Designing and synthesizing specific inhibitors is of fundamental value for understanding the molecular mechanisms involved in the interfacial adsorption step as well as the catalytic activity of lipases. In this Account, we will review and discuss results obtained mostly at our laboratory concerning the covalent inhibition of human gastric and human pancreatic lipases by chiral phosphonates. Rather than presenting an exhaustive list of compounds tested so far with lipases of animal and microbial origin, we selected recent experimental data illustrating well the specific problems encountered during the covalent inhibition of these digestive lipases.
Title: Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer Cavalier JF, Ransac S, Verger R, Buono G Ref: Chemistry & Physic of Lipids, 100:3, 1999 : PubMed
Enantiomerically pure alkylphosphonate compounds RR'P(O)PNP (R = CnH2n + 1, R' = OY with Y = Cn'H2n' + 1 with n = n' or n not equal to n'; PNP = p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (alpha 50) leading to half inhibition, and the chain length, R or Y was observed. (R11Y16)s were the best inhibitor of HPL and (R10Y11)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.
1,2(2,3)-Diradylglycero O-(p-nitrophenyl) n-hexylphosphonates were synthesized, with the diradylglycerol moiety being di-O-octylglycerol, 1-O-hexadecyl-2-O-pyrenedecanylglycerol, or 1-O-octyl-2-oleoyl-glycerol, and tested for their ability to inactivate lipases from Chromobacterium viscosum (CVL) and Rhizopus oryzae (ROL). The experimental data indicate the formation of stable, covalent 1:1 enzyme-inhibitor adducts with the di-O-alkylglycero phosphonates. The differences in reactivity of diastereomeric phosphonates with opposite configuration at the glycerol backbone was less expressed with both enzymes tested as compared to the influence of the stereochemistry at the phosphorus. Both lipases exhibited the same preference for the chirality at the phosphorus that was independent from the absolute configuration at the glycerol backbone. However, with CVL and ROL the inhibitors with the active site serine-directed phosphonate linked at position sn-1 of the glycerol moiety reacted significantly faster than the corresponding sn-3 analogs, reflecting the sn-1 stereopreference of the enzymes towards triacylglycerol analogs with a sn-2 O-alkyl substituent. In contrast, the phosphonates based on the 1-O-octyl-2-oleoylglycerol did not significantly inactivate CVL. Unexpectedly, these substances were hydrolyzed in the presence of lipase.
