Use of protease substrate specificity screening in the rational design of selective protease inhibitors with unnatural amino acids: Application to HGFA, matriptase, and hepsin

• DOI: 10.1002/pro.5110
• Publication Date: 2024-07-29T08:38:58Z
• Authors: Matthew W. Mahoney; Jonathan Helander; Anoopjit S. Kooner; Mariah Norman; Vishnu C. Damalanka; Paolo De Bona; Paulina Kasperkie...; Wioletta Rut; Marcin Poreba; Maithri M. Kaship...; Kevin P. Battaile; Scott Lovell; Anthony J. O'Dono...; Charles S. Craik; Marcin Drag; James W. Janetka
• Keywords: Rational design

Abstract Inhibition of the proteolytic processing hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for drug discovery novel anticancer therapeutics which prevent tumor progression metastasis. Here, we utilized improved expanded version positional scanning substrate combinatorial libraries (PS‐SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors HGF/MSP activating serine proteases, HGFA, matriptase, hepsin. These have electrophilic ketone trapping warhead thus form a reversible covalent bond protease. We demonstrate that by varying P2, P3, P4 positions inhibitor with unnatural amino acids based on protease preferences learned from HyCoSuL, can predictably modify potency selectivity inhibitor. identified tetrapeptide JH‐1144 ( 8 ) as single digit nM matriptase hepsin excellent over Factor Xa thrombin. peptides increased metabolic stability relative natural similar structure. The tripeptide PK‐1‐89 2 has pharmacokinetics in mice good compound exposure out 24 h. In addition, obtained X‐ray structure MM1132 15 bound revealing interesting binding conformation useful future design.