New Antimicrobials that Act Via Membrane Remodeling for Critical Gram-Negative Pathogens

PROJECT SUMMARY Antimicrobial resistance (AMR) is an existential threat to global human health, causing ~1.3M deaths and ~50M years of life lost annually. Antibiotics are the cornerstone of modern medicine, and we stand to lose advances in treating myriad diseases if we lose the arms race with AMR. There is an urgent need for novel antibiotics with unique chemical structures and differentiated mechanisms of action (MOA). The ability of bacteria to rapidly mutate and develop resistance necessitates the selection of targets that are not only essential but also the products of multiple genes. The membrane represents such a target and has been successfully exploited by host immune systems, antimicrobial peptides (AMPs), AMP-like therapeutics such as polymyxins, and antiseptics. Membrane-targeting small molecules have certain favorable properties relative to AMPs, such as simpler manufacturing and the potential for better pharmacokinetics. However, despite the promise, membrane- targeting small molecules have yet to obtain regulatory approval due to challenges with selectivity for bacteria and safety in vivo. We have discovered a novel class of membrane-modifying antimicrobials, called Anti-infective Conjugated Electrolytes (ACEs), that we aim to develop into life-saving treatments for the greatest AMR threats such as lower respiratory infections caused by K. pneumoniae. ACEs are highly selective for bacteria, rapidly bactericidal, active in vivo, and have anti-biofilm activity, low cytotoxicity, and no hemolytic properties. Subtleties of the MOA are still under investigation, but ACEs are not lytic and do not exert their antimicrobial activity through non-specific membrane permeabilization or depolarization. Instead, ACEs induce membrane remodeling, which is suspected to cause mislocalization or dysfunction of essential membrane proteins. ACE structure-activity relationships (SAR) have been elucidated and laid the foundation for our recent partnership with NIH Center for Combating Antibiotic Resistant Bacteria (CC4CARB). New ACE scaffolds co-designed with CC4CARB serve as the initial subject matter for this project. We will assess ~40 ACEs synthesized by CC4CARB to elucidate additional SAR and utilize this information to design an additional ~40 ACE derivatives of promising subfamilies (Aim 1). From this composite set of ACEs, we will identify promising leads via a gated-tier approach (Aim 2). The activity of derivatives will first be assessed against a panel of critical gram-negative and gram-positive pathogens. ACEs with high activity and low cytotoxicity will pass to the second tier of in vitro activity and safety testing. The highest performing 6-8 ACEs will then be assayed for their bactericidal kinetics and antibiofilm activity against K. pneumoniae. Additionally, the activity of these derivates will be determined in host-relevant media as well as a Galleria infection model. 4 ACEs will be selected for assessment of resistance development, efficacy in murine models of lung infection caused by K. pneumoniae, in vivo safety, and pharmacokinetics (Aim 3). Successful completion of this Phase 1 project will validate the ACE platform, identify new lead ACE subfamilies, and provide SAR insights that can be exploited in subsequent lead optimization as part of a Phase 2 project.