Elsevier

Tuberculosis

Volume 89, Issue 5, September 2009, Pages 334-353
Tuberculosis

Drug Discovery and Resistance
High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv

https://doi.org/10.1016/j.tube.2009.05.008Get rights and content

Summary

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.

Section snippets

Bacterial strain, growth conditions and media

M. tuberculosis H37Rv (ATCC 27294) was obtained from the American Type Culture Collection (Manassas, VA). To prepare permanent frozen stocks, H37Rv was grown as five mL subcultures (50 mL conical tubes, 36–37 °C) in Middlebrook 7H9 broth (Becton Dickinson) supplemented with 0.2% glycerol (Becton Dickinson), 0.05% Tween 80 (Becton Dickinson), and 10% ADC enrichment (albumin, dextrose, catalase; Becton Dickinson). The subculture was mixed periodically and used to inoculate (5% inoculum) a second

Assay validation

The assay for H37Rv22 was miniaturized to 384-well format. Initially, experiments were run in 96- and 384-well formats to ensure that the conversion to 384-well plates yielded similar results. CV% for media only, M. tuberculosis, and M. tuberculosis plus either 10 μg/mL ethambutol or 2.5 μg/mL amikacin were <10%. Z-values calculated for either M. tuberculosis alone or M. tuberculosis plus either 10 μg/mL ethambutol or 2.5 μg/mL amikacin were usually >0.7 and were equivalent in either 96- or

Discussion

In an effort to identify specific classes of compounds that show significant antitubercular activity, a structure based clustering analysis was performed on the set of compounds that was considered active with TB IC90 value of less than 10 μg/mL. The clustering analysis was performed using a hierarchical clustering method as implemented in LeadScope (LeadScope, Inc.) to identify common structural elements among this diverse set of structures. Clusters were separated using the ‘Complete Linkage

Conclusions

The discovery of new lead compounds active against M. tuberculosis is only the initial step in the development of a new antitubercular drug. Analysis of cytotoxicity is a rudimentary measure of potential toxicity, but can serve to prioritize compounds for further study. Additional in vitro evaluations that would provide useful information are 1) the evaluation of activity against other non-mycobacterial bacterial pathogens for identification of TB-specific drugs; 2) evaluation of bactericidal

Acknowledgments

This work was supported by contracts N01-AI-95364 (JAS) and N01-AI-15449 (ELW). The complete data sets for the HTS campaign will be deposited in PubChem. The authors thank Dr. Scott Franzblau who generously provided us with his protocol for the Alamar Blue M. tuberculosis assay at the beginning of this program. The authors are appreciative of the assistance of Sara McKellip, Nicole Kushner and Kristen Southworth in running the H37Rv screen, Anna Manouvakhova for assistance in the data analysis

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