Identifying drug targets in M. abscessus through the application of fluorescent probes

About This Grant

Project Summary In the United States, a person is more likely to be infected by a non-tuberculous mycobacteria (NTM) than Mycobacterium tuberculosis (Mtb). Most NTM pulmonary infections are caused by one of two species: Mycobacterium avium or Mycobacterium abscessus (Mab). Of the two, infections with Mab are the most difficult to cure and, consequently, lead to highest rate of mortality among NTM diseases. Mab is intrinsically resistant to most antibiotics and there are no FDA approved drugs to treat this disease. Further challenges come from the pronounced strain-to-strain variability in drug response. New treatment strategies for people infected with Mab are urgently needed. There is emerging evidence that Mab infections can be cured through treatment regimens that include two β-lactam antibiotics at once, termed “dual β-lactam therapy”. Currently there is widespread hesitancy by clinicians to using dual β-lactam therapy for NTMs because such treatment conflicts with decades of guidelines for using β-lactams to treat other bacterial infections. However, those guidelines do not consider the complement of enzyme targets for these drugs that are definitively different in mycobacterial pathogens. For this R21 project, we propose to collect comprehensive data describing the underlying mechanism of action for dual β-lactams in Mab, which is currently unknown. This direct evidence is both urgent and necessary to clearly describe the mechanistic basis of the synergistic activity of dual β-lactams treatment. It is additionally needed to update guidelines for using this novel and effective therapeutic approach to treat NTM infections in patients. We hypothesize that dual β-lactam treatment is more effective than a single drug alone against Mab because the combination of two drugs halts the catalytic activity for a broad set of enzymes used for cell wall biosynthesis. In Mab, this most likely includes more than just the penicillin-binding proteins (PBPs) because L,D-transpeptidases (LDTs), a distinct enzyme class, make the 33 cross-links that predominate in the mycobacterial cell wall. We predict that some dual β-lactams combine to comprehensively inhibit both PBPs and LDTs. We will test our hypothesis through the application of novel chemical probes, called activity-based probes (ABPs). These ABPs are designed to irreversibly label enzyme targets of β-lactams, including mycobacterial PBPs, LDTs, and β-lactamases. We propose to complete two Aims: Aim 1. To identify β-lactam enzyme targets associated with Mab disease. Aim 2. To identify Mab enzymes inhibited potently by dual β-lactam treatment. Successful completion of this R21 project will provide ground-breaking insights into the most efficacious combinations of β-lactam drugs against Mab, information that can be immediately used to design clinical trials and, ultimately, improve treatment regimens for patients infected with Mab and other NTMs.