Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline

Abstract

Background: Whole-cell phenotypic screening is the driving force behind modern anti-tubercular drug discovery efforts. Focus has shifted from screening for bactericidal scaffolds to screens incorporating target deconvolution. Target-based screening aims to direct drug discovery toward known effective targets and avoid investing resources into unproductive lines of enquiry. The protein synthesis pipeline, including RNA polymerase and the ribosome, is a clinically proven target in Mycobacterium tuberculosis. Screening for new hits of this effective target pathway is an invaluable tool in the drug discovery arsenal.

Methods: Using M. tuberculosis H37Rv augmented with anhydrotetracycline-inducible expression of mCherry, a phenotypic screen was developed for the identification of protein synthesis inhibitors in a medium throughput screening format.

Results: The assay was validated using known inhibitors of protein synthesis to show a dose-dependent reduction in mCherry fluorescence. This was expanded to a proprietary screen of hypothetical protein synthesis hits and modified to include quantitative viability measurement of cells using resazurin.

Conclusion: Following the success of the proprietary screen, a larger scale screen of the GlaxoSmithKline anti-tubercular library containing 2799 compounds was conducted. Combined single shot and dose-response screening yielded 18 hits, 0.64% of all screened compounds.

Keywords: RNA polymerase; mCherry; mycobacteria; ribosome; transcription; translation.

Figure 1

Standard drug target validation pathway (A) and target‐based screening pathway (B). The standard method of target validation uses DRM generation and WGS to identify targets, which can be time‐consuming and result in identification of non‐favorable drug targets. Target‐based screening cuts the time investment to target identification and shifts DRM generation and WGS to a position where they can be conducted concurrently with other target validation steps

Figure 2

MIC determination of known protein synthesis inhibitors and control inhibitors. Visual confirmation of drugs MICs using resazurin. Pink colored wells indicate cell survival. Blue wells indicate cell death. Red values in the table show the MIC for each compound

Figure 3

The effect of known inhibitors on the percentage expression of mCherry. Isoniazid and ethambutol negative controls show no mCherry expression decrease. Known protein synthesis inhibitors hygromycin, chloramphenicol, streptomycin, and apramycin show a dose‐dependent decrease in mCherry expression

Figure 4

Dose response of hit compounds found in the preliminary screen. Percentage mCherry expression displayed in yellow. Percentage cell survival displayed in blue

Figure 5

Percentage expression results and cell survival for all 2799 compounds screened. The red line denotes the cutoff threshold for positive results displaying lower than 50% mCherry expression. Panel A shows results for the 1 μmol/L screen, panel B shows the 10 μmol/L screen. The left panels of each figure show the percentage mCherry expression and the right panels show the percentage survival as determined by resazurin

Figure 6

Dose response of hit compounds found in the large‐scale screen. Percentage mCherry expression displayed in yellow. Percentage cell survival displayed in blue. Compound 2 was an additional blind linezolid control

Figure 7

Broken beacon RNA polymerase assay. The plotted lines denote the average fluorescence values from three test wells from 1000 read cycles of 30 seconds. Error bars are shown in lighter colors. The blank shows the base fluorescence from probe dehybridization