Histone acetyltransferases: challenges in targeting bi-substrate enzymes

Abstract

Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to neurological disorders, both through acetylations of histone proteins and non-histone proteins. Several HAT inhibitors, like bi-substrate inhibitors, natural product derivatives, small molecules, and protein-protein interaction inhibitors, have been developed. Despite their potential, a large gap remains between the biological activity of inhibitors in in vitro studies and their potential use as therapeutic agents. To bridge this gap, new potent HAT inhibitors with improved properties need to be developed. However, several challenges have been encountered in the investigation of HATs and HAT inhibitors that hinder the development of new HAT inhibitors. HATs have been shown to function in complexes consisting of many proteins. These complexes play a role in the activity and target specificity of HATs, which limits the translation of in vitro to in vivo experiments. The current HAT inhibitors suffer from undesired properties like anti-oxidant activity, reactivity, instability, low potency, or lack of selectivity between HAT subtypes and other enzymes. A characteristic feature of HATs is that they are bi-substrate enzymes that catalyze reactions between two substrates: the cofactor acetyl coenzyme A (Ac-CoA) and a lysine-containing substrate. This has important-but frequently overlooked-consequences for the determination of the inhibitory potency of small molecule HAT inhibitors and the reproducibility of enzyme inhibition experiments. We envision that a careful characterization of molecular aspects of HATs and HAT inhibitors, such as the HAT catalytic mechanism and the enzyme kinetics of small molecule HAT inhibitors, will greatly improve the development of potent and selective HAT inhibitors and provide validated starting points for further development towards therapeutic agents.

Keywords: Catalytic mechanism; Epigenetics; HAT inhibitors; Histone acetyltransferases; Inhibitor kinetics; Lysine acetylation.

Fig. 1

Lysine acetylation is balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation of lysine residues on the histone tails that protrude from the histone–DNA complex modifies the chromatin structure of the DNA, which allows transcription factors to bind. The transcription factors themselves can be acetylated, which influences promotor activity and specificity. Lysine acetylation of enzymes or nuclear receptors can influence their function. Bromodomain-containing proteins will bind to the acetylated lysine residues. Through lysine acetylations, HATs are involved in many different diseases such as cancer, inflammatory diseases, and neurological disorders. NR nuclear receptor, BRD bromodomain, NE nuclear enzyme, TF transcription factor

Fig. 2

The current HAT inhibitors and activators. Bi-substrate inhibitors mimic the two HAT substrates: Ac-CoA, connected via a linker to a peptide resembling the lysine substrate. Garcinol, curcumin, and anacardic acid are natural product HAT inhibitors. Small molecule inhibitors C646 and thiazinesulfonamide were discovered from a virtual screening. A high throughput screening yielded isothiazolone derivatives. A pentamidine derivative, TH1834, and a benzylidene barbituric acid derivative were developed using a structure-based design. ICG-001 is a protein–protein interaction inhibitor and inhibits the interaction between KAT3A and β-catenin. HAT bromodomain inhibitors have been developed for KAT3A and KAT2B, including the natural product ischemin, a set of cyclic peptides and small molecule N1-aryl-propane-1,3-diamine derivatives. CTPB, TTK21, and SPV106 are salicylic acid-derived HAT activators. CTBP activates KAT3B, TTK21 activates both KAT3B and KAT3A, and SPV106 interestingly is a KAT2B activator and KAT3A/3B inhibitor

Fig. 3

Challenges to get from the concentration of inhibitor that gives 50 % of inhibition (IC₅₀) to the assay independent inhibitory potency (K _i) for a HAT inhibitor. In case of bi-substrate enzymes like HATs, many factors need to be considered when calculating the inhibitory potency from the IC₅₀. Kinetic studies combined with affinity studies, crystal structures, dead-end inhibitors, and studies on the catalytic mechanism of HATs aid in deriving a K _i for HAT inhibitors