HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H3:H4 tetramers in the cytoplasm. It may have a role in cancer metabolism by linking cytoplasmic production of acetyl-CoA to nuclear acetyl flux. This is because the HAT1 di-acetylation mark is not propagated in chromatin and instead is de-acetylated after nascent histone insertion into chromatin. Thus, HAT1 likely provides a nuclear source of free acetate that may be recycled to acetyl-CoA for nuclear acetylation reactions. Correspondingly, suppression of HAT1 protein expression impairs tumor growth. To ascertain whether targeting HAT1 is a viable anti-cancer treatment strategy we sought to identify small molecule inhibitors of HAT1. We developed a high-throughput HAT1 acetyl-click assay to facilitate drug discovery and enzymology. Screening of small molecules computationally predicted to bind the active site led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity by competing with acetyl-CoA binding. These hits were refined by synthesis and testing over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl sidechain improved enzymatic potency and cellular growth suppression. These efforts resulted in a lead compound (JG-2016) that suppressed growth of human cancer cells lines in vitro and impaired tumor growth in vivo. This is the first report of a small molecule inhibitor of the HAT1 enzyme complex and represents a step towards targeting this pathway for cancer therapy.

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H4. To ascertain whether targeting HAT1 is a viable anticancer treatment strategy, we sought to identify small-molecule inhibitors of HAT1 by developing a high-throughput HAT1 acetyl-click assay. Screening of small-molecule libraries led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity. Compounds were refined by synthesis and testing of over 70 analogs, which yielded structure–activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl side chain improved enzymatic potency and cellular growth suppression. One compound (JG-2016 [24a]) showed relative specificity toward HAT1 compared to other acetyltransferases, suppressed the growth of human cancer cell lines, impaired enzymatic activity in cellulo, and interfered with tumor growth. This is the first report of a small-molecule inhibitor of the HAT1 enzyme complex and represents a step toward targeting this pathway for cancer therapy.