Slusky Research Project Summary

Antibiotic resistance is correlated with overexpression of the acridine efflux pump. This pump is the preeminent efflux pump in gram-negative bacteria and is responsible for shuttling out most classes of antibiotics. Previous efforts have led to compounds that disable pumps by inhibiting one of the drug binding sites in the inner membrane component of the pump, but such compounds have proven toxic and overly specific. Here, we propose to create peptides and peptidomimetics that prevent oligomerization of the outer membrane component of the acridine pump. The outer membrane component of the acridine pump is a trimeric β-barrel called TolC. Targeting the outer membrane portion of the pump reduces concerns over toxicity because the target complex is unique to outer membranes and human cells do not possess an outer membrane. Moreover, targeting oligomerization instead of targeting one of the two binding sites broadens the applicability of the inhibitor. Specifically, by targeting oligomerization we can stop all efflux through the pump, not just the antibiotics that interact with one of the multiple acridine pump binding sites. Our long-term goal is to develop compound that resensitize gram-negative antibiotic resistant bacteria to a variety of antibiotics. Our central hypothesis is that we can disrupt assembly of the outer membrane β-barrel component of efflux pumps, by binding their interface strands with β-hairpin peptides or β-hairpin mimetics similar to the interfacial strands’ native binding partners. This will be significant because it represents a step towards enable a revival of existing antibiotics for resistant superbugs, by using helper drugs that would be less likely to suffer from toxicity or over specificity. This work is innovative because it introduces a new type of inhibitor for β-barrels, extending the method of dominant negative fragment inhibition for use in the outer membrane. We plan to carry out this project through pursuit of two aims. In the first aim we will create a screen to find peptides that disrupt TolC drug efflux. We will carry this out by creating a β-hairpin library modeled after the β-strands at the interface of the TolC trimeric interaction. Successful folding and binding of these peptides will be identified through FACS and replica plating, then we will test successful peptides on a broad range of antibiotics. In the second aim we will design peptidomimetics that disrupt TolC oligomerization. We do this by designing β-hairpin mimetics, synthesizing these, and testing their activity against several gram-negative bacteria and antibiotics.