title: Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.
abstract: 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.
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Franco
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Darin E
authors.initials: DE
authors.lastname: Jones
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Peter K W
authors.initials: PK
authors.lastname: Harris
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Zhenfu
authors.initials: Z
authors.lastname: Han
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Wildman
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA.
authors.forename: Cassie M
authors.initials: CM
authors.lastname: Jarvis
authors.affiliation: Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics and Alvin J. Siteman Cancer Center, 660 South Euclid Ave., St. Louis, MO, USA. Electronic address: janetkaj@wustl.edu.
authors.forename: James W
authors.initials: JW
authors.lastname: Janetka
mesh_terms: D000578:Amidines / Q000138:chemical synthesis; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001550:Benzamidines / Q000138:chemical synthesis* / Q000494:pharmacology; D020134:Catalytic Domain; D015195:Drug Design; D057075:Enzyme Assays; D006146:Guanidines / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D057786:Peptidomimetics / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D010879:Piperazines / Q000138:chemical synthesis; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014508:Urea / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D000578:Amidines; D000970:Antineoplastic Agents; D001550:Benzamidines; C548449:CJ 672; D006146:Guanidines; D009363:Neoplasm Proteins; D057786:Peptidomimetics; D010879:Piperazines; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010649:Phenylalanine; D014508:Urea; D001120:Arginine; C081092:HGF activator; D012697:Serine Endopeptidases; C055645:hepsin; C485595:matriptase; C032855:nafamostat
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