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Muhammad, Taj
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Muhammad, T. (2019). LL-37-derived cyclic antimicrobial drug leads: Design, synthesis, activity and different ways of creating them . (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Öppna denna publikation i ny flik eller fönster >>LL-37-derived cyclic antimicrobial drug leads: Design, synthesis, activity and different ways of creating them 
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In an era where last-line antibiotics are failing, one of the powerful approaches to develop novel therapeutic agents is to turn back to nature in order to identify possible drug candidates. Among the potential candidates, antimicrobial peptides (AMPs) have garnered much attention as an antimicrobial. These are broad spectrum host defense molecules produced by all living organisms. LL-37 is such a multitask human defense peptide that mediates various host immune responses and also exerts antimicrobial activity. However, the direct use of this 37-amino acid long α-helical peptide is hampered by protease susceptibility, in particular for antimicrobial applications. A small 12-residues peptide, referred as KR-12, derived from LL-37, has been reported to have selective toxic effect on bacteria. 

Analogues of KR-12 were generated in the form of Alanine and Lysine scans to find out the positions important for improved activity and selectivity. Backbone-cyclised dimers based on KR-12 and KR-12 analogues, tethered by linkers of two to four amino acid residues, were synthesised to explore the concept of cyclisation, dimerisation and cross-linking as means to enhance peptide stability and activity. Antimicrobial activities of the linear peptides and cyclic dimers were assayed against human pathogens, in buffer and/or physiological conditions. Proteolytic stability, permeabilisation efficacy on microbial membranes and, their structures were also characterised.  

From Ala and Lys scans, it was possible to identify two key positions for the enhanced broad-spectrum antibacterial activity: replacement of Gln5 with Lys, and Asp9 with either Ala or Lys. In serum stability assay, KR-12 and analogues were found to be unstable. The backbone-cyclised KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. KR-12 monomers adopt a well-defined α-helical structure in membrane-mimicking environment, while cyclised dimers were unstructured in solution judged by NMR. The KR-12 (Q5K, D9A) cyclised dimers retained antimicrobial activity in physiological conditions. Circular dichroism showed that the cyclic dimer, cd4-PP, had 77% helical content when bound to lyso-phosphatidylglycerol micelles.

Moreover, the limits of cyanobactin-macrocyclase PatGmac were explored to cyclise peptides larger than their natural substrates, namely the PawS derived peptide Sunflower Trypsin Inhibitor-1 (SFTI-1) and the cyclotide kalata B1. PatGmac was used very efficiently to cyclise SFTI-1. In addition, semi-pure butelase 1, isolated from Clitoria ternatea seeds, was immobilised on NHS column. The immobilised column was then used to produce substrates ranging from 16 to 34 varying length.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 281
Nyckelord
antimicrobial peptide, host defense, antimicrobial, LL-37, KR-12, peptide cyclisation, peptide dimerisation, croos-linking, cyanobactin-macrocyclase, PatGmac, butelase 1, enzymatic cyclisation, immobilisation
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Farmakognosi
Identifikatorer
urn:nbn:se:uu:diva-397191 (URN)978-91-513-0813-5 (ISBN)
Disputation
2019-12-19, A1:107a, Biomedical Centrum (BMC), Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-11-27 Skapad: 2019-11-18 Senast uppdaterad: 2020-01-13Bibliografiskt granskad
Mohotti, S., Rajendran, S., Muhammad, T., Strömstedt, A. A., Adhikari, A., Burman, R., . . . Gunasekera, S. (2019). Screening for bioactive secondary metabolites in Sri Lankan medicinal plants by microfractionation and targeted isolation of antimicrobial flavonoids from Derris scandens. Journal of Ethnopharmacology, 246, Article ID 112158.
Öppna denna publikation i ny flik eller fönster >>Screening for bioactive secondary metabolites in Sri Lankan medicinal plants by microfractionation and targeted isolation of antimicrobial flavonoids from Derris scandens
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2019 (Engelska)Ingår i: Journal of Ethnopharmacology, ISSN 0378-8741, E-ISSN 1872-7573, Vol. 246, artikel-id 112158Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Ethnopharmacological relevance: Sri Lanka is known to have very diverse flora. Many of these species are used for plant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurveda and Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification of bioactive natural products. Aim of the study: The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankan medicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim was the progression into defining and characterising potent isolates within targeted compound classes from such plants, i.e. Derris scandens and its antimicrobial flavonoids. Material and methods: The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4 Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and use in Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial disc diffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deep well plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities and masses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with the masses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. The isolated pure compounds were characterised using LC-MS and NMR. Results: Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species and organic extracts from 46 plant species were active against S. aureus (<= 4 mg mL(-1)) in the microdilution assay. Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore, target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin and vicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 mu g mL(-1) against S. aureus. Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 mu g mL(-1)) against Escherichia coli and Pseudomonas aeruginosa). Conclusion: The microfractionation protocol developed in this study enabled time-efficient screening of many plants species, using a small quantity of sample material. In addition, microfractionation served as a guiding tool for identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derris scandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence to Sri Lankan traditional herbal medicine as a source for drug discovery.

