Novel antimicrobials to combat Gram-negative bacteria

Project Summary Drug-resistant Gram-negative pathogens such as Escherichia coli and Klebsiella pneumoniae are life- threatening and challenging to eliminate. It is stunning that they could avoid the killing of even colistin, the last resort antibiotic against these pathogens. While a comprehensive strategy is needed, the development of novel antimicrobials remains an important element. The objective of this project is to discover novel antimicrobials that can effectively eliminate drug-resistant Gram-negative pathogens that escape the killing of conventional antibiotics. Antimicrobial peptides are important candidates and some (e.g., daptomycin and gramicidin) are already in clinical use. However, it is not yet clear how to choose a promising template for antimicrobial developments. This project takes a new avenue to peptide discovery by combining our unique database tool with structural biology. The antimicrobial peptide database is an original resource established and maintained by the PI's laboratory for over a decade. To facilitate our development, we have established universal peptide classification methods, set up criteria for peptide registration, developed the database filtering technology, and discovered a novel concept for peptide design. As exciting preliminary results, we have identified verine, which killed resistant bacteria, disrupted preformed biofilms in vitro and showed systemic efficacy in vivo against colistin-resistant Klebsiella comparable to doripenem. Remarkably, verine possesses a novel amphipathic structure, entirely different from the classic amphipathic helical structure. We hypothesize that verine is potent against antibiotic-resistant Gram-negative bacteria; structure-activity relationship, ADME (absorption, distribution, metabolism, excretion)-toxicity, pharmacokinetic/pharmacodynamics (PK/PD) studies and in vivo efficacy evaluation in different animal models will improve our knowledge and expand its therapeutic potential. To test our hypothesis, we have designed the following specific aims: (1) Elucidate the structure-activity relationship, test antimicrobial susceptibility of verine against Gram-negative pathogens in vitro alone or in combination with antibiotics and study the genetic basis of bacterial response; (2) Investigate the in vitro and in vivo toxicity and pharmacokinetic properties of verine and D-verine; and (3) Evaluate efficacy of verine against antibiotic-resistant Gram-negative pathogens in murine models. We are in an excellent position to pursue this project. Our preliminary results for each Aim support the feasibility of this project. To provide complementary expertise, the PI has assembled a strong team with expertise in bioinformatics, structural biology, peptide chemistry, antimicrobial assays, mechanism of action, microbiology, animal models, PK/PD, pathology, immunology, and industrial antimicrobial development. The completion of this project will foster new ideas to combat antimicrobial resistance, substantially improve our understanding of antimicrobial capability, PK/PD and in vivo efficacy of the novel peptide verine and its analog against various Gram-negative pathogens.