Affinity-Based Proteome Profiling to Identify Targets of Tuberculosis Antibiotics

Abstract Drug-resistant Mycobacterium tuberculosis is an increasing global health crisis that requires the urgent development of new antibiotics. Understanding the mechanism of action of new inhibitors is an essential but challenging component of drug discovery. Affinity-based proteome profiling (AfBP) is a powerful approach for target identification involving the cross-linking and selective pull-down of covalently bound target proteins. Here, we sought to investigate its utility as a technique to identify protein targets of tuberculosis drugs. We constructed photo-cross-linker probes containing a minimalist diazirine and an alkynyl handle of four TB drugs─pretomanid, linezolid, moxifloxacin, and one newly identified inhibitor, BB2-50F, with diverse chemical structures and mechanisms of action. We validated that these probes retain on-target activity against M. tuberculosis in growth and viability assays. Cross-linking and target identification using M. tuberculosis cell lysates produced results consistent with the known mechanism of each compound. The linezolid probe did not identify any proteins, consistent with linezolid binding to rRNA rather than to proteins. The pretomanid probe bound its prodrug activator Ddn, as well as Rv1558, a Ddn homologue that we show through mutagenesis and complementation studies, can bind to PMD but is unable to activate it. In addition to known targets, multiple apparent off-targets were also identified. Overall, this work highlights affinity-based proteome profiling as a useful technique to identify protein targets of tuberculosis drugs and gain insights into the drug mechanism of action.