Abstract:A potent, non-cytotoxic indazole sulfonamide was identified by
high-throughput screening of >100,000 synthetic compounds for activity
against Mycobacterium tuberculosis (Mtb). This
non-cytotoxic compound did not directly inhibit cell wall biogenesis but
triggered a slow lysis of Mtb cells as measured by release of
intracellular green fluorescent protein (GFP). Isolation of resistant mutants
followed by whole-genome sequencing showed an unusual gene amplification of a 40
gene region spanning Rv3371 to Rv3411c a… Show more
“…For agents such as griselimycin, target amplification in the genome confirmed the target of the natural product (Kling et al, 2015). Similarly, resistance to an indazole sulfonamide scaffold was only observed in mutants that resulted in gene amplification, with subsequent structural studies providing an explanation: the inhibitor's interactions were almost exclusively made with the cofactor in the active site of the target, inosine monophosphate dehydrogenase (Park et al, 2016). In some cases, the target is refractory to mutations that confer resistance.…”
Summary
Elucidating the target or mechanism of action of potential drugs in the discovery pipeline is an integral component of most programs. For antibacterial compounds, generation of resistant mutants followed by whole genome sequencing has often been successful in uncovering the proteins involved in regulating compound activation, uptake, efflux and importantly, target processes. When this process succeeds, we are quick to declare a target. In a study reported by Sing and Dhar et al. (in press), the combination of resistant mutant generation, whole genome sequencing and recombineering to identify the target of a Mycobacterium tuberculosis growth inhibitor, pointed to a mechanism involving a scaffolding protein, Wag31, involved in polar elongation of mycobacterial cells. Time‐lapse microscopy and electron microscopy confirmed the view that this inhibitor resulted in interruption of nascent cell wall biosynthesis. However, co‐expression as well as regulated titration of the putative Wag31 target demonstrated that the wild‐type allele was dominant and showed no synergy with the inhibitor. The most plausible explanation from their results was that this inhibitor interfered with the interaction of Wag31 with one of its interacting partners in the elongation complex.
“…For agents such as griselimycin, target amplification in the genome confirmed the target of the natural product (Kling et al, 2015). Similarly, resistance to an indazole sulfonamide scaffold was only observed in mutants that resulted in gene amplification, with subsequent structural studies providing an explanation: the inhibitor's interactions were almost exclusively made with the cofactor in the active site of the target, inosine monophosphate dehydrogenase (Park et al, 2016). In some cases, the target is refractory to mutations that confer resistance.…”
Summary
Elucidating the target or mechanism of action of potential drugs in the discovery pipeline is an integral component of most programs. For antibacterial compounds, generation of resistant mutants followed by whole genome sequencing has often been successful in uncovering the proteins involved in regulating compound activation, uptake, efflux and importantly, target processes. When this process succeeds, we are quick to declare a target. In a study reported by Sing and Dhar et al. (in press), the combination of resistant mutant generation, whole genome sequencing and recombineering to identify the target of a Mycobacterium tuberculosis growth inhibitor, pointed to a mechanism involving a scaffolding protein, Wag31, involved in polar elongation of mycobacterial cells. Time‐lapse microscopy and electron microscopy confirmed the view that this inhibitor resulted in interruption of nascent cell wall biosynthesis. However, co‐expression as well as regulated titration of the putative Wag31 target demonstrated that the wild‐type allele was dominant and showed no synergy with the inhibitor. The most plausible explanation from their results was that this inhibitor interfered with the interaction of Wag31 with one of its interacting partners in the elongation complex.
“…Figure 3.Avoiding resistance hotspots.The GuaB2 inhibitor VCC234718 (represented as yellow sticks [52]) stacks on top of IMP (represented as black sticks) in a similar manner to NAD (represented as orange sticks), but makes different interactions to the neighbouring protomer unit through Y487, which is mutated leading to resistance. Optimisation of a separate scaffold (Compound 6, represented as purple sticks [53]) more closely mimicked the interactions made by NAD, and was active against the Y487C mutant.…”
Section: Mutations and Diseasementioning
confidence: 99%
“…Optimisation of a separate scaffold (Compound 6, represented as purple sticks [53]) more closely mimicked the interactions made by NAD, and was active against the Y487C mutant.…”
For over four decades structural biology has been used to understand the mechanisms of disease, and structure-guided approaches have demonstrated clearly that they can contribute to many aspects of early drug discovery, both computationally and experimentally. Structure can also inform our understanding of impacts of mutations in human genetic diseases and drug resistance in cancers and infectious diseases. We discuss the ways that structural insights might be useful in both repurposing off-licence drugs and guide the design of new molecules that might be less susceptible to drug resistance in the future.
“…In this instance, the lead molecule, 7759844, demonstrated high potency with a K i 0.603 μM and MIC of 0.633 μg.ml −1 , however displayed toxicity in a chronic mouse model18. Several crystal structures of GuaB2 from Mtb have recently been determined in complex with substrate, product and cofactors along with a number of new compounds with anti-mycobacterial activity192021. This enhanced understanding of the biophysics of GuaB2 inhibition can be used for in silico drug discovery and for the assessment of newly discovered anti-mycobacterial compounds targeting GuaB2.…”
High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of ‘hit’ compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a ‘hit’ molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.
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