Nyckelord
Sri Lanka, Medicinal plants, Microfractionation, Antimicrobial activity, Cytotoxicity, Flavonoids
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:uu:diva-397136 (URN)10.1016/j.jep.2019.112158 (DOI)000493211800010 ()31421182 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 2013-06672
Tillgänglig från: 2019-11-28 Skapad: 2019-11-28 Senast uppdaterad: 2019-11-28Bibliografiskt granskad
Gunasekera, S., Muhammad, T., Strömstedt, A. A., Rosengren, K. J. & Göransson, U. (2018). Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity. ChemBioChem (Print), 19(9), 931-939
Öppna denna publikation i ny flik eller fönster >>Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity
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2018 (Engelska)Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, nr 9, s. 931-939Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

Ort, förlag, år, upplaga, sidor
WILEY-V C H VERLAG GMBH, 2018
Nyckelord
antibiotics, cytotoxicity, drug discovery, peptides, structure-activity relationships
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-356391 (URN)10.1002/cbic.201700599 (DOI)000431625100008 ()29430821 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 2011-3403Carl Tryggers stiftelse för vetenskaplig forskning , CTS 10: 126Carl Tryggers stiftelse för vetenskaplig forskning , CTS 11: 169Svenska läkaresällskapet, SLS-254511
Tillgänglig från: 2018-07-25 Skapad: 2018-07-25 Senast uppdaterad: 2020-02-18Bibliografiskt granskad
Hellinger, R., Thell, K., Vasileva, M., Muhammad, T., Gunasekera, S., Kuemmel, D., . . . Gruber, C. W. (2017). Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins. Frontiers in Chemistry, 5, Article ID 73.
Öppna denna publikation i ny flik eller fönster >>Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins
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2017 (Engelska)Ingår i: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 5, artikel-id 73Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Ort, förlag, år, upplaga, sidor
FRONTIERS MEDIA SA, 2017
Nyckelord
cyclotides, cyclic protein, chemical proteomics, peptide-protein interaction, photo-affinity labeling
Nationell ämneskategori
Farmaceutiska vetenskaper
Identifikatorer
urn:nbn:se:uu:diva-338516 (URN)10.3389/fchem.2017.00073 (DOI)000412726000001 ()29075625 (PubMedID)
Forskningsfinansiär
Australian Research Council, FT140100730
Tillgänglig från: 2018-01-15 Skapad: 2018-01-15 Senast uppdaterad: 2018-01-15Bibliografiskt granskad
Uddin, S. J., Muhammad, T., Shafiullah, M., Slazak, B., Rouf, R. & Göransson, U. (2017). Single-step purification of cyclotides using affinity chromatography. Biopolymers, 108(3), Article ID e23010.
Öppna denna publikation i ny flik eller fönster >>Single-step purification of cyclotides using affinity chromatography
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2017 (Engelska)Ingår i: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 108, nr 3, artikel-id e23010Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Mobius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

