Small molecule inhibitors of mesotrypsin from a structure-based docking screen

Abstract

PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no mesotrypsin-selective pharmacological inhibitors which could serve as tools for deciphering the pathological role of this enzyme, and could potentially form the basis for novel therapeutic strategies targeting mesotrypsin. A virtual screen of the Natural Product Database (NPD) and Food and Drug Administration (FDA) approved Drug Database was conducted by high-throughput molecular docking utilizing crystal structures of mesotrypsin. Twelve high-scoring compounds were selected for testing based on lowest free energy docking scores, interaction with key mesotrypsin active site residues, and commercial availability. Diminazene (CID22956468), along with two similar compounds presenting the bis-benzamidine substructure, was validated as a competitive inhibitor of mesotrypsin and other human trypsin isoforms. Diminazene is the most potent small molecule inhibitor of mesotrypsin reported to date with an inhibitory constant (Ki) of 3.6±0.3 μM. Diminazene was subsequently co-crystalized with mesotrypsin and the crystal structure was solved and refined to 1.25 Å resolution. This high resolution crystal structure can now offer a foundation for structure-guided efforts to develop novel and potentially more selective mesotrypsin inhibitors based on similar molecular substructures.

PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma

“…Hypothesizing that the functional significance of PRSS3 expression in lung adenocarcinoma derives from the proteolytic activity of active mesotrypsin, we next tested the ability of pharmacological inhibitors of mesotrypsin to attenuate PC9 cell invasion and proliferation. In a recent study, we identified the small molecule diminazene as an inhibitor of mesotrypsin activity with an inhibitory constant ( K i ) of 3.6 µM 24 . Using this inhibitor over a range of concentrations spanning the K i value, we conducted Matrigel transwell assays to measure cellular invasion and MTT assays to provide a proxy measurement for cell growth of PC9 LAC cells.…”

“…While no currently approved drugs target either mesotrypsin or KLK5, recent research efforts have taken preliminary steps toward identifying selective pharmacological inhibitors of these proteases. For mesotrypsin, small molecule inhibitors identified have so far been limited to relatively weak binders that are poorly selective among trypsin isoforms 24,56 . An alternative approach aims to develop highly selective biologics targeting mesotrypsin, through protein engineering of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) 57 .…”

A Virtual Screening Platform Identifies Chloroethylagelastatin A as a Potential Ribosomal Inhibitor

“…We performed docking using “building up—filtering down” technique iteratively for AA and seven AA analogues and a decoy. Our incremental build-up of the level of accuracy, from SP to XP and GBSA/prime energy calculations has been previously reported [ 5 , 8 , 10 , 11 , 12 , 47 , 48 , 49 , 50 , 51 ]. We docked the AA analogues with the ribosome at the same site as the AA-ribosome X-ray structure to see if similar binding patterns emerged as was shown for AA-ribosome X-ray structure [ 4 ], which has been shown to inhibit the ribosome [ 4 ] ( Figure 3 and Figure 5 ).…”

Proteases: Pivot Points in Functional Proteomics

“…Both processes contribute to the development of human pancreatitis. Crystallization studies identified diminazene analogues as small molecule inhibitors of mesotrypsin, and these structures may form the basis for developing selective, tight-binding drugs [97]. In irritable bowel syndrome (IBS) the intestinal epithelium overproduces mesotrypsin, which increases intestinal epithelium permeability, signals to human submucosal enteric neurons and induces visceral hypersensitivity by a protease-activated receptor-2-dependent mechanism [98].…”