Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors

Abstract

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.

Paralogue-Selective Degradation of the Lysine Acetyltransferase EP300

“…In addition, it is known that ligands targeting the HAT domain of EP300/ CREBBP are sensitive to cellular acetyl-CoA levels. 10,30 While the ability of MC-1 to inhibit acetylation in MCF-7 implies acetyl-CoA is not solely responsible for the limited degradation observed in this cell line, these studies suggest the possibility of leveraging metabolite-competitive degradation to study cellular acetyl-CoA, an application that could provide valuable insights into metabolic signaling. 31 We also note limitations of our study and future directions of inquiry.…”

“…As currently constituted, we envision MC-1 will find utility studies aimed at differentiating the noncatalytic roles of EP300 and CREBBP, particularly in cell lines which are not greatly affected by loss of EP300/CREBBP HAT activity. In addition, it is known that ligands targeting the HAT domain of EP300/CREBBP are sensitive to cellular acetyl-CoA levels. , While the ability of MC-1 to inhibit acetylation in MCF-7 implies acetyl-CoA is not solely responsible for the limited degradation observed in this cell line, these studies suggest the possibility of leveraging metabolite-competitive degradation to study cellular acetyl-CoA, an application that could provide valuable insights into metabolic signaling …”

“…From a small molecule perspective, significant efforts have led to the development of potent and selective inhibitors of the EP300 and CREBBP HAT domains. , One of the most potent of these compounds is CPI-1612. , In biochemical assays CPI-1612 disrupts EP300 and CREBBP catalytic activity at half-maximal inhibitor concentrations of <0.5 and 2.9 nM, respectively (Table S1). Considering how this narrow selectivity window may be leveraged to study individual functions of EP300 and CREBBP, we were inspired by recent examples from the targeted protein degradation literature .…”

Paralogue-selective degradation of the lysine acetyltransferase EP300

“…7,8 One of the most potent of these compounds is CPI-1612. 9,10 In biochemical assays CPI-1612 disrupts EP300 and CREBBP catalytic activity at half-maximal inhibitor concentrations of <0.5 nM and 2.9 nM, respectively (Table S1). Considering how this narrow selectivity window may be leveraged to study individual functions of EP300 and CREBBP, we were inspired by recent examples from the targeted protein degradation literature.…”

“…In addition, it is known that ligands targeting the HAT domain of EP300/CREBBP are sensitive to cellular acetyl-CoA levels. 10,28 This suggests that the sensitivity of EP300 to MC-1 under different metabolic conditions may be a useful proxy for levels of cellular acetyl-CoA, an approach that could provide valuable insights into metabolic signaling. 29 We also note limitations of our study and future directions of inquiry.…”