Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets

Bioorg Med Chem. 2015 May 15;23(10):2328-43. doi: 10.1016/j.bmc.2015.03.072. Epub 2015 Apr 4.


Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.

Keywords: Benzamidine; Cancer; Cell-signaling; Enzyme inhibitor; Growth factor; HGF; HGFA; Hepsin; MSP; Matriptase; Peptidomimetic; RON; Receptor tyrosine kinase; Serine protease; Small-molecule; Structure-based drug design; Therapeutic; c-MET.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidines / chemical synthesis
  • Antineoplastic Agents / chemical synthesis*
  • Antineoplastic Agents / pharmacology
  • Arginine / chemistry
  • Benzamidines / chemical synthesis*
  • Benzamidines / pharmacology
  • Catalytic Domain
  • Drug Design
  • Enzyme Assays
  • Guanidines / chemistry
  • High-Throughput Screening Assays
  • Humans
  • Kinetics
  • Molecular Docking Simulation
  • Neoplasm Proteins / antagonists & inhibitors*
  • Neoplasm Proteins / chemistry
  • Peptidomimetics / chemistry
  • Phenylalanine / analogs & derivatives
  • Phenylalanine / chemical synthesis
  • Piperazines / chemical synthesis
  • Protease Inhibitors / chemical synthesis*
  • Protease Inhibitors / pharmacology
  • Recombinant Proteins / chemistry
  • Serine Endopeptidases / chemistry*
  • Structure-Activity Relationship
  • Urea / analogs & derivatives
  • Urea / chemistry


  • Amidines
  • Antineoplastic Agents
  • Benzamidines
  • CJ 672
  • Guanidines
  • Neoplasm Proteins
  • Peptidomimetics
  • Piperazines
  • Protease Inhibitors
  • Recombinant Proteins
  • Phenylalanine
  • Urea
  • Arginine
  • HGF activator
  • Serine Endopeptidases
  • hepsin
  • matriptase
  • nafamostat