Synthesis and Kinetic Evaluation of Cyclophostin and Cyclipostins Phosphonate Analogs As Selective and Potent Inhibitors of Microbial Lipases

Abstract

New series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. Best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat towards same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these 7-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.

Cyclipostins and cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo

“…These data thus support the formation of a covalent Ag85C-CyC complex as the reaction between the catalytic Ser124 and either CyC7 or CyC8 is expected to yield mass increases of +374.2 Da or +402.25 Da, respectively. Moreover, such results are consistent with the known and irreversible classical mechanism of action of phosphonate compounds, as demonstrated using pure mycobacterial lipolytic enzymes (31).…”

“…Furthermore, no local structural rearrangement was observed (data not shown). As expected, the mode of inhibition of CyC8β consists of blocking the active site (31) and not of destabilizing the overall structure and stability of the protein as reported for ebselen and its analogs (27,45). Furthermore, the mode of action of CyC8β is more related to that of the diethyl p-nitrophenyl phosphate (DEP), a nonspecific α/β hydrolase inhibitor that covalently modifies the Ser124 catalytic residue (17).…”

“…Towards the generation of new lead compounds with unexplored modes of action in M. tuberculosis, the CyC analogs were initially designed to inhibit mycobacterial lipases (31). In particular, by covalently binding to the catalytic serine, they fully inactivated the monoacylglycerol lipase Rv0183 and the triacylglycerol lipase LipY from M. tuberculosis but not the mammalian gastric and pancreatic lipases (31).…”

“…In particular, by covalently binding to the catalytic serine, they fully inactivated the monoacylglycerol lipase Rv0183 and the triacylglycerol lipase LipY from M. tuberculosis but not the mammalian gastric and pancreatic lipases (31). Subsequent biochemical studies involving the selective labeling and enrichment of captured enzymes using appropriate fluorophosphonate probes in combination with CyC17 resulted in the identification of 23 potential target lipolytic enzymes, all of which comprising catalytic serine or cysteine residues (29).…”

“…CyC7, CyC8 and CyC17 were synthesized as previously described (31,50). To study the inhibitory effect on DGAT activity, 30 M of either Ag85A, Ag85B, Ag85C or MPT51 were coincubated with increasing concentrations of CyC7, CyC8 and CyC17 for 1 hr at room temperature in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.6), 10% DMSO and 0.5 × the critical micelle concentration (CMC) of nDodecyl β-D-maltoside (DDM).…”