Antimicrobial Characterization and Therapeutic Applications of Novel Synthetic Thiazole Compounds against Multidrug-Resistant Staphylococci and Enterococci

Haroon Mohammad, Purdue University

Abstract

For more than a century, antibiotics have been valuable allies in combating an array of bacterial infections. However, each year nearly 23,000 people in the United States of America and 25,000 people in Europe die due to infections that are recalcitrant to currently available antimicrobials. The emergence of drug-resistant bacterial species, namely methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has limited the efficacy of several classes of antibiotics. Compounding this problem further is that many large pharmaceutical companies have left the field of antibacterial drug discovery given the high cost of innovation and low return on investment. Collectively, this highlights an urgent, unmet need to identify and develop new antibacterial agents that attack unique molecular targets in bacterial pathogens. Here, we investigate the antibacterial activity of a new series of phenylthiazole antibiotics against a panel of clinically-relevant ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The lead compound 1 was identified through whole-cell screening of libraries of substituted thiazoles and thiadiazoles. Subsequent derivatives were constructed in an attempt to enhance potency, decrease toxicity to host tissues, and improve the lead compound’s drug-like properties. Broth microdilution assay results show that the lead 1 and two derivatives (2 and 3) possess potent activity against Gram-positive bacterial pathogens including MRSA, methicillin-resistant Staphylococcus pseudintermedius (an emerging pathogen of importance in veterinary medicine) and VRE, inhibiting the growth of clinical isolates at concentrations as low as 0.5 µg/mL. The presence of the outer membrane and efflux pumps appears to impede the antibacterial activity of the phenylthiazoles against Gram-negative bacteria. MRSA and VRE mutants resistant to the phenylthiazoles could not be isolated, both via single-step and multi-step resistance selection analysis. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis as deduced from Bacterial Cytological Profiling. Transposon mutagenesis suggested three possible targets: YubA, YubB and YubD. YubB is undecaprenyl diphosphate phosphatase (UPPP) and UPPP as well as undecaprenyl diphosphate synthase (UPPS) were inhibited by 1, as confirmed by traditional enzyme inhibition assays. YubA and YubD are annotated as transporters and may also be targets since 1 collapsed the proton motive force in membrane vesicles. This indicates the phenylthiazole antibacterial agents have a unique mechanism of action that involves inhibition of key enzymes involved in peptidoglycan biosynthesis and potential transporters. This may contribute to the inability to generate bacterial mutants exhibiting resistance to the phenylthiazoles. The compounds were not toxic up to 20-40 µg/mL against different human cell lines including keratinocytes (HaCaT), kidney cells (HEK293), and colorectal cells (HRT-18). Additionally, the compounds were found to be non-toxic (at 20 µg/mL) in a Caenorhabditis elegans animal model. Closer inspection of the physicochemical profile and in silico pharmacokinetic profile of the lead 1 and more metabolically-stable analogue 3 revealed potential application for use topically (for localized skin infections), intravenously (for systemic infections), and as decolonizing agents. Utilizing a murine skin infection model, 1 and 3 were found to significantly reduce the burden of MRSA in infected lesions by more than 96%. Furthermore, both compounds (at 20 µg/mL) were potent in vivo, reducing the burden of VRE in infected C. elegans. Taken altogether, the results indicate that phenylthiazoles 1 and 3 are promising novel topical antibacterial agents and decolonizing agents for use in the treatment of drug-resistant staphylococcal and enterococcal infections.

Degree

Ph.D.

Advisors

Seleem, Purdue University.

Subject Area

Molecular biology|Microbiology|Medicine

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