Lysine-targeting covalent inhibitors

Abstract

Targeted Covalent Inhibitors (TCIs) represent an increasingly important strategy in modern drug discovery. TCIs exploit high affinity, reversible ligands which are then modified with modestly-reactive electrophilic warheads to covalently modify active site, nucleophilic residues within the target enzyme to inhibit their function. Irreversible covalent enzyme inhibition can overcome competition with high abundance, high affinity substrates and low affinity, shallow binding sites. The nucleophilic amino acid cysteine is the most common residue targeted in TCI discovery efforts, but the low abundance of this residue in the proteome limits the scope of cysteine-TCIs. Lysine is a potentially attractive alternative residue to target for TCIs, owing to its nucleophilic amino group and greater natural abundance. The aim of my thesis is to investigate the potential of lysine-TCIs to modulate the function of difficult-to-drug targets. The stress-inducible molecular chaperone, HSP72, is a well-validated oncology target and is considered difficult-to-drug owing to its high affinity for the abundant endogenous ligands ATP and ADP. Previously within our group, nucleoside-derived lysine-TCIs had been developed to target the non-catalytic Lys-56 of the HSP72 nucleotide binding domain. However, we estimated that the potencies of these TCIs, measured through the covalent kinetic parameter kinact/KI, were at least an order of magnitude too weak to attenuate HSP72 function in a cellular context, so required further optimisation. Herein, I detail the design, synthesis and optimisation of a series of high affinity HSP72 lysine-TCIs. This required the development of a novel protecting group strategy to modify the densely-functionalised adenosine core in the presence of the covalent warhead. Extensive characterisation revealed exquisite biochemical potency, but despite substantial improvements in compound stability, the rate of electrophilic warhead hydrolysis under the cell assay conditions unfortunately remained too high to effect downregulation of HSP70 clients at <20 μM total concentration. However, by employing a hydrolytically stable lysine-reactive warhead, I discovered the lysine-TCI CCT400591, the state-of-the-art tool compound for investigating HSP70 inhibition in vitro. Using our strategy, I then designed lysine-TCIs to overcome tertiary resistance mutations in the key oncogenic protein EGFR, leading to the discovery of a series of lysine-TCIs which covalently label the catalytic lysine within the EGFR tyrosine kinase domain.