Peptidomimetic protease inhibitors: activity and mechanism of inhibition

Abstract

The study of protein mechanism and function is central to the development of biosensing tools and therapeutics for the treatment of diseases. This thesis describes an NMR and X-ray crystallography-based characterisation of the mechanism by which a macrocyclic peptidomimetic, the backbone of which is constrained into a β-strand conformation, inhibits α-chymotrypsin. This allowed the development of new peptidomimetic inhibitors that target the 26S proteasome and also inhibitors the activity of which can be modulated photochemically. This then provides a basis for biosensing and therapeutic applications. Chapter one introduces the structures and mechanism of serine, cysteine and threonine proteases, and discusses how theses proteases universally bind ligands in an extended β-strand conformation. In addition, this chapter details the strengths and limitations of current peptidomimetic inhibitors of α- chymotrypsin, calpains and the 26S proteasome and their implications in the treatment of human diseases. Chapter two describes optimisation of the synthesis of two macrocyclic peptidic aldehyde inhibitors 2.12 and 2.13 that target cysteine proteases and α-chymotrypsin, respectively. This allowed the preparation of an analogue of 2.13 containing a ¹³C label in the aldehyde, which was used to confirm the mechanism of inhibition of α-chymotrypsin by ¹³C NMR spectroscopy. This confirmed the formation of a stable hemiacetal intermediate upon the binding of 2.13 with α-chymotrypsin. X-ray crystallography of a complex of 2.13 bound to α-chymotrypsin revealed that the backbone adopts a stable β-strand conformation as per its design. The binding of 2.13 to α-chymotrypsin is further stabilised by the oxyanion hole near the S₁ subsite and multiple hydrogen bonding interactions. Chapter three details the development of new acyclic proteasome inhibitors 3.05-3.08 containing a peptidomimetic backbone and a C-terminal boronate. All analogues showed selectivity for the chymotrypsin-like subunit of the 26S proteasome with IC₅₀ values in the low nanomolar range. Compound 3.08, with an IC₅₀ of 13 nM, was 2-fold more active than the anti-myeloma therapeutics bortezomib and carfilzomib. This inhibitor is more cytotoxic against a range of solid tumour cells and has a larger therapeutic window compared to existing FDA approved drugs. Chapter four presents a new approach to the regulation of the activity of α- chymotrypsin using a new spiropyran-based moiety that can be reversibly switched between an ‘on’ (SP isomer) and ‘off’ (MC isomer) state photochemically. This is demonstrated in solution and also when attached to a microstructured optical fibre (MOF), as a first step to the development of a biosensor. The most active analogue in this series displayed a Kᵢ of 115 nM in solution. The active SP isomer of an analogue 4.07 with a C-terminal Weinreb amide was significantly more active than the corresponding MC isomer both in solution and on fibre.