Unraveling the spectrum of intrabacterial amide hydrolysis to develop new classes of pyrazinoic acid prodrugs against pyrazinamide resistant Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is a global health pandemic claiming 1.5 million lives each year. Current tuberculosis (TB) therapies are challenged by drug resistance, characterized by mutations that render approved drugs increasingly less effective. PZA is a critical component of first-line TB therapy. It allows for shorter treatment regimens and is unrivaled in its efficacy against non-replicating, persister Mtb populations, which decrease the chance of resistance and relapse respectively. If the past predicts the future, the success of many short course and ultra-short course TB therapies may depend on the presence of PZA or other POA releasing prodrugs as part of the treatment regime; thus, the development of a replacement for PZA in the setting of PZA resistance should be among the highest priorities of modern TB drug development. POA itself is relatively polar and has poor penetration into the bacterium. However, PZA crosses the Mtb cell envelope via passive diffusion and is then activated by PncA, an amidase present in the cytosol, to release POA. The amide structure of PZA thus allows the transport and delivery of POA into the cytosol. Unfortunately, PZA resistance is increasing, with approximately half of all isoniazid and rifampin resistant Mtb also showing PZA resistance, usually due to inactivating mutations in the pncA gene. Fortunately, Mtb contains a wide variety of amidases other than pncA that can activate antitubercular compounds which contain a central amide bond. This proposal focuses on the potential for using Mtb amidases as a new class of antitubercular prodrug activators that can circumvent existing forms of resistance. We have identified over 150 Mtb genes that are likely to encode amidases. We have also shown that, like PncA, the Mtb amidase AmiC is involved in the activation of several experimental antitubercular compounds, although AmiC and PncA have different substrate specificities. Conversely, we have shown that approximately 20% of amide containing compounds present in libraries with validated antitubercular activity undergo rapid intrabacterial amide hydrolysis. Thus, different Mtb amidases can specifically activate prodrugs, and diverse antitubercular compounds can be activated by intrabacterial amide hydrolysis. We propose to characterize the diverse set of Mtb amidases and the compound structures that they hydrolyze into paired acids and amines. We will then use this knowledge to rationally design new POA-amide prodrugs that readily diffuse into Mtb where they can then become activated by intrabacterial amidases, releasing POA. We will then confirm that these compounds have activities similar to PZA but are effective against both PZA susceptible and resistant Mtb. Finally, we will perform hit-to-lead optimization and confirmatory pharmaco-kinetic studies in a mouse model. Project Number: 1R01AI189899-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: David Alland | Institution: RUTGERS BIOMEDICAL AND HEALTH SCIENCES, Newark, NJ | Award Amount: $813,373 | Activity Code: R01 | Study Section: Drug Discovery and Molecular Pharmacology A Study Section [DMPA] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18989901