Prospects of In vivo Incorporation of non-canonical amino acids for the chemical diversification of antimicrobial peptides

The incorporation of non-canonical amino acids (ncAA) is an elegant way for the chemical diversification of recombinantly produced antimicrobial peptides (AMPs). Residue- and site-specific installation methods in several bacterial production hosts hold great promise for the generation of new-to-nature AMPs, and can contribute to tackle the ongoing emergence of antibiotic resistance in pathogens. Especially from a pharmacological point of view, desirable improvements span pH and protease resistance, solubility, oral availability and circulation half-life. Although the primary focus of this report is on ribosomally synthesized and post-translationally modified peptides (RiPPs), we have included selected cases of peptides produced by solid phase peptide synthesis to comparatively show the potential and impact of ncAA introduction. Generally speaking, the introduction of ncAAs in recombinant AMPs delivers novel levels of chemical diversification. Cotranslationally incorporated, they can take part in AMP biogenesis either through direction interaction with elements of the post-translational modification (PTM) machinery or as untargeted sites with unique physicochemical properties and chemical handles for further modification. Together with genetic libraries, genome mining and processing by PTM machineries, ncAAs present not a mere addition to this process, but a highly diverse pool of building blocks to significantly broaden the chemical space of this valuable class of molecules. This perspective summarizes new developments of ncAA containing peptides. Challenges to be resolved in order to reach large-scale pharmaceutical production of these promising compounds and prospects for future developments are discussed

Peptides and Peptidomimetics for Antimicrobial Drug Design

The purpose of this paper is to introduce and highlight a few classes of traditional antimicrobial peptides with a focus on structure-activity relationship studies. After first dissecting the important physiochemical properties that influence the antimicrobial and toxic properties of antimicrobial peptides, the contributions of individual amino acids with respect to the peptides antibacterial properties are presented. A brief discussion of the mechanisms of action of different antimicrobials as well as the development of bacterial resistance towards antimicrobial peptides follows. Finally, current efforts on novel design strategies and peptidomimetics are introduced to illustrate the importance of antimicrobial peptide research in the development of future antibiotics

Novel antimicrobial peptides for enhanced antimicrobial activity against methicillin resistant Staphylococcus aureus: design, synthesis and formulation.

Doctoral Degree. University of KwaZulu-Natal, Durban.Abstract available in pdf

DE NOVO DESIGN OF ANTIMICROBIAL PEPTIDES FOR APPLICATION AS ANTI-INFECTIVE AGENTS

Ph.DNUS-ICL JOINT PH.D. (FoS

Bionano-Interfaces through Peptide Design

The clinical success of restoring bone and tooth function through implants critically depends on the maintenance of an infection-free, integrated interface between the host tissue and the biomaterial surface. The surgical site infections, which are the infections within one year of surgery, occur in approximately 160,000-300,000 cases in the US annually. Antibiotics are the conventional treatment for the prevention of infections. They are becoming ineffective due to bacterial antibiotic-resistance from their wide-spread use. There is an urgent need both to combat bacterial drug resistance through new antimicrobial agents and to limit the spread of drug resistance by limiting their delivery to the implant site. This work aims to reduce surgical site infections from implants by designing of chimeric antimicrobial peptides to integrate a novel and effective delivery method. In recent years, antimicrobial peptides (AMPs) have attracted interest as natural sources for new antimicrobial agents. By being part of the immune system in all life forms, they are examples of antibacterial agents with successfully maintained efficacy across evolutionary time. Both natural and synthetic AMPs show significant promise for solving the antibiotic resistance problems. In this work, AMP1 and AMP2 was shown to be active against three different strains of pathogens in Chapter 4. In the literature, these peptides have been shown to be effective against multi-drug resistant bacteria. However, their effective delivery to the implantation site limits their clinical use. In recent years, different groups adapted covalent chemistry-based or non-specific physical adsorption methods for antimicrobial peptide coatings on implant surfaces. Many of these procedures use harsh chemical conditions requiring multiple reaction steps. Furthermore, none of these methods allow the orientation control of these molecules on the surfaces, which is an essential consideration for biomolecules. In the last few decades, solid binding peptides attracted high interest due to their material specificity and self-assembly properties. These peptides offer robust surface adsorption and assembly in diverse applications. In this work, a design method for chimeric antimicrobial peptides that can self-assemble and self-orient onto biomaterial surfaces was demonstrated. Three specific aims used to address this two-fold strategy of self-assembly and self-orientation are: 1) Develop classification and design methods using rough set theory and genetic algorithm search to customize antibacterial peptides; 2) Develop chimeric peptides by designing spacer sequences to improve the activity of antimicrobial peptides on titanium surfaces; 3) Verify the approach as an enabling technology by expanding the chimeric design approach to other biomaterials. In Aim 1, a peptide classification tool was developed because the selection of an antimicrobial peptide for an application was difficult among the thousands of peptide sequences available. A rule-based rough-set theory classification algorithm was developed to group antimicrobial peptides by chemical properties. This work is the first time that rough set theory has been applied to peptide activity analysis. The classification method on benchmark data sets resulted in low false discovery rates. The novel rough set theory method was combined with a novel genetic algorithm search, resulting in a method for customizing active antibacterial peptides using sequence-based relationships. Inspired by the fact that spacer sequences play critical roles between functional protein domains, in Aim 2, chimeric peptides were designed to combine solid binding functionality with antimicrobial functionality. To improve how these functions worked together in the same peptide sequence, new spacer sequences were engineered. The rough set theory method from Aim 1 was used to find structure-based relationships to discover new spacer sequences which improved the antimicrobial activity of the chimeric peptides. In Aim 3, the proposed approach is demonstrated as an enabling technology. In this work, calcium phosphate was tested and verified the modularity of the chimeric antimicrobial self-assembling peptide approach. Other chimeric peptides were designed for common biomaterials zirconia and urethane polymer. Finally, an antimicrobial peptide was engineered for a dental adhesive system toward applying spacer design concepts to optimize the antimicrobial activity

