Establishment of an N-terminal acetylation enzymatic assay to validate novel substrates and to identify inhibitors of NAA40

Abstract

NAA40 (N-alpha acetyltransferase 40) is a member of the extended family of NATs (N-terminal acetyltransferases) which deposit acetyl groups on the N-terminal ends of proteins. NAA40 is a highly selective NAT, and its two established substrates are the N-terminal ends of the core histones H4 and H2A. It has been highlighted by recent work of ours and other groups, that NAA40 is upregulated in cancer, and it is considered as a potential target for cancer treatment. In this study we have mainly established an in vitro NAA40 acetylation assay, a powerful tool for investigation of new substrates and inhibitors of the enzyme. This assay was used to characterize in vitro the kinetic properties of NAA40 with three candidate substrates predicted in silico: the histone variants H2A.X and H2A.J and the chromatin remodeler SMARCD2. Additionally, we utilized the acetylation assay for an unbiased drug screening and for the characterization of the inhibitory potential of small-molecules which can act as inhibitors of NAA40. These small-molecules were initially identified using a specific targeted screening methodology which included in silico screening and differential scanning fluorimetry assay. Thus, we provide proof of principle that the assay can be used for the characterization of novel substrates and inhibitors of NAA40, paving the way for future research into investigations of NAA40’s activity and the development of novel treatments.