Exploring protein stabilization as an approach to drug discovery

Abstract

Certain genetic mutations reduce the lifetimes of encoded proteins by destabilizing the functional folded form, preventing them from performing their cellular roles. In many cases, significant loss of functionality due to protein destabilization leads to the initiation and progression of human disease. This can be intercepted by a strategy known as pharmacological chaperoning, that employs small molecules that bind and stabilize their targets in order to restore native function. This thesis describes the discovery of novel small molecule stabilizers of thermally-unstable disease associated proteins as candidates for development towards bioactive drugs that operate through pharmacological chaperoning. Herein is reported steps towards the application of this strategy to two model proteins that are mutationally deactivated in disease: the tumour suppressor p53, which is central to the natural physiological defence against cancer, and formylglycine-generating enzyme (FGE), the inactivation of which leads to the rare lysosomal storage disorder multiple sulfatase deficiency (MSD). Novel protein stabilizers were discovered by primary screening and structurally-characterized ligands were optimized by medicinal chemistry. Development of ligands for cancer mutant p53- Y220C by chemical synthesis and biophysical evaluation led to the discovery of new chemical probes that bind with nanomolar affinity, crossing a significant milestone in the field of Y220C chaperoning. Through rational design a new, tractable Y220C-binding scaffold was discovered that has favourable physical properties and activity in vitro. Biophysical screening of fragments and high-throughput virtual screening of almost 1 million lead-like compounds led to the identification of the first small molecules to bind and stabilize FGE and attained low micromolar affinity. Further, pharmacological chaperoning was validated against a novel protein target (FGE), demonstrating the broad and currently underrepresented potential of protein stabilization as a therapeutic strategy. Thus far, protein stabilization remains a relatively unexplored approach within the world of drug discovery, despite the key benefits of high selectivity and broad target scope. This work seeks to demonstrate the applicability and prosperity of the new modality of pharmacological chaperoning. We address a target for which no approved treatment currently exists, and present preliminary hit discovery and lead optimization data that demonstrate the in vitro efficacy of protein stabilizers. With only one marketed chaperone drug (migalastat) produced so far and a sparingly small proportion of the known proteome exploited, we aspire to contribute to the establishment of the young field of pharmacological chaperoning and provide a basis for a new generation of pharmaceutical drugs for the treatment of human disease through protein structural stabilization.

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ORCID for Matthias Baud: orcid.org/0000-0003-3714-4350

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