Investigation of Small Molecule Adjuvants that Augment Antibiotic Potency Against Gram-Negative Pathogens

Three years after the COVID-19 pandemic, The Centers for Disease Control and Prevention (CDC) published a special report indicating setbacks in combatting antimicrobial resistance due to pandemic related challenges. Even though antibiotics are not effective against viruses, they were prescribed to upwards of 80% of patients hospitalized with COVID-19 between March and October 2020, with an increase in hospital-acquired antibiotic resistant-infections of 20% reported in 2024. Advantageously, the CDC has simultaneously provided a robust investment in combating the spread of COVID-19, as well as antimicrobial stewardship guidelines to the public and healthcare providers through supporting programs such as Project Firstline to stop the spread of these drug-resistant pathogens. The CDC also provided new data in their Antimicrobial Resistance Threats in the United States reports, with the latest being released July 2024, that describes their key findings on the burden of antimicrobial resistance in 2021 and 2022 compared to 2019. The report explains how during the height of the COVID-19 pandemic healthcare facilities were overwhelmed with longer hospitalizations, struggled with implementing infection prevention and control practices, and increased inappropriate antibiotic usage. While the antibiotic development pipeline features a low return on investment, antibacterial development is still being pursed in both academia and start-up companies to aid in reducing the incidence of antimicrobial resistant infections; however, other approaches are needed to combat this critical threat to human health. One such approach to combat bacterial infections is by using small molecule adjuvants. Adjuvants are non-microbicidal compounds that allow for continued use of already U.S. Food and Drug Administration (FDA) approved antibiotics whose efficacy is diminished due to resistance and also expand the spectrum of antibiotics to include bacteria that are intrinsically resistant. The development of adjuvants that circumvent bacterial resistance mechanisms is a promising orthogonal approach to the development of new antibiotics. This document describes multiple projects focused on the use of small molecule adjuvants in combination with FDA-approved antibiotics against multidrug resistant (MDR) gram-negative pathogens. Initial efforts described in this document include an extensive structure activity relationship (SAR) study on a known IKK-ß inhibitor, IMD-0354, which effectively suppresses colistin resistance in multiple highly colistin resistant clinical bacterial isolates. This SAR study led to the identification of several compounds with more potent colistin adjuvant activity than IMD-0354 against chromosomally resistant Acinetobacter baumannii and Klebsiella pneumoniae, as well as strains that harbor a plasmid-borne mobile colistin resistance (mcr) gene. Four of the compounds uncovered during this project abrogate colistin resistance at nanomolar concentrations, making them some of the most potent colistin adjuvants to date. Another project in this work describes utilizing small molecule adjuvants to augment the activity of gram-positive selective antibiotics against gram-negative bacteria to expand current treatment options for these infections. Comprehensive discussions outline the discovery and SAR investigation of compounds containing a 2-aminobenzimidazole (2-ABI) moiety as adjuvants for the macrolide class of antibiotics. The lead 2-ABI adjuvant lowers the clarithromycin minimum inhibitory concentration (MIC) 512-fold at 30 µM against K. pneumoniae and 16-fold at 5 µM against A. baumannii. Preliminary investigations into the mechanism of action of lead 2-ABI adjuvants suggest the compounds are binding to lipopolysaccharide (LPS) in K. pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, transport, or assembly in A. baumannii. Furthermore, synthetic lethal interactions in the presence of a lead 2-ABI adjuvant were mapped using transposon sequencing (Tn-seq) to aid in uncovering the adjuvant target(s). Lastly, this document details collaborative efforts with North Carolina State University (NCSU) and Purdue University towards uncovering novel scaffolds for use as colistin adjuvants against highly colistin resistant gram-negative bacteria. In collaboration with NCSU, a series of dipyrrins were examined for their potential as colistin adjuvants, where lead compounds increase the activity of colistin against A. baumannii and Pseudomonas aeruginosa. In collaboration with Purdue University, lead compounds were investigated against several colistin-resistant strains and were found to lower the colistin MIC 32,768-fold when dosed at 60 µM against P. aeruginosa.