CPI-1612

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that simultaneously bind an E3 ligase and a protein of interest, inducing degradation of the latter via the ubiquitin-proteasome system. Here we present the development of degraders targeting CREB-binding protein (CBP) and E1A-associated protein (EP300)-two homologous multidomain enzymes crucial for enhancer-mediated transcription. Our PROTAC campaign focused on CPI-1612, a reported inhibitor of the histone acetyltransferase (HAT) domain of these two proteins. A novel asymmetric synthesis of this ligand was devised, while PROTAC-SAR was explored by measuring degradation, target engagement, and ternary complex formation in cellulo. Our study demonstrates that engagement of Cereblon (CRBN) and a sufficiently long linker between the E3 and CBP/EP300 binders (≥21 atoms) are required for PROTAC-mediated degradation using CPI-1612 resulting in a new active PROTAC dCE-1. Lessons learned from this campaign, particularly the importance of cell-based assays to understand the reasons underlying PROTAC performance, are likely applicable to other targets to assist the development of degraders.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.