# Studies of the antibacterial activity of and resistance to molecules targeting the ClpP peptidase

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $457,161

## Abstract

This proposal outlines experiments that will provide mechanistic insights into and accelerate the
medicinal development of small molecules that perturb the function of the ClpP peptidase in M. tuberculosis,
the deadliest bacterial pathogen. The Mtb ClpP peptidase is an atypically complex, barrel-shaped assemblage
of fourteen subunits that degrades proteins through associations with ATP-dependent chaperones that
recognize and unfold substrates. In the past five years, my collaborator, Prof. Robert Sauer at MIT and I have
made significant investments of time and effort in the functional reconstitution of the heterotetradecameric ClpP
and accessory ATPases (ClpX and ClpC1) from Mtb. With that success, we have been able to characterize our
rationally designed modulators of ClpP activity. The voluminous preliminary data (much of which is published)
that we have generated provide a clear roadmap for the proposed research. They will yield insights into protein
homeostasis in bacteria that can be exploited in drug development and molecules that could be much needed
additions to the dwindling armamentarium used in the fight against multi-drug resistant Mtb.
Aim 1. Develop and characterize small molecule modulators of the Mtb ClpP system. This aim is
centered in chemical synthesis and enzymology. One objective is the multi-gram synthesis of an optimized,
mycobactericidal ADEP that inhibits the ClpP system for use in murine models of tuberculosis. Another is
optimization of a novel ADEP fragment that kills Mtb by activation of its ClpP system. We will also synthesize
rationally designed ClpP inhibitors lacking the pharmacological liabilities of the only known mechanism-based
inhibitor (developed in our laboratories). Compounds will be evaluated in in vitro assays of Clp system activity.
Aim 2. Assess activities of and resistance to ClpP modulators in living cultures of mycobacteria. This
aim is focused on comparative evaluations of ClpP modulators in living cultures of mycobacteria- including
assays of minimal inhibitory and bactericidal concentrations. We believe that identification of the substrates of
the Mtb Clp system will reveal insights into the mechanisms of these compounds. By applying new proteomics
technologies along with a well-established in vivo ClpP substrate trapping method to the M. smegmatis Clp
system for the first time, we will identify its substrates in the protease's native state and as it is perturbed by
modulators. We will also investigate the mechanisms of small molecules that potentiate ADEP activity against
Mtb by as much as 16-fold (i.e., hypothetical suppressors of efflux and the tuberculosis drug bedaquiline).
Aim 3. Assess activities of ClpP modulators in mouse models of tuberculosis. Though ADEPs cured
certain bacterial infections in animals, they have not been studied in tuberculosis. In murine models of
tuberculosis, Dr. William Bishai at Johns Hopkins will assess the efficacies of an optimized ADEP alone and in
combination...

## Key facts

- **NIH application ID:** 10238901
- **Project number:** 5R01AI123400-06
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Jason K Sello
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $457,161
- **Award type:** 5
- **Project period:** 2017-09-25 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10238901

## Citation

> US National Institutes of Health, RePORTER application 10238901, Studies of the antibacterial activity of and resistance to molecules targeting the ClpP peptidase (5R01AI123400-06). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10238901. Licensed CC0.

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