Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening

Abstract

Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.

Figure 1. Screening validation and cross-species comparison of hits identified in primary screens against M. tuberculosis, M. smegmatis, and M. bovis BCG

A. Reproducibility of a GFP reporter based screen for identifying inhibitors of M. tuberculosis replication. To determine the reproducibility of the GFP based assay, the same set of 4308 unique compounds was screened against M. tuberculosis on two different days. A high degree of correlation is observed for z-scores obtained using the same assay against M. tuberculosis on different screening days. B. Correlation of hits identified in primary screens against M. tuberculosis and M. smegmatis. 11,147 unique compounds were screened against M. tuberculosis using the GFP assay and M. smegmatis using an OD600 based assay. Plotted are composite z-scores calculated for each compound in both the M. tuberculosis (x-axis) and M. smegmatis (y-axis) screens. C. Correlation of hits identified in primary screens against M. tuberculosis and M. bovis BCG. 13,715 unique compounds were screened against M. tuberculosis and M. bovis BCG using GFP based assays. Plotted are composite z-scores calculated for each compound in both the M. tuberculosis (x-axis) and M. bovis BCG (y-axis) screens. All screens were conducted using duplicate wells for each compound, and composite z-scores for the duplicates are plotted. R square values were calculated using the two-tailed Pearson product moment correlation test.

Figure 2. Structures of gliotoxin analogs

A. Gliotoxin B. Dimethylthioether of reduced gliotoxin.

Figure 3. Identification and characterization of compounds with glycerol dependent killing mechanisms

A. Structure of compound A039. B. Dose response curves for mutants with high-level resistance to compound A039. Black triangles represent wild-type M. tuberculosis and open circles represent four independently generated resistant mutants. Points represent the average of duplicates. The experiment was repeated three times. C. Compound A039 killing of M. tuberculosis requires glycerol. Bacteria were cultured on media containing either glycerol or acetate as the sole carbon source for a period of 7d prior to dilution into media containing acetate or glycerol as the sole carbon source with the addition of 2μM of compound A039 or DMSO as a control. After 14 days of growth the OD600 was measured. Columns represent the average of duplicates with error bars representing standard deviation. The experiment was repeated three times. D. Comparison of Z-scores obtained from retesting hits using acetate (x-axis) or glycerol (y-axis) as the sole carbon source. Plotted is the composite z-score from each screen calculated by screening in duplicate.

Figure 4. The benzimidazole C215 kills M. tuberculosis by targeting MmpL3

A. Structure of C215. B. Dose response curves of resistant mutants generated to inhibitor C215. Black triangles represent wild-type M. tuberculosis and open circles represent four independently generated resistant mutants. Bacterial numbers are determined by OD600 and points represent the average twelve replicates from three independent experiments. * p < 0.0001 by Mann-Whitney U. C. Treatment with C215 inhibits mycolic acid transport. Fatty Acid Methyl Esters (FAMEs) and Mycolic Acid Methyl Esters (MAMEs) are labeled. The experiment was repeated twice.

Figure 5. Nitro-triazole inhibitors kill M. tuberculosis by targeting DprE1

A. Structure of nitro-triazole inbibitor 377790. B. Dose response curves for two high-level resistant mutants independently generated to inhibitor 377790. Black triangles represent wild type M. tuberculosis and open circles represent resistant mutants. C. Dose response curves for DprE1 overexpression and wild-type M. tuberculosis tested against inhibitor 377790. Black triangles represent wild type M. tuberculosis and open squares represent overexpression of DprE1. Points represent the average of quadruplicates with error bars representing standard deviation. The experiments were repeated three times.