Project Grant F31AI203132

PROFILING CYSTEINE LIGANDABILITY AND ESSENTIALITY IN MYCOBACTERIUM TUBERCULOSIS - PROJECT SUMMARY TUBERCULOSIS (TB) IS A LEADING CAUSE OF DEATH GLOBALLY. TREATMENT IS HAMPERED BY PERSISTENCE OF THE CAUSATIVE BACTERIUM, MYCOBACTERIUM TUBERCULOSIS (MTB), IN DRUG-TOLERANT, NONREPLICATING STATES IN THE HOST. THE DEVELOPMENT OF NEW DRUGS IS COMPLICATED BY AN INCOMPLETE UNDERSTANDING OF DISEASE-RELEVANT PROCESSES IN THE PATHOGEN. CYSTEINE RESIDUES IN THE MTB PROTEOME OFFER A HANDLE ON BOTH CHALLENGES: PLAYING MANY IMPORTANT ROLES IN BACTERIAL PHYSIOLOGY, THEY REPRESENT A LARGELY UNTAPPED LANDSCAPE OF DRUGGABLE SITES IN MTB THAT CAN BE EXPLOITED TO COVALENTLY INHIBIT GROWTH, ADAPTATION, OR PERSISTENCE. THE OBJECTIVE OF THIS PROJECT IS TO ADVANCE TARGET- BASED DRUG DISCOVERY FOR TB BY IDENTIFYING KEY DRUGGABLE CYSTEINES IN MTB, INCLUDING IN NONREPLICATING STATES, AND ELUCIDATING THE MECHANISTIC ROLES THEY PLAY IN ESSENTIAL BACTERIAL PROCESSES. THE PROPOSED RESEARCH APPLIES ACTIVITY-BASED PROTEIN PROFILING TO MAP CYSTEINE LIGANDABILITY ACROSS THE MTB PROTEOME IN BOTH STANDARD CULTURE AND A DISEASE-RELEVANT CASEUM MODEL OF NONREPLICATION. THESE ATLASES WILL REVEAL CYSTEINE SITES SELECTIVELY TARGETABLE BY STEREOSPECIFIC ELECTROPHILES AND IDENTIFY HOW BACTERIAL ADAPTATION TO THE HOST ENVIRONMENT RESHAPES COVALENT DRUGGABILITY. FUNCTIONAL GENETIC SCREENING IN MYCOBACTERIUM SMEGMATIS AND MTB WILL THEN HIGHLIGHT WHICH LIGANDABLE CYSTEINES ARE REQUIRED FOR FITNESS. FINALLY, TAILORED BIOCHEMICAL AND FUNCTIONAL ASSAYS WILL DEFINE THE MECHANISMS BY WHICH HIGH-PRIORITY CYSTEINES GOVERN ESSENTIAL PROCESSES. THIS WORK WILL SHED NEW LIGHT ON THE MTB PROTEOME TO YIELD BOTH FUNDAMENTAL MYCOBACTERIOLOGY INSIGHTS AND PINPOINT NEW HANDLES FOR DRUG DISCOVERY. THE TRAINING PLAN WILL ALSO DEVELOP BROAD EXPERTISE IN GENETIC, BIOCHEMICAL, AND CHEMICAL-PROTEOMIC APPROACHES CENTRAL TO MECHANISTIC INFECTIOUS DISEASE RESEARCH. TRAINING INCLUDES CLOSE COLLABORATION WITH EXPERTS IN MICROBIAL GENETICS, CHEMICAL BIOLOGY, MASS SPECTROMETRY, BIOCHEMISTRY, AND MORE; REGULAR MENTORSHIP FROM DIVERSE SOURCES; AND DEDICATED PRACTICE IN SCIENTIFIC MENTORSHIP, LEADERSHIP, AND COMMUNICATION. THE RESEARCH ENVIRONMENT AT THE ROCKEFELLER UNIVERSITY AND IN THE LAB OF DR. JEREMY ROCK, CHARACTERIZED BY INTERDISCIPLINARY COLLABORATION, STATE-OF-THE-ART FACILITIES, AND SUPPORT FOR PROFESSIONAL DEVELOPMENT, PROVIDES AN EXCEPTIONAL SETTING FOR RIGOROUS SCIENTIFIC TRAINING AND FOR PREPARATION TOWARD AN INDEPENDENT CAREER INVESTIGATING MECHANISMS OF HOST-PATHOGEN INTERACTIONS AND THERAPEUTIC VULNERABILITIES IN INFECTIOUS DISEASE.