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Small peptides mimicking substance P (1-7) and encompassing a C-terminal amide functionality
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
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2008 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 42, no 1, 31-37 p.Article in journal (Refereed) Published
Abstract [en]

Some of the biological effects demonstrated after administration of substance P (SP) in vivo can indirectly be attributed to the fragmentation of the undecapeptide to its N-terminal bioactive fragment SP1–7. This heptapeptide (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is a major bioactive metabolite from SP that frequently exerts similar biological effects as the parent peptide but also, in several cases, completely opposite actions. Specific binding sites for the heptapeptide SP1–7 that are separate from the SP preferred NK receptors have been identified. In this study we demonstrate that (a) the C-terminal part of the SP metabolite SP1–7 is most important for binding as deduced from an Ala scan and that a replacement of Phe7 for Ala is deleterious, (b) truncation of the N-terminal amino acid residues of SP1–7 delivers peptides with retained binding activity, although with somewhat lower binding affinities than SP1–7 and (c) a C-terminal amide group as a replacement for the terminal carboxy group of SP1–7 and for all of the truncated ligands synthesized affords approximately 5–10-fold improvements of the binding affinities.

Place, publisher, year, edition, pages
2008. Vol. 42, no 1, 31-37 p.
Keyword [en]
Substance P (1–7), SP (1–7), SP1–7, Substance P, Structure–activity relationships (SAR), Ala scan, Peptidomimetics
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97823DOI: 10.1016/j.npep.2007.11.002ISI: 000254231100003PubMedID: 18093649OAI: oai:DiVA.org:uu-97823DiVA: diva2:172906
Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Characterization of Substance P (SP) Aminoterminal SP (1-7) Binding in Brain Regions and Spinal Cord of the Male Rat: Studies on the Interaction with Opioid Related Pathways
Open this publication in new window or tab >>Characterization of Substance P (SP) Aminoterminal SP (1-7) Binding in Brain Regions and Spinal Cord of the Male Rat: Studies on the Interaction with Opioid Related Pathways
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Binding sites for substance P(1-7), SP(1-7) have been identified and characterized for the first time in crude membrane fraction from rat CNS using tritiated ([3H]) SP(1-7) as tracer. These putative receptors were investigated in relation to their affinity for tachykinins, opioid peptides and sigma receptor ligands. [3H]-SP(1-7) specifically binds to high affinity binding sites identified as receptor targets for the heptapeptide SP (1-7). Two distinct binding sites were observed in the spinal cord. One site is recognized by high affinity for SP(1-7) with a Kd of 0.5 nM, whereas the other site showed low affinity for the heptapeptide (Kd=12 nM). In the brain, the binding of SP(1-7) fitted a single site binding model with a Kd of 4.4 nM and a Ki of 4.2 nM. Further, using the spinal cord membranes the binding of [3H]-SP (1-7) was weakly displaced by SP and other N-terminal fragments thereof and no or negligible affinity was observed for ligands of the NK-1, NK-2 and NK-3 tachykinin receptors, C-terminal SP(5-11), Tyr-w-MIF-1 or the mu-opioid receptor antagonists naloxone and naloxonazine. On the other hand it was significantly displaced by endomorphin-2, DAMGO, and Try-MIF-1 and exhibit some affinity for MIF-1, ß-casomorphin and endomorphin-1. However, only endomorphin-2, DAMGO and Tyr-MIF-1 showed affinity in the close range of the native peptide SP(1-7). The affinity of endomorphin-2 for the spinal cord site was 10 times lower than that of SP(1-7) but more than 100 times higher than the affinity recorded for endomorphin-1. Tyr-MIF-1 but not Tyr-w-MIF-1 showed similar affinity as endomorphin-2 for SP(1-7) site. All peptides exhibiting high affinity at the SP(1-7) site, have a phenylalanine or a leucine residue in their C-terminal structure.

