Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase
Autor: | Sunita M. de Sousa, Prashanti Madhavapeddi, Aishwarya Sundaram, Md. Gulebahar Sheikh, Kakoli Mukherjee, Reetobrata Basu, Vaishali Humnabadkar, Rajendra Rane, Halesha D. Basavarajappa, Prateek Verma |
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Rok vydání: | 2015 |
Předmět: |
ATPase
Mycobacterium smegmatis Antitubercular Agents Microbial Sensitivity Tests Biology Biochemistry DNA gyrase Analytical Chemistry Mycobacterium tuberculosis Inhibitory Concentration 50 chemistry.chemical_compound Enzyme Inhibitors IC50 Enzyme Assays Adenosine Triphosphatases chemistry.chemical_classification Reproducibility of Results bacterial infections and mycoses biology.organism_classification Molecular biology High-Throughput Screening Assays Kinetics Enzyme chemistry DNA Gyrase biology.protein Molecular Medicine DNA supercoil DNA Biotechnology |
Zdroj: | SLAS Discovery. 20:265-274 |
ISSN: | 2472-5552 |
Popis: | 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. |
Databáze: | OpenAIRE |
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