob, Nathalie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Skjeldal, Lars
    Norwegian Univ Life Sci, Dept Chem Biochem & Food Sci, N-1432 As, Norway.
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Selective membrane disruption by the cyclotide kalata B7: complex ions and essential functional groups in the phosphatidylethanolamine binding pocket2016In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1858, no 6, p. 1317-1327Article in journal (Refereed)
    Abstract [en]

    The cyclic cystine knot plant peptides called cyclotides are active against a wide variety of organisms. This is primarily achieved through membrane binding and disruption, in part deriving from a high affinity for phosphatidylethanolamine (PE) lipids. Some cyclotides, such as kalata B7 (kB7), form complexes with divalent cations in a pocket associated with the tyrosine residue at position 15 (Tyr15). In the current work we explore the effect of cations on membrane leakage caused by cyclotides kB1, kB2 and kB7, and we identify a functional group that is essential for PE selectivity. The presence of PE-lipids in liposomes increased the membrane permeabilizing potency of the cyclotides, with the potency of kB7 increasing by as much as 740-fold. The divalent cations Mn(2+), Mg(2+) and Ca(2+) had no apparent effect on PE selectivity. However, amino acid substitutions in kB7 proved that Tyr15 is crucial for PE-selective membrane permeabilization on various liposome systems. Although the tertiary structure of kB7 was maintained, as reflected by the NMR solution structure, mutating Tyr into Ser at position 15 resulted in substantially reduced PE selectivity. Ala substitution at the same position produced a similar reduction in PE selectivity, while substitution with Phe maintained high selectivity. We conclude that the phenyl ring in Tyr15 is critical for the high PE selectivity of kB7. Our results suggest that PE-binding and divalent cation coordination occur in the same pocket without adverse effects of competitive binding for the phospholipid.

  • 298.
    Strömstedt, Adam A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Vikeved, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Cárdenas, Paco
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Alsmark, Cecilia
    Uppsala University.
    Chen, Yung Hsuan
    National Museum of Marine Biology and Aquarium.
    Backlund, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Aaptamines from Haliclona and bromopyrroles from Agelas — marine sponge alkaloids with distinct modes of action against bacteria and protozoaManuscript (preprint) (Other academic)
  • 299.
    Stubberud, Karin
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Studies of Micellar Electrokinetic Chromatography as an Analytical Technique in Pharmaceutical Analysis - an Industrial Perspective2002Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Studies have been performed to evaluate the use of micellar electrokinetic chromatography (MEKC), one mode of capillary electrophoresis (CE), as an analytical technique in industrial pharmaceutical analysis. The potential for using chemometrics for the optimisation of MEKC methods has also been studied as well as the possibilities of coupling MEKC with mass spectrometry (MS).

    Two methods were developed, one for the determination of ibuprofen and codeine and another for pilocarpine, together with their degradation products and impurities in both cases. MEKC was found to be the most suitable mode of CE for the methods. Both methods were optimised by means of experimental design. Valuable information was gathered and optimum conditions were defined which resulted in fast systems with baseline-separated peaks. The ibuprofen-codeine method was validated according to the recommended validation procedures of the International Conference of Harmonisation. The validation was performed on a commercially available tablet formulation to verify the suitability of the method, i.e. for quantification of the two main compounds and to determine the degradation products and impurities in area% of each main peak. The following parameters were determined: selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range. The results confirm that the method is highly suitable for its intended purpose, i.e. as a routine method for assay and impurity determination. The MEKC method for ibuprofen-codeine was coupled to a mass spectrometer in order to evaluate the potential of partial filling (PF)-MEKC-MS for identification of impurities in pharmaceutical substances and products. The so-called partial-filling technique was used to prevent the non-volatile micelles from entering the MS and was shown to fulfil its purpose of providing detection limits of about 10 pg.

    The study clearly shows that micellar electrokinetic chromatography is well-suited as an analytical technique in industrial pharmaceutical analysis.

