Elucidating the bactericidal mechanism of action of the linear antimicrobial tetrapeptide BRBR-NH2.

Linear antimicrobial peptides, with their rapid bactericidal mode of action, are well-suited for development as topical antibacterial drugs. We recently designed a synthetic linear 4-residue peptide, BRBR-NH2, with potent bactericidal activity against Staphylococcus aureus (MIC 6.25 µM), the main causative pathogen of human skin infections with an unknown mechanism of action. Herein, we describe a series of experiments conducted to gain further insights into its mechanism of action involving electron microscopy, artificial membrane dye leakage, solution- and solid-state NMR spectroscopy followed by molecular dynamics simulations. Experimental results point towards a SMART (Soft Membranes Adapt and Respond, also Transiently) mechanism of action, suggesting that the peptide can be developed as a topical antibacterial agent for treating drug-resistant Staphylococcus aureus infections.

Antifungal peptides: a potential new class of antifungals for treating vulvovaginal candidiasis caused by fluconazole-resistant Candida albicans.

Vulvovaginal candidiasis/candidosis is a common fungal infection afflicting approximately 75% of women globally caused primarily by the yeast Candida albicans. Fluconazole is widely regarded as the antifungal drug of choice since its introduction in 1990 due to its high oral bioavailability, convenient dosing regimen and favourable safety profile. However, its widespread use has led to the emergence of fluconazole-resistant C. albicans, posing a universal clinical concern. Coupled to the dearth of new antifungal drugs entering the market, it is imperative to introduce new drug classes to counter this threat. Antimicrobial peptides (AMPs) are potential candidates due to their membrane-disrupting mechanism of action. By specifically targeting fungal membranes and being rapidly fungicidal, they can reduce the chances of resistance development and treatment duration. Towards this goal, we conducted a head-to-head comparison of 61 short linear AMPs from the literature to identify the peptide with the most potent activity against fluconazole-resistant C. albicans. The 11-residue peptide, P11-6, was identified and assayed against a panel of clinical C. albicans isolates followed by fungicidal/static determination and a time-kill assay to gauge its potential for further drug development. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2-P4 study.

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 µM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.

Designing an ultra-short antibacterial peptide with potent activity against Mupirocin-resistant MRSA.

Staphylococcus aureus is the pathogen responsible for the majority of human skin infections. In particular, the methicillin-resistant variety, MRSA, has become a global clinical concern. The extensive use of mupirocin, the first-line topical antibacterial drug of choice, has led to the emergence of mupirocin-resistant MRSA globally, resulting in the urgent need for a replacement. Antimicrobial peptides are deemed plausible candidates. Herein, we describe a structure-activity relationship approach in the design of an ultra-short peptide with potent anti-MRSA activity with a rapid, bactericidal mode of action. Coupled to a low cytotoxic activity, we believe our lead compound can be developed into a topical antibacterial agent to replace mupirocin as the first-line drug for treating MRSA skin infections.

Structure-activity relationship studies of ultra-short peptides with potent activities against fluconazole-resistant Candida albicans.

Vulvovaginal candidiasis (VVC) is a genital fungal infection afflicting approximately 75% of women globally and is primarily caused by the yeast Candida albicans. The extensive use of fluconazole, the first-line antifungal drug of choice, has led to the emergence of fluconazole-resistant C. albicans, creating a global clinical concern. This, coupled to the lack of new antifungal drugs entering the market over the past decade, has made it imperative for the introduction of new antifungal drug classes. Peptides with antifungal properties are deemed potential drug candidates due to their rapid membrane-disrupting mechanism of action. By specifically targeting and rapidly disrupting fungal membranes, they reduce the chances of resistance development and treatment duration. In a previous screening campaign involving an antimicrobial peptide library, we identified an octapeptide (IKIKIKIK-NH2) with potent activity against C. albicans. Herein, we report a structure-activity relationship study on this peptide with the aim of designing a more potent peptide for further development. The lead peptide was then tested against a panel of fluconazole-resistant C. albicans, subjected to a fungicidal/static determination assay, a human dermal fibroblast viability assay and a homozygous profiling assay to gain insights into its mechanism of action and potential for further development as a topical antifungal agent.

Preliminary investigations into developing all-D Omiganan for treating Mupirocin-resistant MRSA skin infections.

Staphylococcus aureus is the primary pathogen responsible for the majority of human skin infections, and meticillin-resistant S. aureus (MRSA) currently presents a major clinical concern. The overuse of Mupirocin, the first-line topical antibacterial drug over 30 years, has led to the emergence of Mupirocin-resistant MRSA, creating a clinical concern. The antimicrobial peptide Omiganan was touted to be a promising antibacterial drug candidate due to its rapid membrane-disrupting bactericidal mode of action, entering clinical trials in 2005 as a topical gel to prevent catheter site infections. However, drug development ceased in 2009 due to a lack of efficacy. We postulate this to be due to proteolytic degradation caused by endogenous human skin proteases. Herein, we tested our hypothesis using Omiganan and its all-D enantiomer in a human skin protease stability assay, followed by anti-MRSA activity assay against of a panel of clinical MRSA isolates, a bactericidal/static determination and a time-kill assay to gauge all-D Omiganan's potential for further topical antibacterial drug development.

Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.

Discovery of an ultra-short linear antibacterial tetrapeptide with anti-MRSA activity from a structure-activity relationship study.

The overuse and misuse of antibiotics has resulted in the emergence of drug-resistant pathogenic bacteria, including meticillin-resistant Staphylococcus aureus (MRSA), the primary pathogen responsible for human skin and soft-tissue infections. Antibacterial peptides are known to kill bacteria by rapidly disrupting their membranes and are deemed plausible alternatives to conventional antibiotics. One advantage of their membrane-targeting mode of action is that bacteria are unlikely to develop resistance as changing their cell membrane structure and morphology would likely involve extensive genetic mutations. However, major concerns in using peptides as antibacterial drugs include their instability towards plasma proteases, toxicity towards human cells due to their membrane-targeting mode of action and high manufacturing cost. These concerns can be mitigated by developing peptides as topical agents, by the judicial selection of amino acids and developing very short peptides respectively. In this preliminary report, we reveal a linear, non-hemolytic tetrapeptide with rapid bactericidal activity against MRSA developed from a structure-activity relationship study based on the antimicrobial hexapeptide WRWRWR-NH2. Our finding opens promising avenues for the development of ultra-short antibacterials to treat multidrug-resistant MRSA skin and soft tissue infections.

An FDA-Drug Library Screen for Compounds with Bioactivities against Meticillin-Resistant Staphylococcus aureus (MRSA).

The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA), a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA) for bioactivities against MRSA in a 10 µM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections.

Application of Fragment-Based Drug Discovery against DNA Gyrase†B.

Bacterial resistance to antibiotics remains a serious threat to global health. The gyrase B enzyme is a well-validated target for developing antibacterial drugs. Despite being an attractive target for antibiotic development, there are currently no gyrase B inhibitory drugs on the market. A fragment screen using 1,800 compounds identified 14 fragments that bind to Escherichia coli (E. coli) gyrase B. The detailed characterization of binding is described for all 14 fragments. With the aid of X-ray crystallography, modifications on a low-affinity fragment (KD =253 µM, IC50 =634 µM) has led to the development of a new class of potent phenyl aminopyrazole inhibitors against E. coli gyrase B (IC50 =160 nM). The study presented here combines the use of a set of biophysical techniques including differential scanning fluorimetry, nuclear magnetic resonance, isothermal titration calorimetry, and X-ray crystallography to methodically identify, quantify, and optimize fragments into new chemical leads.