Design, Synthesis and Formulation by Mbuso Faya
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Novel Antimicrobial Peptides for enhanced antimicrobial activity against Methicillin Resistant Staphylococcus aureus: Design, Synthesis and Formulation by Mbuso Faya (MSc Health Science – University of KwaZulu-Natal) Submitted as fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmaceutics at the Discipline of Pharmaceutical Sciences of the School of Health Sciences at the University of KwaZulu-Natal Supervisor: Professor Thirumala Govender (PhD, University of Nottingham, United Kingdom) Date submitted: December 6th 2018 “A candle loses nothing by lighting another candle’’ - James Keller - I “This thesis is dedicated to my family who have consistently pushed beyond boundaries and refused earthly definitions to press on to discover new realities”. ii Declaration 1 – Plagiarism I, Mr. Mbuso Faya, declare that 1. The research data reported in this thesis, except where otherwise indicated is my own original work. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain data, pictures, graphs, or other information belonging to other people, unless specifically acknowledged as being sourced from other people. 4. This thesis does not contain any other persons’ writing, unless specifically acknowledged as being sources from other researchers. Where other written sources have been quoted, then: a. Their words have been rephrased but the general information attributed to them has been referenced; b. Where their exact words have been used, their writing has been placed inside quotation marks, and referenced. 5. Where I have reproduced a publication of which I am an author, co-author, or editor, I have indicated in detail which part of the publication was written by myself alone and have fully referenced such publications. 6. This thesis does not contain any graphics, text or tables copied from the internet, unless specifically acknowledged, and the source being detailed in the reference sections of the dissertation. th Signed: Date: 6 December 2018 I, Professor Thirumala Govender as supervisor of the Ph.D. studies hereby consent to the submission of this Ph.D. thesis. th Signed: Date: 6 December 2018 III Declaration 2 –Publications Details of contribution to publications that form part and/or include research presented in this thesis: The following publications were submitted to their respective reputable high impact factor peer reviewed journals. Conjugates and nano-delivery of antimicrobial peptides for enhancing therapeutic activity M. Faya, R.S. Kalhapure, H.M. Kumalo, A.Y. Waddad, C. Omolo, T. Govender, Conjugates and nano-delivery of antimicrobial peptides for enhancing therapeutic activity, Journal of Drug Delivery Science and Technology. 44 (2018) 153–171. doi:https://doi.org/10.1016/j.jddst.2017.12.010. Antimicrobial cell penetrating peptides with bacterial cell specificity: Pharmacophore modelling, Quantitative Structure Activity Relationship and Molecular Dynamics Simulation M. Faya, R.S. Kalhapure, D. Dhumal, N. Agrawal, C. Omolo, K.G. Akamanchi, T. Govender, Antimicrobial cell penetrating peptides with bacterial cell specificity: Pharmacophore modelling, Quantitative Structure Activity Relationship and Molecular Dynamics Simulation, Journal of Biomololecular Structure and Dynamics. (2018) 1–31. Supramolecular Lipidation of Novel Antimicrobial Peptides Enhances Antimicrobial Activity Against methicillin-resistant Staphylococcus aureus (MRSA) Mbuso Faya, Calvin A. Omolo, Fernando Albericio, Beatriz G. de la torres, Heba A. Hazzah, Ruma Maji, Pavan Walwaker, Chunderika Mocktar, Bongani Nkambule, Thirumala Govender*, European Journal of Pharmaceutics and Biopharmaceutics (manuscript ID: EJPB_2018_1382) The published, accepted and submitted first author manuscripts can be found in Chapters three to five of this thesis. IV Mr. Mbuso Faya contributed to the conceptualization and design of the papers. In addition, he was responsible for analysis and data collection and wrote the papers and undertook all revisions. He also contributed to the synthesis, and characterization of the AMPs and formulation, and characterization of the nanostructured liposomal carriers in terms of particle size, polydispersity index, zeta potential, surface morphology, entrapment efficiency, in vitro and antimicrobial activity and analysis of Molecular dynamics (MD) simulations studies. Dr. R.S. Kalhapure assisted with the inception, overall design of the project. Mr. Dinesh Dhumal was responsible for proving the QSAR data. Mr. Calvin Omolo assisted with the conceptualization and minor revisions of the paper. Mr. Pavan Walwaker assisted in performing the cytotoxicity studies. Dr Ruma Maji assisted with the formulation of liposomes. Dr Hezekiel