Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site

POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH(2)OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (K(i)=11.8–0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, k(on)=(2.43±0.12)×10(5) M(−1)·s(−1), whereas the inhibitor with a CN group exhibited the fastest binding, k(on)=(12.0±0.08)×10(5) M(−1)·s(−1). In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH(2)OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the K(i) values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors

SIMPLE, DIRECT AND INFORMATIVE METHOD FOR THE ASSESSMENT OF CYP2C19 ENZYME INACTIVATION KINETICS

Dibenzylfluorescein (DBF) is widely used as pro-fluorescent probe substrate for CYP activity and inhibition assays, but its use has been considered to be limited to traditional end-point assays. We monitored CYP2C19 catalyzed metabolism of DBF using synthesized fluorescein benzyl and 1.96 ± 0.5 µM and 0.135 ± 0.009 min -1 , respectively. Tranylcypromine did not display any time-dependent inhibition which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization