Development of bifunctional small molecules to induce acetylation by CBP/p300

Thesis

Lysine acetylation is a common, reversible, highly dynamic post-translational modification which occurs at thousands of sites in the human proteome. Lysine acetyltransferases (KATs) catalyse the transfer of an acetyl group onto lysine, while lysine deacetylases (KDACs) catalyse the removal of the modification.

Heat shock protein 90 (Hsp90ɑ and Hsp90β) are ATP-dependent molecular chaperones that maintain active conformations of client proteins. Previous studies involving the knockdown or chemical inhibition of KDACs (particularly HDAC6) have shown hyper-acetylation on Hsp90ɑ attenuates chaperone function, while Hsp90ɑ K/Q mutants (acetyl-lysine mimetic) have unique interaction profiles with co-chaperones and client proteins. Induced proximity between an effector enzyme and a target protein with a bifunctional molecule promotes post-translational modification on the target. This concept is the basis of target protein degradation and has recently been shown to be applicable to lysine acetylation.

The aim of this thesis was to develop a bifunctional molecule, termed Acetylation Directing Chimera (AcDC), that recruits the acetyl transferases CBP/p300 towards Hsp90 and induces an increase in lysine acetylation on that specific target protein. Starting with the CBP/p300 bromodomain inhibitor, inobrodib, and Hsp90 N-terminal domain inhibitor, BIIB021, we designed and synthesised AcDCs comprising varied linkers (AcDC-1–4). We demonstrated specific AcDC-induced proximity with recombinant proteins in an AlphaScreen based assay and validated a co-operative binding model with isothermal calorimetry (ITC). NanoBRET based assays demonstrated target engagement and induced proximity with full-length proteins in a cellular system. Co-immunoprecipitation of endogenous Hsp90 from showed AcDC-2 forms a stable complex with CBP (but not necessarily p300). We studied whether this induced proximity results in a significant change in lysine acetylation on Hsp90, and determined the observed phenotypic effects were predominantly driven by inhibition of the proteins’ binding domains.

Authors: Stratton, OJ

• DOI: 10.5287/ora-b77zypyek
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: CREBBP; CBP; lysine acetylation; p300; HSP90; EP300; chemically induced proximity; targeted protein acetylation
• Subjects: Chemical biology; Chemistry