The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis.

8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 µg/ml against M. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 µM and present favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

Towards a new combination therapy for tuberculosis with next generation benzothiazinones.

The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.

Mechanism of action of 5-nitrothiophenes against Mycobacterium tuberculosis.

On using the streptomycin-starved 18b strain as a model for nonreplicating Mycobacterium tuberculosis, we identified a 5-nitrothiophene compound as highly active but not cytotoxic. Mutants resistant to 5-nitrothiophenes were found be cross-resistant to the nitroimidazole PA-824 and unable to produce the F420 cofactor. Furthermore, 5-nitrothiophenes were shown to be activated by the F420-dependent nitroreductase Ddn and to release nitric oxide, a mechanism of action identical to that described for nitroimidazoles.

Finding of the low molecular weight inhibitors of resuscitation promoting factor enzymatic and resuscitation activity.

BACKGROUND: Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC(50) 1-7 microg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC(50). The candidate compounds suppressed resuscitation of dormant ("non-culturable") cells of M. smegmatis at 1 microg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 microg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. CONCLUSIONS/SIGNIFICANCE: NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.

Structural characterization of prazosin hydrochloride and prazosin free base.

The three-dimensional solid-state structures of prazosin hydrochloride, C19H22N5O4+.Cl- (A), and prazosin free base, C19H21N5O4 (B), have been determined by synchrotron X-ray powder diffraction. A and B crystallize in triclinic P-1 and monoclinic Cc space groups, respectively, with one structural unit per asymmetric part. In A and B, the prazosin molecule adopts different conformations, which do not correspond to those obtained by DFT optimizations of protonated and free prazosin.