Kumalo and Dr Ayman Waydad assisted in the revisions of the review paper. Dr Nikhil Agrawal assisted in MD simulation studies. Dr Heba Hazzah assisted with synthesis and purification of the AMPs. Dr. Chunderika Mocktar supervised the in vitro antibacterial activity studies. Dr. Bongani Nkambule assisted with all flow cytometric work. Prof K.G Akamanchi, Prof Fernando Albericio and Prof Beatriz G. de la Torre served as collaborators on the project. Prof. Thirumala Govender served as supervisor and was responsible for project conceptualization, problem- solving, paper and abstract editing and general supervision of the study. Research output from the dissertation 1. First authored Publications The following research papers were published as results generated from specific objectives from this study and they include: - • Faya, M., Kalhapure, R. S., Kumalo, H. M., Waddad, A. Y., Omolo, C., & Govender, T. (2017). Conjugates and nano-delivery of antimicrobial peptides for enhancing therapeutic activity. Journal of Drug Delivery Science and Technology. https://doi.org/10.1016/j.jddst.2017.12.010 • Faya, M., Kalhapure, R. S., Dhumal, D., Agrawal, N., Omolo, C., Akamanchi, K. G., & Govender, T. (2018). Antimicrobial cell penetrating peptides with bacterial cell specificity: Pharmacophore modelling, Quantitative Structure Activity Relationship and Molecular Dynamics Simulation. Journal of Biomolecular Structure and Dynamics, (just-accepted), 1-31. Mbuso Faya1, Heba A. Hazzah, Calvin A. Omolo1, Ruma Maji1, Pavan Walwaker1, Chunderika Mocktar1, Bongani Nkambule2, Fernando Albericio, Beatriz G. de la Torre, Thirumala Govender*,1. Supramolecular Lipidation of Novel Antimicrobial Peptides Enhances Antimicrobial Activity Against methicillin-resistant Staphylococcus aureus (MRSA). European Journal of Pharmaceutics and Biopharmaceutics (manuscript ID: EJPB_2018_1382) V 2. Conference Presentations Poster Presentations The following conference presentations were produced from data generated during this study: 1. Antimicrobial cell penetrating peptides with bacterial cell specificity: Pharmacophore modelling, Quantitative Structure Activity Relationship and Molecular Dynamics Simulation. Mbuso Faya, Rahul S. Kalhapure, Dinesh Dhumal, Nikhil Agrawal, Calvin Omolo, Krishnacharya G. Akamanchi & Thirumala Govender. Journal of Biomolecular Structure and Dynamics. Nano Africa, 23-25 April 2018, Durban South Africa, (senior Author. Oral presentations 2. Antimicrobial cell penetrating peptides with bacterial cell specificity: Pharmacophore modelling, Quantitative Structure Activity Relationship and Molecular Dynamics Simulation. Mbuso Faya, Rahul S. Kalhapure, Dinesh Dhumal, Nikhil Agrawal, Calvin Omolo, Krishnacharya G. Akamanchi & Thirumala Govender. Journal of Biomolecular Structure and Dynamics. 38th Annual Conference of the Academy of Pharmaceutical Sciences,06-08 July 2017, Johannesburg, South Africa. 3. Supramolecular Lipidation of Novel Antimicrobial Peptides Enhances Antimicrobial Activity Against methicillin-resistant Staphylococcus aureus (MRSA). Mbuso Faya1, Calvin A. Omolo 1, Fernando Albericio1, Beatriz G. de la torres1, Heba A. Hazzah3, Ruma Maji1, Pavan Walwaker1, Chunderika Mocktar1, Bongani Nkambule2, Thirumala Govender*,1 University of KwaZulu-Natal Nanotechnology Platform Workshop, 22nd November 2017. The posters can be found in Appendix A. VI Abstract The control of infectious diseases is seriously threatened by the steady increase in the number of microorganisms that are resistant to antimicrobial agents. Some of the interventions to address the problem of resistance include the use of drug combinations and improvements in patient compliance with dosing. The discovery of antimicrobial peptides (AMPs) has given hope to the problem of drug resistance. Therefore, the aim of this study was to design, synthesize and evaluate novel AMPs for their bacterial membrane penetration and activity followed by their co- encapsulation with vancomycin (VCM) and oleic acid (OA) in a liposomal system to enhance their antimicrobial activity. In this study a QSAR model which can simultaneously estimate antimicrobial potential (MIC) and bacterial cell penetrating ability (TI) of antimicrobial cell penetrating peptides (aCPPs) against S. aureus was developed and novel AMPs were designed, synthesized and employed to decorate vancomycin and oleic acid containing liposomes to achieve pH responsiveness for enhanced antimicrobial activity. The QSAR study proved the viability of the therapeutic index of aCPPs in relation to their cell penetrating ability and antimicrobial potential by building a QSAR model which outlined specific descriptors responsible for their potency. The synthesized novel AMPs were found to be biosafe, exhibiting cell viability of