Project Summary As part of the program “Modulation of Protein Production and Degradation as an Integrated Approach to Rapid Sterilization of Drug Sensitive and Resistant Mycobacterium tuberculosis (Mtb)”, Project 2 will focus on “Discovery of inhibitors that target the Mtb ClpP1P2 protease”. Genetic studies suggest that the Clp system represents a uniquely attractive drug target. It is essential under all conditions, both growing and non-growing. And it is tightly regulated – even small changes in activity result in cell death. Upon depletion of Clp proteins, cells rapidly die, both in vitro and in a mouse model of tuberculosis. In fact, the death is much more rapid than is seen with depletion of traditional drug targets. Thus, an antibiotic that targeted Clp could provide a path to a sought-after treatment-shortening regimen. Several compounds have been found that target the Clp complex. The vast majority of those compounds target one of the ATPase components, ClpC1, and these compounds will be optimized as part of Project 1. Project 2 will instead focus on modulators of the ClpP1P2 protease, the core function of Clp. In other bacteria, activators of Clp proteolytic activity are lethal. Genetic studies have shown that inhibition of Clp protease in mycobacteria also results in cell death. Moreover, a series of modified peptides inhibit Clp-mediated proteolysis in vitro and kill Mtb in a Clp-dependent manner. Thus, the goal of Project 2 is to work together with all the Scientific Cores to develop these promising compounds to improve their activity and pharmacology, towards the program’s ultimate goal of developing new antituberculous agents. In particular, the project will work with Cores A and C to help design and synthesize new inhibitors, and rely on Cores B and D for testing and product development. Specifically, Project 2 will: 1. Identify the sequence determinants of Clp-inhibitory peptides. Mapping of the sequence specificity of the Clp protease has been used to design first generation inhibitors. This project will use purified ClpP1P2 complexes in vitro to further define peptide characteristics to enable design of improved inhibitors. 2. Develop optimized mechanism-based peptide inhibitors. The project will use information from substrate identification to produce modified peptides that act as inhibitors. These will initially be tested for their ability to inhibit the enzyme and then their ability to kill Mtb. For those that are optimal, pharmacologic and toxicologic parameters will be determined as an initial step toward drug development. 3. Identify non-peptidic analogues that act as Clp inhibitors. Peptides often have pharmacologic issues that could make them non-optimal. Non-peptidic inhibitors will be designed using optimized substrates and co-crystals. These will be tested with both purified enzymes and in whole cell assays.

Project Narrative Tuberculosis remains a difficult disease to treat, with current therapy requiring a six-month course of supervised treatment – expensive and logistically challenging to implement, particularly in endemic areas that include some of the most poverty-stricken in the world. Because of this, and the significant burden of drug resistant disease worldwide, drugs that worked more rapidly and that were active against drug resistant strains could have a considerable impact on a disease that still kills more than 1.3 million people each year. This project brings together experts in mycobacteriology and drug discovery and development, to pursue modulation of protein production and degradation in an integrated approach to rapid sterilization.