Tsotetsi, K. | bioRxiv Science

bioRxiv2022

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

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.

microbiology↗

bioRxiv2020

Disulfiram inhibits M. tuberculosis growth by altering methionine pool, redox status and host-immune response

Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis (Mtb) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb, which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb. In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb-infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy.

microbiology↗