Substrate Activity Screening:  A Fragment-Based Method for the Rapid Identification of Nonpeptidic Protease Inhibitors

Abstract

A new fragment-based method for the rapid development of novel and distinct classes of nonpeptidic protease inhibitors, Substrate Activity Screening (SAS), is described. This method consists of three steps: (1) a library of N-acyl aminocoumarins with diverse, low molecular weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay, (2) the identified N-acyl aminocoumarin substrates are optimized by rapid analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. The SAS method was successfully applied to the cysteine protease cathepsin S, which is implicated in autoimmune diseases. Multiple distinct classes of nonpeptidic substrates were identified upon screening an N-acyl aminocoumarin library. Two of the nonpeptidic substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency for each class. Select nonpeptidic substrates were then directly converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity to cathepsin S. This study demonstrates the unique characteristics and merits of this first substrate-based method for the rapid identification and optimization of weak fragments and provides the framework for the development of completely nonpeptidic inhibitors to many different proteases.

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

“…1) can be performed before converting substrates into inhibitors because transition-state theory for enzyme-catalyzed reactions predicts that the inhibitory activity (K i ) of mechanism-based inhibitors can be correlated with the inverse of the catalytic efficiency of the corresponding substrates (K m /k cat ) 10 . Other researchers have confirmed this correlation for peptide substrates 11,12 , and we have observed this correlation for non-peptidic substrates and inhibitors for both cathepsin S and chymotrypsin [1][2][3] . To obtain accurate relative substrate cleavage efficiencies (k cat /K m ) in the substrate optimization step, assays should be conducted at substrate concentrations below the K m of the substrates (Michaelis-Menten equation:…”

“…We have developed the first substrate-based fragment identification method, called 'substrate activity screening' (SAS) [1][2][3] . This method addresses two key challenges in fragment-based screening: (i) the accurate and efficient identification of weak binding fragments and (ii) the rapid optimization of the initial weak binding fragments into higher-affinity compounds 4,5 .…”

“…We have successfully applied the SAS method to both Cys and Ser protease targets to identify novel, low-molecular-weight substrates and have subsequently optimized and converted the substrates into potent, non-peptidic inhibitors [1][2][3] . The SAS method was initially successfully applied to the identification of two distinct classes of novel, non-peptidic inhibitors with nanomolar affinity to cathepsin S, which is an enzyme implicated in a number of autoinflammatory diseases (Fig.…”

“…Direct conversion of the substrates into inhibitors was then accomplished simply by replacing the aminocoumarin with a hydrogen atom to introduce the aldehyde mechanism-based pharmacophore 1 . The novel inhibitors that resulted from this process are distinct from previously reported inhibitors of cathepsin S 6,7 .…”

Identification of a New Class of Nonpeptidic Inhibitors of Cruzain

“…10,12 Substrate analogues were therefore first evaluated and optimized before conversion to inhibitors. A triazole-based substrate library consisting of more than 150 substrates was screened against cruzain.…”