Targeting the ClpC1 ATPase for antibiotic development in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis (Mtb). It remains a leading cause of death due to infectious disease globally, with over ten million new cases yearly. TB treatment requires months-long antibiotic combination therapy regimens, which complicates the delivery of care; compounding this problem is the increasing spread of multidrug resistant TB (MDR-TB), which renders current first-line antibiotics ineffective. These factors highlight the urgent need to develop new therapeutics for TB and invite the exploration of novel target classes and modalities. Toward these objectives, we explored proteolytic systems in mycobacteria as a target class distinct from those currently targeted by TB antibiotics used clinically; specifically, the ClpC1 ATPase which, together with ClpP1 and ClpP2, form the multimeric ClpC1P1P2 proteolytic complex. Specifically, we examine previously identified cyclic peptide modulators of ClpC1 activity and investigate endogenous mycobacterial targets for their suitability in a targeted protein degradation (TPD) therapeutic approach tailored for bacteria.

Chapter 1 of this dissertation provides an overview of proteolytic complexes as drug targets and highlights what is known about the ClpC1 ATPase in mycobacteria. We also review the ClpC1 modulatory cyclic peptides and foundational work for developing TPD in bacteria. Finally, we outline the major aims of this work. In Chapter 2, we create an in vivo reporter system to validate the on-target activity of cyclic peptide ClpC1 modulators in Mtb cells to complement a suite of biochemical assays measuring in vitro activity. We find that the cyclic peptides block degradation of the ClpC1-directed reporter at concentrations that inhibit growth, suggesting that the growth-inhibitory potency of these compounds arises through disruption of protease activity, as predicted in vitro. We also discover that the different cyclic peptides exert differential effects on protease and ATPase activity and induce differential subcellular localization of ClpC1 in live cells. In Chapter 3, we develop a genetic system to model the induced proximity of a set of native mycobacterial proteins to ClpC1 and measure their ability to be targeted for degradation. We observe a gradient of degradability among the targets examined and demonstrate profound bacterial growth inhibition when select targets are redirected for degradation. We further find that targeted degradation of specific targets can potentiate antibacterial effects and accelerate the killing kinetics of existing antibiotics which inhibit the same pathways and complexes.

Together, these studies expand what is known about targeting ClpC1 in mycobacteria for the development of new antibiotics. Our work contributes a set of tools that enable the functional interrogation of how existing ClpC1 modulators work and the rational selection of suitable targets for targeted degrader antibiotics for Mtb. In Chapter 4, we discuss the implications of the work presented in this dissertation and outline further work necessary to aid continued efforts to discover ClpC1 modulators and to develop TPD as an emerging modality for TB therapy. Finally, because we believe these findings belong to the whole world and especially to the millions of patients TB afflicts, a summary of this work that is accessible to a general audience is provided as the final appendix. Through this work, we aim to share new knowledge on targeting ClpC1 therapeutically with the scientific community and hope to ultimately contribute to the global pursuit of developing powerful new therapeutics against this deadly pathogen.