Further, synthetic analogues of SP(1-7) were tested for their affinity for the SP(1-7) receptor in the rat spinal cord. An important finding here was that the receptor-ligand-interaction was favoured by the C-terminal region of SP(1-7). Residues at positions 5-7 appeared crucial for binding to the specific SP(1-7) site. The presence of the amidated Phe7 residue was extremely critical for binding to the SP(1-7) site.The analogue Gln5-Gln6-Phe7-NH2 was almost equipotent with the parent peptide in the SP (1-7) receptor binding assay.

Furthermore, the SP(1-7)-amide potently and dose dependently reduced several signs of the reaction to morphine withdrawal and was significantly attenuated by the addition of the sigma agonist SK-10047.

In conclusion, the work presented in this thesis has contributed the characterization of the properties of highly selective binding sites for SP(1-7) in the rat spinal cord and VTA. These sites appear to be distinct from the µ-opioid receptor or any of the known neurokinin receptors. The study further indicates that the SP(1-7)-amide mimics the effect of the nativ heptapeptide and that the mechanisms for its action involve a sigma receptor site.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 81 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 85
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-9401 (URN)978-91-554-7349-5 (ISBN)
Public defence
2008-12-12, B41, BMC, Husargatan, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-11-21 Created: 2008-11-21Bibliographically approved
2. Discovery of Small Peptides and Peptidomimetics Targeting the Substance P 1-7 Binding Site: Focus on Design, Synthesis, Structure-Activity Relationships and Drug-Like Properties
Open this publication in new window or tab >>Discovery of Small Peptides and Peptidomimetics Targeting the Substance P 1-7 Binding Site: Focus on Design, Synthesis, Structure-Activity Relationships and Drug-Like Properties
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biologically active peptides are important for many physiological functions in the human body and therefore serve as interesting starting points in drug discovery processes. In this work the neuropeptide substance P 1–7 (SP1–7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), which has been demonstrated to reduce neuropathic pain and attenuate opioid withdrawal symptoms in animal models, has been addressed in a medicinal chemistry program with the overall aim of transforming this bioactive peptide into more drug-like compounds. Specific binding sites for this neuropeptide have been detected in the brain and the spinal cord. Interestingly, the smaller neuropeptide endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) also interacts with these binding sites, although 10-fold less efficient.

In this work the structure–activity relationship of SP1–7 and EM-2, regarding their affinity to the SP1–7 binding site was elucidated using alanine scans, truncation, and terminal modifications. The C-terminal part of both peptides, and especially the C-terminal phenylalanine, was crucial for binding affinity. Moreover, the C-terminal functional group should preferably be a primary amide. The truncation studies finally resulted in the remarkable discovery of H-Phe-Phe-NH2 as an equally good binder as the heptapeptide SP1–7. This dipeptide amide served as a lead compound for further studies. In order to improve the drug-like properties and to find a plausible bioactive conformation, a set of rigidified and methylated dipeptides of different stereochemistry, and analogs with reduced peptide character, were synthesized and evaluated regarding binding, metabolic stability and absorption. Small SP1–7 analogs with retained affinity and substantially improved permeability and metabolic stability were identified.

Beside peptide chemistry the synthetic work included the development of a fast and convenient microwave-assisted protocol for direct arylation of imidazoles. Furthermore, microwave-assisted aminocarbonylation using Mo(CO)6 as a solid carbon monoxide source was investigated in the synthesis of MAP amides and for coupling of imidazoles with amino acids.

In a future perspective the present findings, together with the fact that some of the SP1–7 analogs discovered herein have been shown to reproduce the biological effects of SP1-7 in animal studies related to neuropathic pain and opioid dependence, can ultimately have an impact on drug discovery in these two areas.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 88 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 143
Keyword
substance P 1-7, peptidomimetics, structure-activity relationship, drug-like properties, phenylalanine, imidazole, MAP aryl amides, carbonylation
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-149480 (URN)978-91-554-8040-0 (ISBN)
Public defence
2011-05-06, B42, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-04-15 Created: 2011-03-20 Last updated: 2011-05-05Bibliographically approved

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Fransson, RebeccaNyberg, FredLindeberg, GunnarSandström, AnjaHallberg, Mathias

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