Discovery of Modified Peptides and Proteins Targeting Proteases

Abstract

Coagulation factor XI (FXI(a)) deficiency and inhibition has been shown to induce an anticoagulant effect, without an associated increase in severe bleeding risk. Although a few therapeutic candidates are undergoing evaluation, the FXI(a) inhibitor pool remains small and is yet to produce an agent with clinical use approval. Towards addressing this vacancy, mRNA display with genetic code reprogramming was employed to discover novel macrocyclic peptide inhibitors of FXI(a) that demonstrate potent anticoagulant activity. Described in Chapters 2 and 3, the discovery, chemical synthesis and in vitro evaluation of several catalytic and allosteric modulators of FXIa activity presents promising lead candidates for future development. Chemical protein synthesis offers an attractive alternative to library generation by mRNA display or recombinant expression, as large libraries of disordered proteins can be assembled rapidly through a convergent peptide ligation approach, permitting near unlimited scope for chemical diversity. Chapter 4 describes the application of diselenide-selenoester ligation (DSL) chemistry to establish a combinatorial protein synthesis platform that facilitates rapid assembly and tandem in vitro screening of novel disordered proteins. To this end, potent thrombin protein inhibitors derived from hematophagous organisms served as promising proof-of-concept libraries and delivered several novel, bifunctional inhibitors of thrombin activity worthy of future investigation. In response to the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Chapter 5 recounts the discovery of SARS-CoV-2 main protease inhibitors by mRNA display. Following chemical synthesis and in vitro evaluation of several anti-viral agents, a lead candidate was selected for successful co-crystallisation with the SARS-CoV-2 main protease during the height of the COVID-19 pandemic.