Discovering accumulation guidelines and applying them to develop new antibiotics for Gram-negative pathogens - Abstract Antibiotics were wonder drugs of the 1900s and largely responsible for the dramatic extension of life-span observed during that century. Unfortunately, drug-resistance is now rampant, limiting options for treating certain infections. This problem is exacerbated for Gram-negative pathogens, and there are only a handful of antibiotic drug classes for treating Enterobacterales (including E. coli, K. pneumoniae, and E. cloacae) and Acinetobacter, with even fewer options for P. aeruginosa. Discovering novel antibiotics for Gram-negative pathogens has been challenging, due to the ability of these organisms to severely restrict the types of compounds that traverse the outer membrane and evade efflux. We have pioneered a novel workflow that has focused on understanding small molecule accumulation in Gram-negative bacteria, leading to the discovery of the eNTRy rules (for E. coli), and very recently the PASsagE rules (for P. aeruginosa). The eNTRy rules have been used by researchers all over the world to design and discover >25 novel Gram-negative-active antibiotics, including several that are advancing toward clinical trials. However, it is clear that the majority of antibiotics – even ones with excellent influx – have an ‘efflux liability’, that is, are pumped out of the Gram-negative cell by promiscuous efflux pumps, reducing their ability to act as antibiotics. If there were rules for efflux pump evasion, then highly potent antibiotics from multiple compound classes could be readily designed. Here we propose to apply our successful experimental workflow to identify physicochemical traits that allow efflux evasion. For this work we will measure the accumulation of thousands of structurally diverse compounds in efflux-deficient and efflux-proficient isogenic Gram-negative strains, allowing calculation of an Efflux Ratio. This data will be analyzed with the aid of sophisticated chemoinformatics, followed by validation experiments, allowing the derivation of the ‘EXPEL rules’, chemical features that allow compounds to evade efflux. We will conduct separate experiments and devise separate EXPEL rules for E. coli, P. aeruginosa, and A. baumannii; in important preliminary results we have already made an excellent start to this for E. coli. With an excellent understanding of both influx and efflux in place, we will apply the combined learnings from the eNTRy, PASsagE, and EXPEL rules to develop novel antibiotics from 4 different compound classes. We anticipate at least 4 different optimized antibiotics will arise from this work, compounds that can then advance to help patients. As importantly, these demonstrations will provide a blueprint for other researchers to implement these learnings to develop maximally potent Gram-negative active antibiotics. Our team has expertise in all facets of this work, and collaborating together we have made breakthrough discoveries in understanding compound accumulation and have developed novel antibiotics for the most difficult-to-treat Gram-negative pathogens. We will now bring this experience to bear on the efflux problem, an understanding of which will greatly facilitate Gram-negative antibiotic discovery and development.
