Masters Thesis

Enhancing efficacy of protease drugs through site directed mutagenesis

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 while maintaining catalytic 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, four trypsin variants were produced with amino acid substitutions at position 39 (Y39E, Y39S, Y39V and Y39L). Single variants Y39S and Y39L display activity that is comparable to wild type [kcat= (8.5 ± 47) x 103 min'1), (9.1 ± 143) x 10'3 min*1 and (4.9 ± 100) x 103 min'1] respectively.

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