Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

Levine, C.; Jadhav, R.; Yan, P.; Tsotetsi, K.; Wang, X.; Awasthi, D.; Grady, C.; Shelke, A.; Daher, S. S.; Richmann, T.; Shrestha, R.; Sukheja, P.; Patel, J.; Barnett, P. R.; Dikdan, R. J.; Kim, T.; Russo, R.; Hanna, J.; Zimmerman, M.; Dartois, V.; Freundlich, J.; Alland, D.; Kumar, P.

doi:10.1101/2022.07.05.496887

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bioRxiv · 10.1101/2022.07.05.496887

Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

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Abstract

Rapid emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is one of the most significant healthcare challenges of our time. The cause of drug resistance is multifactorial, with the long course anti-tubercular therapy required to treat tuberculosis (TB) constituting a major contributing factor. Introduction of pyrazinamide (PZA) resulted in shortening of TB treatment from twelve to six months and consequently played a critical role in curbing drug resistance that developed over long course therapy. Nevertheless, because PZA is a prodrug activated by a nonessential amidase, PncA, resistance to PZA develops and frequently results in treatment failure. Here, we leveraged a whole cell drug screening approach to identify anti-tuberculars with unconventional mechanisms of action or activation that could be further developed into compounds effective at killing Mtb resistant to PZA. We discovered an amide containing prodrug, DG160, that was activated by the amidase, Rv2888c (AmiC). This amidase was capable of metabolizing a variety of amide containing compounds including a novel pyrazinoic acid-isoquinolin-1-amine prodrug, JSF-4302, which we developed as a potential PncA-independent replacement for PZA. As predicted, AmiC activation of JSF-4302 led to the generation of POA in Mtb including in a PZA resistant clinical isolate, thereby successfully delivering the active component of PZA while bypassing the need for activation by PncA. This work provides a framework for a new approach to drug development and prodrug activation in Mtb. SIGNIFICANCEPyrazinamide (PZA) is a vital component of Mycobacterium tuberculosis (Mtb) treatment since its inclusion shortened tuberculosis therapy by six months. However, PZA is a prodrug and resistance develops at a high frequency due to mutations in its activator PncA. Here, we present the discovery of amide-containing anti-tubercular prodrugs that are activated intracellularly by the Mtb amidase, AmiC. Taking advantage of this finding, we successfully designed and synthesized pyrazinoic acid (POA) prodrugs that were activated by AmiC and found that these compounds delivered intracellular POA to PZA- resistant Mtb isolates that contained a nonfunctional PncA. This new approach to prodrug development provides a method for delivering conjugated drugs into Mtb with the potential to overcome clinical drug resistance.

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Levine, C., Jadhav, R., Yan, P., Tsotetsi, K., Wang, X., Awasthi, D., Grady, C., Shelke, A., Daher, S. S., Richmann, T., Shrestha, R., Sukheja, P., Patel, J., Barnett, P. R., Dikdan, R. J., Kim, T., Russo, R., Hanna, J., Zimmerman, M., Dartois, V., Freundlich, J., Alland, D., Kumar, P.. 2022-07-05. Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis. https://doi.org/10.1101/2022.07.05.496887

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