1. Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl oligopeptidase and its tight binding inhibition by JTP-4819
Jarkko I Venäläinen, Risto O Juvonen, Markus M Forsberg, Arturo Garcia-Horsman, Antti Poso, Erik A A Wallen, Jukka Gynther, Pekka T Männistö Biochem Pharmacol. 2002 Aug 1;64(3):463-71. doi: 10.1016/s0006-2952(02)01184-x.
Prolyl oligopeptidase (POP) is a cytosolic serine protease that hydrolyses small peptides at the carboxyl end of the proline residue. It has raised pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties. We studied prolyl oligopeptidase kinetics with two 7-amino-4-methylcoumarin derivatives: Z-Gly-Pro-AMC and Suc-Gly-Pro-AMC. Z-Gly-Pro-AMC was found to obey standard Henri-Michaelis-Menten kinetics with a K(m) of 30+/-3 microM, whereas Suc-Gly-Pro-AMC exhibited substrate inhibition kinetics with K(m) and K(is) of 510+/-150 and 270+/-90 microM, respectively. Autodock simulations revealed that either the succinyl or the AMC-end of Suc-Gly-Pro-AMC may bind to the S'1 subsite of the active site. We believe that non-specifically bound Suc-Gly-Pro-AMC allows the simultaneous binding of second substrate molecule to the active site and this leads in substrate inhibition. In addition, we demonstrated that the inhibition type of a well characterized prolyl oligopeptidase inhibitor, JTP-4819, is competitive tight binding with a K(ic) of 0.045+/-0.008 nM. We suggest that due to the high concentration of prolyl oligopeptidase in the brain (0.12 nmol/g pig brain), the tight binding nature of the inhibition should be considered when using brain homogenate as the enzyme source in prolyl oligopeptidase inhibition measurements. This is of importance in studying structure-activity relationships of potent prolyl oligopeptidase inhibitors.
2. A cell-based fluorescent assay for FAP inhibitor discovery
Bingye Zhang, Futao Liu, Min-Fu Yang, Jianfeng Xu, Zheng Wang, Jianhua Zhang, Rongfu Wang, Xing Yang Bioorg Med Chem Lett. 2020 Jul 15;30(14):127253. doi: 10.1016/j.bmcl.2020.127253. Epub 2020 May 11.
To facilitate the discovery of FAP inhibitors, a convenient cell-based fluorescent assay was developed by using a commonly available U87MG cell line and a FAP-specific substrate Suc-Gly-Pro-AMC. The assay enabled the fast determination of multiple IC50s by simply incubating a solution of phosphate-buffered saline in a 96-well plate within 30 min. The substrate specificity, cross-reaction and other related conditions were systematically optimized. This method was successfully applied to determine the IC50s of seven known inhibitors. The results are in consistence with the trend reported, which indicating that this practical assay is a valuable method to accelerate the discovery of FAP inhibitor.
