Table 1.
List of primers used in this publication.
Figure 1.
Development of a single-time point assay with a diaphorase-coupled fluorescence readout.
A. Reaction schematic. Reaction 1: DHFR, in a NADPH-dependent reaction, converts dihydrofolate (DHF) to tetrahydrofolate (THF), causing a depletion of reduced NADPH. Reaction 2: Diaphorase utilizes NADPH that was unused from the DHFR reaction to generate fluorescence. Enzymes are italicized and bold. B. Kinetic NADPH-depletion assay. The rates of NADPH depletion in reactions performed with 200 µM NADPH, 0.2 mM DHF and varied levels of DHFR were monitored by measuring absorbance at 340 nm. C. Fluorescence coupling. The fluorescence generated after 4 minutes of incubating diaphorase and resazurin with various concentrations of NADPH were measured. D. End-point fluorescence assay. The NADPH-depletion assay run as in (B) was coupled to diaphorase and resazurin after 30 minutes, and the resulting fluorescence measured at excitation and emission wavelengths of 560 and 590 nM, respectively.
Figure 2.
Confirming the robustness of the assay for HTS.
A. Standard curve of methotrexate (MTX). The concentration-dependent inhibitory activity of MTX was measured in the coupled assay. B. Statistical Factors. Sixteen replicates of each control reaction were performed in the optimized assay conditions. High-throughput screening (HTS) parameters, including Z’-factor (Z’) and signal to background ratio (S/B) were studied. Raw fluorescence units (right Y-axis) from each of the controls and % inhibition (left Y-axis) are shown. The (-) DHFR controls were used as a measure of complete inhibition, in order to calculate % inhibition.
Figure 3.
A collection of 32,000 compounds were tested against the optimized DHFR assay at a final concentration of 10 µM. Sixteen positive (5 µM MTX shown in pale grey symbols) and negative (DMSO alone shown in dark grey symbols) control reactions were plated on each of the 100 screening plates in order to calculate % inhibition. Controls from the entire screening are shown clustered together. An initial threshold for hits was set at 30% inhibition (dashed line).
Figure 4.
Characterization of NC00094221.
A. Dose-response curves in low-throughput Mtb DHFR enzyme assay. NC00094221 (chemical structure shown inset into the graph) and MTX were tested at varied concentrations in the kinetic assay performed with 10 µM DHF and 125 ng/mL recombinant DHFR. Data were fitted to a non-linear least-squares curve and IC50 values were calculated using GraphPad Prism™.B. Quanitification of DHFR transcript levels in a recombinant Mtb strain. The levels of DHFR transcript were assayed by qRT-PCR in wild-type Mtb strain H37Rv (wt- pale grey bar) and the engineered Mtb strain (H37Rv:dfrA-TetON, referred to as DHFR kd- dark grey bars) in the presence of varied levels of tetracycline (ATc). DHFR transcript levels from each sample were first normalized to SigA transcript levels and then shown as the fold change when compared to wild-type (wt) Mtb. C. Live Mtb growth inhibition assay. Varied concentrations of methotrexate (MTX, white symbols) or NC00094221 (black symbols) were incubated with wild-type Mtb (triangles) or the DHFR kd (circles) and grown for 6 days prior to assessment of cell growth using the commercially available Bactiter-Glo assay kit. For each strain, controls including wells with no drug (DMSO carrier was added instead), 1.22 µM rifampicin (10× MIC99) and a 1/100 dilution of the starting culture were used to calculate 0%, 100% and 99% inhibition, respectively. Data were fitted to a non-linear least-squares curve and MIC99 calculations were performed using GraphPad Prism™. D. Target-specific drug sensitivity in DHFR kd. The sensitivity of the wild-type Mtb and DHFR kd was tested against various inhibitors and displayed as the ratio of a compound’s MIC99 when measured in wild-type Mtb over the MIC99 measured in DHFR kd in the absence of ATc.