Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis

Abstract

A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC ₁₇ exhibited the best extracellular antitubercular activity (MIC₅₀ = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC ₁₇ may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.

Figure 1

Chemical structure of CyC compounds. Structure of (A) natural Cyclophostin (CyC ₁), Cyclipostins P (CyC _18(β)) and its trans diastereoisomer (CyC _18(α)); as well as (B) the related enolphosphorus analogues: Cyclophostin phosphonate analogs (CyC ₂); monocyclic enolphosphorus analogs to either Cyclophostin (CyC _3-10;15-16) or Cyclipostins (CyC _11-14;17). CyC _5-10 and CyC ₁₃ were best described by the relationship between the OMe on phosphorus and the H-substituent on the C-5 carbon atom as being either in a trans (α-isomer) or cis (β-isomer) relationship. (C) Mode of action of CyC analogs. All CyC compounds are able to form a covalent adduct with the nucleophilic serine or cysteine catalytic residues present at the active site of α/β-hydrolase enzymes family.

Figure 2

In vitro and ex vivo dose-response activity of the CyC analogs against M. tb H37Rv. (A) Activity of CyC _7(α), CyC _7(β), CyC ₁₇, and CyC _18(β) against GFP-expressing M. tb replicating in broth medium, expressed as normalized relative fluorescence units (RFU%). (B) Activity of CyC _7(α) and CyC _7(β) against M. tb replicating inside Raw264.7 macrophages. Results are expressed as the percentage of infected macrophages after 5 days post-infection. For each concentration, data are means ± SD of at least two independent assays performed in duplicate. The MIC₅₀ of CyC ₁₇, CyC _18(β), CyC _7(β) and CyC _7(α) replicating in culture broth medium were 0.5 μM, 1.7 µM, 16.6 µM and 92.6 μM, respectively. The MIC₅₀ of CyC _7(α) and CyC _7(β) replicating inside macrophages were 4.5 μM and 3.1 μM, respectively. Values are means ± SD of three independent assays performed in triplicate (CV% < 5%).

Figure 3

Activity based protein profiling (ABPP) workflow for the identification of the proteins covalently bound to CyC₁₇ inhibitor. Cell lysates of M. tb mc² 6230 were either (A) pre-treated with CyC₁₇ prior to incubation with Desthiobiotin-FP probe or (B) incubated with Desthiobiotin-FP alone. Both samples were further treated with streptavidin-magnetic beads for the capture and enrichment of labelled proteins. (C) Uncompetitive binding assay using streptavidin-magnetic beads on cell lysate. (D) Detection of all potential serine/cysteine enzymes in total cell lysate using fluorescent TAMRA-FP probe. (E) Equal amounts of proteins obtained in A to D were separated by SDS-PAGE and visualized by Coomassie staining (right panel – lanes A–C) or in-gel fluorescence (left panel - lane D: TAMRA detection). Enzymes whose labelling is impeded because of the presence of CyC₁₇ in the active-site are circled in red and shown by arrowheads. The corresponding bands were excised form the gel and subjected to triptic digestion and tandem mass spectrometry analysis. The SDS gel presented in panel E is representative of three independent ABPP experiments.

Figure 4

In silico molecular docking experiments. (A) In silico molecular docking of CyC ₁₇ into the crystallographic structure of HsaD in a van der Waals surface representation. Hydrophobic residues (alanine, leucine, isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline and methionine) are highlighted in white. (B) Superimposition of the top-scoring docking position of CyC ₁₇ (yellow) with the crystal structure of 3,5-dichloro-4-hydroxybenzoic acid (cyan) found to bind in the vicinity of the catalytic Ser¹¹⁴ of HsaD. Each inhibitor is in stick representation with the following atom color-code: oxygen, red; phosphorus, orange; carbon, yellow or cyan; chloride, green. The catalytic Serine residue is colored in magenta. Structures were drawn with PyMOL Molecular Graphics System (Version 1.4, Schrödinger, LLC) using the PDB file 5JZS. (C) Ligplot + analyses results: 2D representation of schematic ligand-protein interactions of CyC ₁₇ in HsaD active site showing both hydrogen-bonds and hydrophobic interactions.