Search

Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1 thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ back-up program we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulphonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ and covalent adducts with the active site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 as compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the h istone-like n ucleoid- s tructuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis , as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δ rv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δ rv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δ rv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis . We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein. IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes.

New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis. AX compounds target the QcrB subunit of the cytochrome bc1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development., New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis. We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro. All five compounds showed good activity against M. tuberculosis in vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc1-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis. Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.