Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase

Abstract

Mycobacterium tuberculosis (Mtb) DNA gyrase ATPase was the target of a tuberculosis drug discovery program. The low specific activity of the Mtb ATPase prompted the use of Mycobacterium smegmatis (Msm) enzyme as a surrogate for lead generation, since it had 20-fold higher activity. Addition of GyrA or DNA did not significantly increase the activity of the Msm GyrB ATPase, and an assay was developed using GyrB alone. Inhibition of the Msm ATPase correlated well with inhibition of Mtb DNA gyrase supercoiling across three chemical scaffolds, justifying its use. As the IC50 of compounds approached the enzyme concentration, surrogate assays were used to estimate potencies (e.g., the shift in thermal melt of Mtb GyrB, which correlated well with IC(50)s >10 nM). Analysis using the Morrison equation enabled determination of K(i)(app)s in the sub-nanomolar range. Surface plasmon resonance was used to confirm these IC(50)s and measure the K ds of binding, but a fragment of Mtb GyrB had to be used. Across three scaffolds, the dissociation half life, t1/2, of the inhibitor-target complex was ≤ 8 min. This toolkit of assays was developed to track the potency of enzyme inhibition and guide the chemistry for progression of compounds in a lead identification program.

The discovery of penta-peptides inhibiting the activity of the formylglycine-generating enzyme and their potential antibacterial effects against Mycobacterium tuberculosis

“…To prove our hypothesis, we chose Mycolicibacterium smegmatis as a tuberculosis model. 11 Different concentrations of the hit peptides were incubated with the culture media, from 12.5 μM to 100 μM, for 3 days, and growth inhibition was measured via bacterial counting. Interestingly, both SMMMC and SCGMM exhibited a dose-dependent growth inhibition trend, and SMMMC and SCGMM inhibited the growth of M. smegmatis by up to 40% and 25%, respectively, at 100 μM (Fig.…”

Predictive Power of In Silico Approach to Evaluate Chemicals against M. tuberculosis: A Systematic Review

“…4B6C codes for the structure of M. smegmatis GyrB ATPase domain in complex with an aminopyrazinamide, a known inhibitor of the GyrB [71]. The use of M. smegmatis as a surrogate for M. tuberculosis has been previously demonstrated in the literature, as a result of Mtb’s low specific activity [72] and slow growth [71]. Also, Saxena et al [45] did a sequence alignment of the M. smegmatis and M. tuberculosis DNA GyrB and found that they share 87.4% similarity.…”