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Enhancing efficacy of protease drugs through site‐directed mutagenesis
Author(s) -
Amorello Angela Nicole,
Batt Anna,
Baird Teaster
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.918.15
Subject(s) - proteases , trypsin , protease , serine protease , biochemistry , serine , mutagenesis , chemistry , enzyme , protease inhibitor (pharmacology) , site directed mutagenesis , trypsin inhibitor , biology , mutant , genetics , virus , gene , antiretroviral therapy , viral load
A modern approach to drug development draws from nature to treat disease. Proteases, historically targeted for the development of inhibitors, show therapeutic potential. Development of protease therapeutics is complicated by the presence of endogenous inhibitors, which result in reduced serum half‐lives of these agents. Determining which residues influence inhibitor binding can inform the design of efficacious protease therapeutics that have less favorable interactions with inhibitors through mutagenesis. Trypsin‐fold serine proteases account for the majority of commercially available protease therapeutics. Consequently, substitutions that confer inhibitor resistance without compromising activity in trypsin may be applied to prospective serine protease drugs. Examination of crystal structures of trypsin and its macromolecular inhibitors have shown a conserved hydrogen bond interaction between Tyr 39, Lys 60 and inhibitor. Using PCR mutagenesis, three trypsin variants were produced with amino acid substitutions at position 39 (Y39E, Y39S, and Y39L). We have demonstrated that certain Tyr 39 substitutions confer weaker enzyme‐inhibitor interactions. Y39A trypsin had a lower association rate (4.1 × 10‐3 μM‐1 s‐1) and higher dissociation rate (3.3 × 10‐3 s‐1) compared to wild type (5.5 × 10‐3 μM‐1 s‐1 and 2.5 × 10‐3 s‐1) respectively with bovine pancreatic trypsin inhibitor and also demonstrated catalytic activity similar to that of wild type. Substitutions at position 39 in trypsin show promise to retain catalysis and reduce inhibition.