title: 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.
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Vaishali
authors.initials: V
authors.lastname: Humnabadkar
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Prashanti
authors.initials: P
authors.lastname: Madhavapeddi
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India Indiana University School of Medicine, Indianapolis, USA.
authors.forename: Halesha
authors.initials: H
authors.lastname: Basavarajappa
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India Enzene Bioscience Ltd., Bangalore, India.
authors.forename: Md Gulebahar
authors.initials: MG
authors.lastname: Sheikh
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Rane
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Reetobrata
authors.initials: R
authors.lastname: Basu
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Prateek
authors.initials: P
authors.lastname: Verma
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Sundaram
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India Alkem Laboratories, Bangalore, India.
authors.forename: Kakoli
authors.initials: K
authors.lastname: Mukherjee
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India sunitadesousa@gmail.com.
authors.forename: Sunita M
authors.initials: SM
authors.lastname: de Sousa
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000995:Antitubercular Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology*; D015203:Reproducibility of Results
chemical_list: D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase
keywords: Morrison plot; Mycobacterium smegmatis; T
                 m shift; gyrase ATPase; residence time; surface plasmon resonance; tuberculosis