The p300/CBP histone acetyltransferase (HAT) catalyzes the lysine acetylation of more than 75 different cellular proteins [1
] including itself [6
]. Beyond histones, such acetylation events have wide-ranging effects on cell growth and gene expression, influencing cancer, the immune system, endocrine and metabolic pathways, host-pathogen interactions, and cardiovascular physiology [7
]. Within the field of epigenetics, there has been increasing interest in developing potent and specific inhibitors of p300/CBP HAT as potential pharmacologic agents for a number of disease indications [11
Compared to other HATs, p300/CBP has been reported to follow an unusual Theorell-Chance kinetic mechanism [16
]. The well-characterized HATs including PCAF/GCN5 and Esa1 employ a sequential mechanism in which acetyl-CoA binding to enzyme precedes peptide/protein substrate binding to form a ternary complex [19
]. There is then direct transfer of the acetyl group from acetyl-CoA to the Lys side chain with ordered product release. The Theorell-Chance mechanism proposed for p300/CBP also involves initial binding of acetyl-CoA and direct acetyl transfer to peptide/protein substrate but does not form a detectable ternary complex. In the Theorell-Chance mechanism, often called hit and run, the peptide/protein binds in to the p300/CBP-acetyl-CoA complex in too fleeting a fashion to be measured but does allow for covalent chemistry to occur [16
]. The Theorell-Chance mechanism is deduced from a characteristic pattern of product inhibition in which acetylated peptide product is competitive versus peptide substrate [18
A number of years ago, several bisubstrate analogs including H4-CoA-20 and Lys-CoA were tested as p300 HAT inhibitors and it was found that Lys-CoA, with Ki* 20 nM, was highly potent and selective whereas the more elaborate H4-CoA-20, containing 20 residues from the histone H4 tail, was far weaker as a p300 HAT inhibitor [21
]. This was unexpected because p300 HAT preferentially acetylates lysine residues in H4 tail peptides rather than in isolation and because the Lys-CoA moiety is a constituent of the H4-CoA-20 framework. An X-ray crystal structure of the p300 HAT domain in complex with Lys-CoA revealed that Lys-CoA sits in an extended conformation in a narrow tunnel formed within p300 HAT and the Lys alpha amino and carboxyl groups are near the walls of the tunnel [18
] (see ). This suggested a model of steric blockade in which the additional residues of H4-CoA-20 attached to the Lys would limit its p300 binding affinity.
Crystal structure of p300 histone acetyltransferase complexed to Lys-CoA (PDB ID: 3BIY). The highlighted distances extend from the alpha carbon of the Lys moiety in Lys-CoA to the Asp side chains of the proposed histone tail binding residues.
A separate, negatively charged shallow pocket lined by the side chains Asp-1625 and Asp-1628 of p300 spaced about 10 Å from the Lys-CoA tunnel appears to be important for peptide substrate interaction. Mutation of Asp-1625 and Asp-1628 to Arg reduces p300's acetyltransferase efficiency toward positively charged peptide substrates but has much less of an effect on negatively charged peptide substrates [18
]. Taken together with the proposed Theorell-Chance mechanism and Lys-CoA binding selectivity, it has been suggested that the p300-Lys-CoA X-ray structure captures a late stage of the reaction coordinate which represents further destabilization of H4 tail peptide interactions after a predicted weak encounter complex between p300 and a peptide substrate.
Our hypothesis emerging from these earlier studies is that it may be possible to generate more potent versions of H4-CoA-20 in which the linker between the CoA and the peptide backbone is lengthened. Such stretched analogs of H4-CoA-20 could accommodate dual interactions on p300 engaging both the Lys-CoA channel and the potential peptide substrate binding groove. Below, we describe the synthesis and experimental analysis of this novel class of analogs.