Simultaneous ¹⁹F NMR Screening of Prolyl Oligopeptidase and Dipeptidyl Peptidase IV Inhibitors

Abstract

Prolyl oligopeptidase (POP) and dipeptidyl peptidase IV (DPP IV) are serine proteases that belong to the same family of enzymes. These peptidases are relevant because of their association with the pathophysiology of serious illnesses, such as type 2 diabetes (DPP IV), and those related to cognitive disorders (POP). Several NMR-based screening methods are being used to find and validate new hit scaffolds. In particular, (19)F NMR-based screening methods have proven to be powerful tools for the discovery and development of new inhibitors. Here we present an accurate and reliable (19)F NMR-based simultaneous assay that is used to screen for new selective POP and DPP IV inhibitors in compound mixtures. This activity assay consists of the simultaneous performance of POP and DPP-IV (19)F NMR activity assays in the presence of their fluorine-containing substrates. Furthermore, the assays were conducted in the presence of 0.01 % v/v of Triton X-100, which is a detergent that disrupts micelle formation, thereby preventing unspecific aggregate-based inhibition. Finally, this (19)F NMR methodology was applied to screen for ligands in plant extracts. Our results indicate that this method allows the simultaneous and accurate identification of selective POP and DPP IV inhibitors in these compound mixtures.

19F NMR: a valuable tool for studying biological events

“…This advantage has been harnessed to screen selective inhibitors from plant extracts of traditional Chinese medicine, of prolyl oligopeptidase and dipeptidyl peptidase. 41 In addition, FABS has been used to determine the function of novel proteins and explore their potential as drug targets, 39 an important step in the pharmaceutical development of new drugs to treat various diseases.…”

Ligand-Based Fluorine NMR Screening: Principles and Applications in Drug Discovery Projects

“…Similar screening experiments can be performed with 19 F labeled RNA or DNA (see, for example, refs − and references cited therein). The substrate/cofactor observed technique, also known as n -fluorine atoms for biochemical screening (n-FABS), − is a functional assay and requires the selective labeling of the cofactor or substrate of an enzymatic reaction with a fluorinated group. − The principles and several applications of the n-FABS to simple and complex biological and chemical systems have been described in a comprehensive review article . Recently, the versatility of the n-FABS has allowed its application to the screening and IC 50 measurements in intact human living cells .…”

Fluorine NMR‐Based Screening on Cell Membrane Extracts

“…n-FABS has been successfully applied to several pharmaceutically relevant targets resulting in the identification of novel inhibitors. [5][6][7][8][9][10][11] In a recent work, we reported its application to the screening of chemical fragments against a purified recombinant membrane enzyme. [12] Indeed, membrane proteins are challenging targets for biochemical assays.…”