    List of papers
    1. Fractional Factorial Design Optimization of the Separation of Pilocarpine and its Degradation Products by Capillary Electrophoresis
    Open this publication in new window or tab >>Fractional Factorial Design Optimization of the Separation of Pilocarpine and its Degradation Products by Capillary Electrophoresis
    1997 (English)In: Journal of Chromatography B: Biomedical Sciences and Applications, ISSN 1387-2273, E-ISSN 1878-5603, Vol. 697, no 1-2, p. 207-215Article in journal (Refereed) Published
    Abstract [en]

    The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, its trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89801 (URN)10.1016/S0378-4347(97)00108-4 (DOI)9342671 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    2. Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: I. Method Development and Optimization with Fractional factorial Design
    Open this publication in new window or tab >>Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: I. Method Development and Optimization with Fractional factorial Design
    1998 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 798, no 1-2, p. 307-314Article in journal (Refereed) Published
    Abstract [en]

    A capillary electrophoresis method has been developed and optimized for the separation of ibuprofen, codeine phosphate and their main degradation products and impurities. In the course of developing the method, it was found that micellar electrokinetic capillary chromatography was necessary for the separation of the eleven peaks. A fractional factorial design was used for the optimization of the experiments. Six process parameters were varied at two levels: the concentration of sodium dodecyl sulfate (SDS), the pH, the concentration of acetonitrile, the concentration of boric acid, the field strength and the temperature. All these factors had a significant effect on the migration time and resolution. The optimum conditions were found to be a borate buffer of 40 mM H3BO3 at pH 10 with the addition of 40 mM SDS and 9% acetonitrile, a field strength of 515 V/cm and a temperature of 25 degrees C. This resulted in baseline separation of the eleven peaks within 12 min.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89802 (URN)10.1016/S0021-9673(97)00955-2 (DOI)9542142 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    3. Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: II. Validation
    Open this publication in new window or tab >>Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: II. Validation
    1998 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 826, no 1, p. 95-102Article in journal (Refereed) Published
    Abstract [en]

    A micellar electrokinetic chromatography method for the determination of ibuprofen and codeine phosphate hemihydrate and their degradation products and impurities in a commercial tablet formulation has been validated. The validation has been performed according to the International Conference of Harmonisation's guidance on the validation of analytical methods, and selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range test were performed to determine the suitability of the method. It was possible to use the fractional factorial design model from the optimisation of the method to draw conclusions about its robustness. The results confirm that the method is highly suitable for its intended purpose.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89803 (URN)10.1016/S0021-9673(98)00711-0 (DOI)9882139 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    4. Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part I
    Open this publication in new window or tab >>Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part I
    2002 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 4, p. 572-577Article in journal (Refereed) Published
    Abstract [en]

    Studies have been performed to evaluate whether an on-line partial filling-micellar electrokinetic chromatography (PF-MEKC) system could be applied to a recently developed MEKC method for the separation of ibuprofen, codeine and one of the degradation products. Attempts to couple the PF-MEKC system to MS have also been performed. SDS concentration, micellar zone length and concentration of acetonitrile in the buffer were optimized using factorial design. When a small micelle zone was injected directly after the sample introduction, the results improved markedly. The MS parameters have not been optimized, but the studies show promising results for the use of PF-MEKC-mass spectrometry for identification of the degradation products.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89804 (URN)10.1002/1522-2683(200202)23:4<572::AID-ELPS572>3.0.CO;2-# (DOI)11870767 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    5. Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part II
    Open this publication in new window or tab >>Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part II
    2003 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 6, p. 1008-1015Article in journal (Refereed) Published
    Abstract [en]

    We describe the use of partial filling-micellar electrokinetic chromatography-mass spectrometry (PF-MEKC-MS) on the pharmaceutical ingredients ibuprofen and codeine phosphate as well as their degradation products and impurities. The study focuses on the change of the borate buffer to the volatile ammonium acetate and the optimization of critical MS parameters. The sensitivity of the method is also evaluated. The results are compared to an existing MEKC-UV method that is used for quantitative determination of the two main substances as well as for the analysis of the degradation products. It is concluded that the PF-MEKC-MS system is suitable for separation and identification.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89805 (URN)10.1002/elps.200390116 (DOI)12658689 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
  • 300.
    Sundell, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Vögeli, Beat
    Department of Biochemistry and Molecular Genetics, University of Colorado at Denver, 12801 East 17th Avenue, Aurora, Colorado 80045, United States.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ12018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 1, p. 66-71Article in journal (Refereed)
    Abstract [en]

    The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

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