Inhibitor Report for: CyC-17

The progression of mycobacterial diseases requires the development of new therapeutics. This study evaluated the efficacy and selectivity of a panel of Cyclophostin and Cyclipostins analogues (CyCs) against various bacteria and mycobacteria. The activity 26 CyCs was first assayed by the agar plate method. Compounds exhibiting 50-100% growth inhibition were then selected to determine their minimum inhibitory concentrations (MICs) by the resazurin microtiter assay (REMA). The best drug candidate was further tested against clinical mycobacterial isolates and bacteria responsible for nosocomial infections, including 6 Gram-negative bacteria, 5 Gram-positive bacteria, 29 rapid-growing mycobacteria belonging to the Mycobacterium chelonae-abscessus clade and 3 slow-growing mycobacteria (Mycobacterium marinum, Mycobacterium bovis BCG and Mycobacterium tuberculosis). Among the 26 CyCs tested, 10 were active and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate (CyC17) was further tested against 26 clinical strains and showed high selectivity for mycobacteria, with MICs (<2-40 microg/mL) comparable with those of most classical antimicrobials used to treat M. abscessus infections. Together, these results support the fact that such CyCs represent a new family of potent and selective inhibitors against mycobacteria. This is of particular interest for future chemotherapeutic developments against mycobacterial-associated infections, especially against M. abscessus, the most drug-resistant mycobacterial species.

An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7beta, CyC8beta, and CyC17 bind covalently to the catalytic Ser(124) residue in Ag85C; inhibit mycolyltransferase activity (i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8beta disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.

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.

The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening -200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.

The progression of mycobacterial diseases requires the development of new therapeutics. This study evaluated the efficacy and selectivity of a panel of Cyclophostin and Cyclipostins analogues (CyCs) against various bacteria and mycobacteria. The activity 26 CyCs was first assayed by the agar plate method. Compounds exhibiting 50-100% growth inhibition were then selected to determine their minimum inhibitory concentrations (MICs) by the resazurin microtiter assay (REMA). The best drug candidate was further tested against clinical mycobacterial isolates and bacteria responsible for nosocomial infections, including 6 Gram-negative bacteria, 5 Gram-positive bacteria, 29 rapid-growing mycobacteria belonging to the Mycobacterium chelonae-abscessus clade and 3 slow-growing mycobacteria (Mycobacterium marinum, Mycobacterium bovis BCG and Mycobacterium tuberculosis). Among the 26 CyCs tested, 10 were active and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate (CyC17) was further tested against 26 clinical strains and showed high selectivity for mycobacteria, with MICs (<2-40 microg/mL) comparable with those of most classical antimicrobials used to treat M. abscessus infections. Together, these results support the fact that such CyCs represent a new family of potent and selective inhibitors against mycobacteria. This is of particular interest for future chemotherapeutic developments against mycobacterial-associated infections, especially against M. abscessus, the most drug-resistant mycobacterial species.

An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7beta, CyC8beta, and CyC17 bind covalently to the catalytic Ser(124) residue in Ag85C; inhibit mycolyltransferase activity (i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8beta disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.

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.

Cyclipostins are bicyclic lipophilic phosphate natural products. We report here that synthesized individual diastereomers of cyclipostins P and R have nanomolar IC50s toward hormone sensitive lipase (HSL). The less potent diastereomers of these compounds have 10-fold weaker IC50s. The monocyclic phosphate analog of cyclipostin P is nearly as potent as the bicyclic natural product. Bicyclic phosphonate analogs of both cyclipostins exhibit IC50s similar to those of the weaker diastereomer phosphates (about 400nM). The monocyclic phosphonate analog of cyclipostin P has similar potency. A series of monocyclic phosphonate analogs in which a hydrophobic tail extends from the lactone side of the ring are considerably poorer inhibitors, with IC50s around 50muM. Finally cyclophostin, a related natural product inhibitor of acetylcholinesterase (AChE) that lacks the hydrocarbon tail of cyclipostins, is not active against HSL. These results indicate a critical SAR for these compounds, the hydrophobic tail. The smaller lactone ring is not critical to activity, a similarity shared with cyclophostin and AChE. The HSL kinetics of inhibition for the cyclipostin P trans diastereomer were examined in detail. The reaction is irreversible with a KI of 40nM and a rate constant for inactivation of 0.2min(-1). These results are similar to those observed for cyclophostin and AChE.