The alarming emergence of multidrug-resistant (MDR) pathogenic microorganisms worldwide and the lack of next-generation portfolios of novel antimicrobials threaten human and public health. Therefore, it is a worldwide priority to expedite the development of novel antimicrobial therapies to control MDR bacteria effectively. Natural and synthetic antimicrobial peptides (AMPs) exhibit great potential as therapeutic agents because of their unique modes of action in fast-killing bacteria through membrane permeation. However, several barriers to AMP development limit its clinical application. This application aims to overcome current AMP limitations to develop a safe and effective broad-spectrum antimicrobial against MDR Gram-negative bacterial infection. Our novel peptide therapeutics A4-AMP antibiotics (A4X) is a new generation of computationally engineered AMPs (eAMPs) derived from the antimicrobial motif, alpha-4, of a natural human host defense protein SPLUNC1 with negligible toxicity to mammalian cells. The extensive results from our studies demonstrate that our current lead candidate displays superior antibacterial activity to standard of care (SoC) antibiotics in over 500 clinical isolates of difficult-to-kill MDR Gram-negative pathogens obtained from hospitals and the CDC & FDA Antibiotic Resistance Isolate Bank. Our A4X lead also has a much lower tendency to develop resistance than SoC antibiotics. The A4X lead is safe and well tolerated when intravenously administered to mice and rats, with a four times higher maximum tolerated dosage than colistin, a last resort antibiotic, in mouse blood circulation. Moreover, we have demonstrated the efficacy of the A4X lead against Klebsiella pneumoniae and Acinetobacter baumannii in mouse models of bacteremia and respiratory infection. In this project, we will carry out preclinical and pre-IND non-clinical development activities and perform structure-activity relationship (SAR) based optimization of the current A4X lead to advance the preclinical development and to determine the clinical utility. We will extensively examine the safety, pharmacokinetic/pharmacodynamic, and efficacy of these novel antimicrobial agents in small and large animals of the most effective A4X. The targeting bacteria are the MDR strains of Gram-negative species on the CDC's urgent pathogen threats list and WHO's the most critical global priority 1 pathogens list (carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumonia, and Escherichia coli) and, including resistant strains to colistin. This proposal targets the urgent unmet global medical need for novel antibiotics and addresses the U.S. National Action Plan for Combating Antibiotic-Resistant Bacteria in a timely manner. Successful completion of these studies will have an enormous impact on developing a novel class of antibiotics capable of fighting MDR "superbugs."

Public Health Relevance Statement Antimicrobial resistance is an urgent problem that will result in millions of unnecessary deaths in the near future if the development of new antimicrobials is not significantly increased. We have worked diligently in developing a novel class of peptide-based antibiotics named A4X that successfully overcomes the limitations of current antimicrobial peptides and the urgent antimicrobial resistance problem associated with existing standard-of-care antibiotics. Our proposal outlines a rationalized design with the milestone-driven approaches to carry out a series of mechanistic and preclinical testing studies to further develop A4X. The goal of this proposal is to deliver a safe and effective new antimicrobial drug into IND-enabling ready at the end of the study to help initiate future clinical trial and overcome the crisis of antimicrobial and antibiotic resistance.