A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

Abstract

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1-P1 0 ) but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors-namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.

Complement, a target for therapy in inflammatory and degenerative diseases

“…Several broad-spectrum small-molecule protease inhibitors, including dichloroisocoumarin, bind to and inhibit FD; structures obtained from these complexes have identified unique properties of FD that offer the prospect of designing specific inhibitors 94,95 . Novartis recently reported the evolution of small-molecule inhibitors of FD that are active in the nanomolar range 96 .…”

Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH ‐dependent specificity switch of legumain

“…Especially under in vivo conditions, other determinants, such as spatiotemporal location of a protease and its substrates, exosites, and three‐dimensional structure of native substrates, additionally influence substrate proteolysis (Timmer et al , ; Turk et al , ). Protease cleavage specificity therefore facilitates our understanding of the dynamic interactions of proteases with their substrates in homeostasis or disease and provides additional information for designing selective substrates, probes for imaging and activity assays as well as specific inhibitors (Turk, ; Sukuru et al , ; Kasperkiewicz et al , ; Sanman & Bogyo, ).…”

Protease Inhibitors in View of Peptide Substrate Databases

“…The advantage compared with the method of Sukuru et al 18 is that we directly transfer the information about the known peptide substrates for a protease to the small-molecule space. Thus, to find new inhibitors no prior knowledge of small-molecule ligands is required.…”

“…Recently it was shown in our group that proteases that are close in substrate space are often targeted by the same small molecules. 17 Sukuru et al 18 developed a lead discovery strategy based on the similarity of proteases in the protease substrate space. They recovered the known inhibitors of proteases that are highly correlated.…”