Design of advanced materials and nano delivery approaches for enhancing activity against Methicillin resistant Staphylococcus aureus.

Doctoral Degree. University of KwaZulu-Natal, Durban.Infectious diseases, including bacterial infections, continue to be a significant cause of morbidity and mortality globally, antimicrobial resistance has further made them fatal. Limitations of conventional dosage forms have been found to be one of the contributing factors to antimicrobial resistance. Novel nano delivery systems are showing potential to combat antimicrobial resistance. The search for novel materials for efficient delivery of antibiotics is an active research area. The aim of the study was to design and synthesize advanced materials and explore nano-based strategies for preparations of novel drug delivery systems to treat SA and MRSA infections. In this study two novel materials; a linear polymer dendrimer hybrid star polymer (3-mPEA) comprising of a generation one poly (ester-amine) dendrimer (G1-PEA) and copolymer of methoxy poly (ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) and oleic acid based quaternary lipid (QL) were synthesized and characterized and Poloxamer 188 (P188) material available in the market were employed to formulate three nano drug delivery systems for efficient and targeted delivery of antibiotics. The synthesized materials and the drug delivery system were found to be biosafe after exhibiting cell viability above 75% in all the cell lines tested on using MTT assay. The formulated nano based systems were evaluated for sizes, polydispersity indices (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibacterial activity. Nanovesicles were formulated from 3-mPEA and they had sizes, PDI, ZP and entrapment efficiency of 52.48 ± 2.6 nm, 0.103 ± 0.047, -7.3 ± 1.3 mV and 76.49 ± 2.4%. respectively. QL lipid was employed to formulate vancomycin (VCM) loaded liposomes with Oleic acid based ‘On’ and ‘Off’” pH responsive switches for infection site and intracellular bacteria targeting. They were found to have the size of 98.88 ± 01.92 at pH 7.4. and exhibited surface charge switching from negative at pH 7.4 to positive charge accompanied by faster drug release at pH 6.0. Fusidic acid nanosuspension (FA-NS) with size, PDI and ZP of 265 ± 2.25 nm, 0.158 ± 0.026 and -16.9 ± 0.794 mV respectively was formulated from P188. The drug release profile from both the nanovesicles and liposomes was found to have sustained release. In vitro antibacterial activity for the nanovesicles, FA-NS and liposomes showed 8, 6 and 4-fold better activity at pH 7.4, while the liposome being a pH responsive antibacterial system at pH 6 showed 8- and 16- fold better activity against both Methicillin susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) respectively when compared with the bare drugs. An in vivo BALB/c mice, skin infection model revealed that treatment with VCM-loaded nanovesicles, liposomes and FA-Ns significantly reduced the MRSA burden compared to bare drugs and untreated groups. There was a 20, 6.33 and 76-fold reduction in the MRSA load in mice skin treated with nanovesicles, liposomes and FA-NS respectively compared to those treated with bare VCM and fusidic acid. In summary, synthesized material showed to be biosafe and potential for the development of nano-based drug delivery systems of antibiotics against bacterial infections. The data from this study has resulted in one book chapter and 3 first authored and 3 co-authored research publications

