High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors

Abstract

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Figure 1. Monitoring of intracellular growth of tubercle bacilli inside macrophages by automated confocal microscopy and HT/CS screening results and hit profile.

(A) Pictures of Raw264.7 cells infected with M. tuberculosis H37Rv-GFP at different time points after infection. NI: Non infected. Scale bar: 50 µm. (B) Infected Cell segmentation: 1: 2-color image; 2: cell mask detection, 3: purple cells correspond to infected cells. (C) Pictures of M. tuberculosis H37Rv-GFP infected Raw264.7 cells at day 5 in presence of INH, RIF at 1 µg/mL or DMSO control (D–G) INH and RIF pharmacology in intracellular (D, E) and in vitro (F ,G) assays. Bacterial load (arbitrary units, black circles), host cell number (gray circles) and percentage of infected cells (black squares) represent main parameters determined by our customized image analysis for the intracellular assay. In vitro M. tuberculosis growth is given in Relative Fluorescent Units (gray triangles). Results are representative of three independent experiments with standard deviation (SD) and have been reproduced more than 50 times during the screening. (H) Distribution of primary screening results after PCA-1x analysis. Yellow: INH 7 µM; Green: RIF 1.2 µM, blue: DMSO, red: screened compounds. (I) MIC range of the 486 confirmed hits.

Figure 2. MIC (µM) for different nitrobenzamides against M. tuberculosis H37Rv growth.

Extracellular and Intracellular correspond to growth in broth and in macrophages. DNB1 and DNB2 are compound 8 and 7 respectively.

Figure 3. DNB1 activity confirmation and target identification.

(A) Structure of DNB1 (B) Profile in intracellular assay from DNB1 of the benzamide scaffold; black squares and gray circles correspond to percentage of infected cells and host cell number (C) inhibition of the in vitro growth fluorescence assays by DNB1; black triangles correspond to relative fluorescent units. Results (mean+/−SD from 5 independent experiments) were normalized according to DMSO and INH control values. (D) Representative pictures of human primary macrophages infected with M. tuberculosis H37Rv-GFP (MOI 2.5∶1) at day 7 for DNB1 and INH (5 µM) and DMSO. NI: non infected. Scale bar corresponds to 50 µm. (E) Dose-response curve for DNB1 on M tuberculosis H37Rv-GFP infected mouse bone marrow-derived macrophages and human primary macrophages after 5 days of infection. Results are representative of 2 independent experiments. (F) Effect of DNB1 on the synthesis of decaprenyl-phospho-arabinose (DPA) by cell-free extracts of M. smegmatis. Bacterial extracts were treated with 4 µM (1.4 µg/mL) of DNB1 for 30 min at 37°C prior to the addition of p(¹⁴C)Rpp and ATP and further incubation for 90 min at 37°C. The same amount of reaction material was loaded as for the control (CTL). PRPP, 5-phosphoribosyl 1-pyrophosphate; DPPR, decaprenylphosphoryl 5-phosphoribose; DPR, decaprenyl-phospho-ribose.