Materials and methods br Results and discussion br Acknowled
Materials and methods
Results and discussion
Acknowledgments This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grants JP21300174 and JP25282144 (H.K.).
Introduction Epigenetic modifications of histones, such a lysine acetylation, play a key role in the regulation of gene transcription. Histone acetyltransferases (HATs) are a class of acetyltransferases that catalyze the acetylation of ε-amino groups on lysine residues in both histone and non-histone proteins. Through histone acetylation, they play a regulatory role in the chromatin structure, thereby influencing gene transcription. The acetylation of non-histone proteins, for example transcription factors, is involved in the regulation of many processes, such as cell growth and inflammatory signaling . This enzyme class has been linked to the pathology of various diseases, including cancer , , , inflammatory diseases , , , viral infections  and neurological diseases , . However, knowledge on their role in specific diseases and drug discovery efforts towards this class of enzymes are still limited. In particular the HAT lysine acetyltransferase 8 (KAT8) is marginally explored in drug discovery projects. The HATs are a disparate group of enzymes that can be divided into different families based on their structural homology. The three main families are GNAT (Gcn5-related N-acetyltransferases), MYST (MOZ, YBF2/SAS3, SAS2 and TIP60) and p300/CBP (CREB binding protein). The HAT of our interest, KAT8, is a member of the MYST family. This enzyme was originally discovered in Drosophila, where it is involved in dose-compensation of the X-chromosome gene transcription in male flies. KAT8 functions in two protein complexes, MSL and MSL1v1, that are conserved throughout the eukaryotic kingdom, including humans . Both KAT8 complexes have been described to be responsible for acetylating lysine 16 on histone H4 (H4K16) and were shown to play a role in 2871 progression . However, only the MSL1v1 complex seems to be involved in acetylation of the tumor suppressor protein p53 . KAT8 has also been shown to play a role in embryonic stem cell renewal. Embryonic stem cells lacking KAT8 lose differentiation potential and show changes in morphology and gene expression of essential transcription factors . Thus, it is clear that KAT8 plays a very important role in normal physiology and disease. KAT8 is a bi-substrate enzyme that binds two substrates; acetyl coenzyme A (Ac-CoA) and histone H4 containing free lysine ε-amino groups. Development of inhibitors for bi-substrate enzymes requires knowledge of the catalytic mechanism. It is important to understand if the substrates, acetyl coenzyme A and histone H4, bind simultaneously or consequently and if the individual binding events are inter-dependent. In addition, knowledge on the catalytic mechanism combined with inhibitor kinetics, enables the calculation of the assay-independent inhibition constant (Ki) from the assay-dependent inhibitory concentration (IC50) as described by Cheng and Prusoff . Therefore, we investigated the catalytic mechanism of KAT8 using enzyme kinetic studies based on models described by Copeland . We demonstrated that the non-selective HAT inhibitor anacardic acid (AA)  also inhibits KAT8 and performed kinetic studies to further investigate this inhibitor. Based on the results, we proposed a model comprising the catalytic activity of KAT8 and the inhibitory action of AA. We employed this knowledge to study the inhibitory potency of a small collection of anacardic acid derived inhibitors and to calculate their respective binding constants (Ki). Inhibition studies on p300 did not reveal selectivity between both enzymes for the compound collection that was investigated.
Results and discussion
Conclusions In this study, the catalytic mechanism of KAT8 histone acetyltransferase has been investigated. Enzyme kinetic experiments indicate that this bi-substrate enzyme operates by a ping-pong mechanism. This mechanism is supported by the observation that binding of the first substrate Ac-CoA is required for binding of the second substrate histone H4 as determined by ITC measurements. The presence of cysteine 143 in the KAT8 active site combined with the previous evidence that this residue is essential for catalysis further supports the evidence for a ping-pong mechanism for acetyltransferase activity of KAT8. We employed this model for calculation of the Ki values of inhibitors of this enzyme. In order to generate small molecule inhibitors for KAT8 we assembled a focused compound collection around the known non-selective HAT inhibitor AA. This compound collection was tested for inhibition of KAT8. Kinetic studies were performed with the reference compound AA and based on both inhibitor kinetics and mechanistic studies, a catalytic model was proposed involving two different conformations of the enzyme. The equilibrium binding constant Ki was calculated using an adaptation of the Cheng–Prusoff equation based on the catalytic mechanism and the proposed model. AA and its derivatives inhibited KAT8 and both the aliphatic tail and the salicylate functionality proved to be important for binding. The inhibitors were tested for activity on p300 and showed a similar SAR as on KAT8, suggesting a similar binding mode even though the two enzymes are structurally different.