Expanding the Genetic Code of and to Incorporate Non-canonical Amino Acids for Production of Modified Lantibiotics

The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium Lactococcus lactis, bears great potential to expand the chemical space of various antimicrobials. The ncAA Nε-Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase–tRNAPyl pair to establish orthogonal translation in L. lactis for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in Escherichia coli cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in E. coli and L. lactis presents a promising tool to create new-to-nature nisin derivatives

Current and emerging therapies for corneal infection: a clinical and laboratory study

Corneal infection or infectious keratitis (IK) is a major cause for corneal blindness worldwide. Broad-spectrum antimicrobial therapy is currently the mainstay of treatment for IK, but the efficacy is being challenged by the emergence of antimicrobial resistance. Host defense peptides (HDPs), also known as antimicrobial peptides (AMPs), are evolutionarily conserved molecules of innate immune system that are found in all kingdoms of life. HDPs have shown promise as a novel class of antimicrobial therapeutics due to their broad-spectrum and rapid antimicrobial activity against a wide array of infection with minimal risk of developing resistance. At the ocular surface, HDPs, particularly human cathelicidin (LL-37) and human beta-defensins (HBDs), have been shown to play a vital role during IK. The first part of this work (Chapter 2 to Chapter 4) consisted of a body of work examining the epidemiology, causes, clinical characteristics, outcomes, and prognostic factors of IK in Nottingham, UK. IK was shown to be a persistent burden in Nottingham over the past decade, with ocular surface diseases, contact lens wear and systemic immunosuppression being the most common risk factors. More than 50% of the patients with IK required hospitalisation for intensive treatment, highlighting the burden of the disease on the patients and the healthcare system. Poor clinical outcome was significantly affected by older age, large infiltrate size and poor presenting vision. The second part (Chapter 5 and Chapter 6) systematically examined the effectiveness and safety of adjuvant therapeutic corneal collagen cross-linking (PACK-CXL) and amniotic membrane transplant for treating IK, in addition to standard antimicrobial therapy. The meta-analyses demonstrated that both interventions significantly expedited the healing of IK, though the overall quality of evidence was low, highlighting the need for further high-quality randomised controlled trials. The third part (Chapter 7 and Chapter 8) highlighted a body of work in developing a new class of HDP-based antimicrobial therapy for IK based on hybrid derivatives of human cathelicidin (LL-37) and human beta-defensins-1 to -3. CaD23, derived from LL-37 and HBD-2, exhibited good in vitro efficacy against Gram-positive bacteria and moderate efficacy against Gram-negative bacteria. It demonstrated a rapid antimicrobial activity, which was likely attributed to its membrane-permeabilising activity, supported by SYTOX green dye uptake assay and molecular dynamics simulation study. CaD23 was also shown to exhibit a strong additive effect when used in combination with conventional antibiotics against Gram-positive bacteria. Finally, CaD23 exhibited good antimicrobial efficacy against Gram-positive bacteria (1.2 logCFU or 94% reduction in the bioburden) in a murine bacterial keratitis model. The discovery of CaD23 has provided a new scaffold for future development of newer generations of hybrid peptides

Antimicrobial Peptides from Venom: Structure, Function and Toxicity

The need for new antimicrobials with novel mechanisms of action is becoming one of the most urgent requirements in modern medicine. Antimicrobial peptides (AMPs) are naturally occurring compounds which possess a rapid killing mechanism and low resistance potential. Consequently, they are being viewed as potential alternatives to traditional antibiotics. One of the major factors limiting further development of AMPs is off target toxicity. Enhancements to antimicrobial peptides which can maximise antimicrobial activity whilst reducing mammalian cytotoxicity would make these peptides more attractive as future pharmaceuticals. Smp24 and Smp43 are AMPs derived from the venom of the scorpion Scorpio maurus palmatus. This study sought to better understand the relationship between structure, function and bacterial selectivity of these peptides by performing single amino acid substitutions. The structure-function relationship of the two AMPs has been investigated by performing N-terminal, mid-chain and Cterminal amino acid substitutions and determining the effect this has on the antimicrobial and cytotoxic activity of the peptides. The structural implications of the amino acid substitutions have been investigated via homology modelling and circular dichroism spectroscopy. Functional improvements have been made to modified peptides when compared with native Smp24 and native Smp43, which have produced peptides with enhanced therapeutic indices