Nyckelord
affinity chromatography, cyclotides, cycloviolacin O2, immobilized IgG
Nationell ämneskategori
Biokemi och molekylärbiologi Biofysik
Identifikatorer
urn:nbn:se:uu:diva-334107 (URN)10.1002/bip.23010 (DOI)000407992300005 ()
Forskningsfinansiär
Vetenskapsrådet, 2012-5063
Tillgänglig från: 2017-11-24 Skapad: 2017-11-24 Senast uppdaterad: 2017-11-24Bibliografiskt granskad
Mohotti, S., Rajendran, S., Muhammad, T., Strömstedt, A. A., Burman, R., Hellman, B., . . . Gunasekera, S. (2016). A bioactivity-guided screening of Sri Lankan plants in the search for novel antibacterial and anticancer agents. Paper presented at 9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK. Planta Medica, 82
Öppna denna publikation i ny flik eller fönster >>A bioactivity-guided screening of Sri Lankan plants in the search for novel antibacterial and anticancer agents
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2016 (Engelska)Ingår i: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 82Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Nyckelord
Ayurvedic, MIC, NMR, FMCA, antibacterial, anticancer
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:uu:diva-346857 (URN)10.1055/s-0036-1596561 (DOI)000411789300385 ()
Konferens
9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK
Tillgänglig från: 2018-03-27 Skapad: 2018-03-27 Senast uppdaterad: 2018-03-27Bibliografiskt granskad
Muhammad, T., Gunasekera, S., Strömstedt, A. A. & Göransson, U. (2016). Engineering Of A Minimalized Domain Derived From Human Host Defense Peptide LL-37 Into A Stable And Potent Antimicrobial Drug Lead. Journal of Peptide Science, 22(S2), S184-S186
Öppna denna publikation i ny flik eller fönster >>Engineering Of A Minimalized Domain Derived From Human Host Defense Peptide LL-37 Into A Stable And Potent Antimicrobial Drug Lead
2016 (Engelska)Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 22, nr S2, s. S184-S186Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-331384 (URN)10.1002/psc.2950 (DOI)000398216100347 ()
Tillgänglig från: 2017-10-17 Skapad: 2017-10-17 Senast uppdaterad: 2017-10-17Bibliografiskt granskad
Muhammad, T., Gunasekera, S., Strömstedt, A. A. & Göransson, U. (2016). Engineering of KR-12: A minimalized domain derived from human host defense peptide LL-37 into a potent antimicrobial drug lead. Paper presented at 9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK. Planta Medica, 82
Öppna denna publikation i ny flik eller fönster >>Engineering of KR-12: A minimalized domain derived from human host defense peptide LL-37 into a potent antimicrobial drug lead
2016 (Engelska)Ingår i: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 82Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Ort, förlag, år, upplaga, sidor
GEORG THIEME VERLAG, 2016
Nyckelord
LL-37, KR-12, dimerization, membrane permabilization, NMR
Nationell ämneskategori
Medicinska och farmaceutiska grundvetenskaper
Identifikatorer
urn:nbn:se:uu:diva-346858 (URN)10.1055/s-0036-1596780 (DOI)000411789300604 ()
Konferens
9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK
Tillgänglig från: 2018-03-23 Skapad: 2018-03-23 Senast uppdaterad: 2018-03-23Bibliografiskt granskad
Anwar, J., Muhammad, T., Göransson, U. & Iqbal, Z. (2016). Fungi as a source for antibacterial compounds. Paper presented at 9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK. Planta Medica, 82
Öppna denna publikation i ny flik eller fönster >>Fungi as a source for antibacterial compounds
2016 (Engelska)Ingår i: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 82Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Nyckelord
Dual culture technique, culture media optimization, in-vitro antibacterial bioassay
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:uu:diva-346849 (URN)10.1055/s-0036-1596690 (DOI)000411789300514 ()
Konferens
9th Joint Meeting of AFERP, ASP, GA, JSP, PSE and SIF, JUL 24-27, 2016, Copenhagen, DENMARK
Tillgänglig från: 2018-03-27 Skapad: 2018-03-27 Senast uppdaterad: 2018-03-27Bibliografiskt granskad
Gunasekera, S., Muhammad, T., Strömstedt, A. A. & Göransson, U. (2014). Backbone-Cyclized Peptide Dimers Derived from Human Cathelicidin Peptide LL-37 Mediate Potent Antimicrobial Activity. Journal of Peptide Science, 20, S270-S271
Öppna denna publikation i ny flik eller fönster >>Backbone-Cyclized Peptide Dimers Derived from Human Cathelicidin Peptide LL-37 Mediate Potent Antimicrobial Activity
2014 (Engelska)Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 20, s. S270-S271Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Nyckelord
dimer, cyclic, anti-bacterial, LL-37
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
urn:nbn:se:uu:diva-233367 (URN)000340876400402 ()
Tillgänglig från: 2014-10-02 Skapad: 2014-10-02 Senast uppdaterad: 2017-12-05
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