Rubin, Eric JZinga, Samuel2026-06-0920262026-06-052026Zinga, Samuel. 2026. Targeting the Clp Protease for Mycobacterial Drug Development. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32702593https://dash.harvard.edu/handle/1/42740473Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), remains one of the deadliest and most widespread infectious diseases worldwide. Although antibiotic therapy for TB has existed for several decades, drug resistance has made the disease increasingly difficult to treat and cure. Today, there are more than 400,000 estimated cases of drug-resistant TB annually, underscoring the need for therapies that exploit previously untargeted vulnerabilities in Mtb. One particularly compelling area for new antibiotic development is protein homeostasis (proteostasis). Proteostasis is critical to the survival of all organisms, enabling repair of damage from environmental stress, regulation of dosage-sensitive proteins, and remodeling the proteome during adaptation to new environments. This is particularly true for Mtb, which relies on these processes for successful transmission and survival within the host. As such, proteostasis—and protein degradation in particular—may provide a key therapeutic vulnerability. In this thesis, we focus on proteolysis mediated by the essential caseinolytic protease system (Clp) as an emerging target, using two distinct but complementary approaches. First, we evaluate the potential of directly inhibiting this essential protease and characterize how mycobacteria can evolve resistance to a specific class of Clp inhibitors, with the goal of informing efforts to advance these compounds toward clinical studies. We identify two mutations in Mtb that confer resistance to these inhibitors and assess their consequences for bacterial fitness. These data inform how likely such mutations are to arise in clinical settings and clarify the implications of resistance for future inhibitor design and deployment. Second, we expand on an emerging modality for antibiotic development— targeted protein degradation. In this approach, essential proteins are inducibly degraded by cell-intrinsic proteolytic systems rather than inhibited by a classical small molecule. This approach holds the potential of enabling inactivation of several essential proteins which were previously considered difficult to inhibit. However, in mycobacteria, this approach is limited by an incomplete understanding of which proteins are amenable to this strategy. Here, we explore the capacity of the Clp protease to degrade native mycobacterial proteins using a chemical-genetic, induced-proximity platform. We show that several native proteins can be targeted for degradation in this manner and use these results to define protein-intrinsic features that predict degradation potential. Finally, we demonstrate that degrading certain proteins can not only inhibit mycobacterial growth but can also potentiate the effects of existing antibiotics and resensitize drug-resistant bacteria to clinically useful compounds. Taken as a whole, these findings identify genetic routes of resistance to Clp inhibitors and provide a foundation for exploiting Clp-mediated proteolysis as a therapeutic strategy, thereby laying the groundwork for the rational development of a new class of TB therapeutics.application/pdfenAntibioticClpDegradationPROTACTargeted protein degradationTuberculosisMicrobiologyBiologyThesis or Dissertation2026-06-090000-0002-4633-8299