VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Conspectus “ Drug resistance is an unavoidable consequence of the use of drugs; however, the emergence of multi-drug resistance can be managed by accurate diagnosis and tailor-made regimens. ” Antimicrobial resistance (AMR), is one of the most paramount health perils that has emerged in the 21st century. The global increase in drug-resistant strains of various bacterial pathogens prompted the World Health Organization (WHO) to develop a priority list of AMR pathogens. Mycobacterium tuberculosis ( Mtb ), an acid-fast bacillus that causes tuberculosis (TB), merits being one of the highest priority pathogens on this list since drug-resistant TB (DR-TB) accounts for ∼29% of deaths attributable to AMR. In recent years, funded collaborative efforts of researchers from academia, not-for-profit virtual R&D organizations and industry have resulted in the continuous growth of the TB drug discovery and development pipeline. This has so far led to the accelerated regulatory approval of bedaquiline and delamanid for the treatment of DR-TB. However, despite the availability of drug regimes, the current cure rate for multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) treatment regimens is 50% and 30%, respectively. It is to be noted that these regimens are administered over a long duration and have a serious side effect profile. Coupled with poor patient adherence, this has led to further acquisition of drug resistance and treatment failure. There is therefore an urgent need to develop new TB drugs with novel mechanism of actions (MoAs) and associated regimens. This Account recapitulates drug resistance in TB, existing challenges in addressing DR-TB, new drugs and regimens in development, and potential ways to treat DR-TB. We highlight our research aimed at identifying novel small molecule leads and associated targets against TB toward contributing to the global TB drug discovery and development pipeline. Our work mainly involves screening of various small molecule chemical libraries in phenotypic whole-cell based assays to identify hits for medicinal chemistry optimization, with attendant deconvolution of the MoA. We discuss the identification of small molecule chemotypes active against Mtb and subsequent structure–activity relationships (SAR) and MoA deconvolution studies. This is followed by a discussion on a chemical series identified by whole-cell cross-screening against Mtb , for which MoA deconvolution studies revealed a pathway that explained the lack of in vivo efficacy in a mouse model of TB and reiterated the importance of selecting an appropriate growth medium during phenotypic screening. We also discuss our efforts on drug repositioning toward addressing DR-TB. In the concluding section, we preview some promising future directions and the challenges inherent in advancing the drug pipeline to address DR-TB.

A combination of chemical genetic and biochemical assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was associated with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo production of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the observed incorporation of 5-FU into RNA and DNA and the reduced amount of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug.

Tuberculosis (TB) is predominantly an airborne disease. Background However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, , has proven very Mycobacterium tuberculosis (Mtb) challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of bacilli. Mtb : We monitored each of 35 newly diagnosed, GeneXpert Methods sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m ) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect in each of the bio-aerosol collection Mtb devices.: was detected in 27/35 (77.1%) of aerosol samples; 15/35 Results Mtb (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity. A correlation was found between cough rate and culturable bioaerosol.was Mtb detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5μm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x10 CFU/ml (IQR: 2.9x10 -5.6x10 ) of exhaled particulate bio-aerosol.: was identified in bio-aerosols exhaled by the majority of Conclusions Mtb untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of Page 1 of 25Gates Open Research 2018, 1:11 Last updated: 09 JUL 2018 Gates Open Research Discuss this article(1) Comments than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable bacilli in these samples.