Elsevier

Bioorganic Chemistry

Volume 39, Issue 1, February 2011, Pages 42-47
Bioorganic Chemistry

Probing the reaction coordinate of the p300/CBP histone acetyltransferase with bisubstrate analogs

https://doi.org/10.1016/j.bioorg.2010.10.004Get rights and content

Abstract

Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell–Chance or “hit-and-run” catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail.

Graphical abstract

We describe here the synthesis and evaluation of bisubstrate analogs as mechanistic probes for p300/CBP histone acetyltransferase. These analogs vary in the linker that conjoins the peptide backbone and coenzyme A.

  1. Download : Download full-size image
.

Research highlights

► A novel set of bisubstrate analogs with increasing linker lengths was made as mechanistic probes for p300 histone acetyltransferase. ► Longer linker compounds were more potent. ► Mutation confirmed peptide site interactions.

Introduction

The p300/CBP histone acetyltransferase (HAT) catalyzes the lysine acetylation of more than 75 different cellular proteins [1], [2], [3], [4], [5] 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], [8], [9], [10]. 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], [12], [13], [14], [15].

Compared to other HATs, p300/CBP has been reported to follow an unusual Theorell–Chance kinetic mechanism [16], [17], [18]. 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], [20]. 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 into 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 Fig. 1). 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.

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.

Section snippets

General

All materials for Fmoc solid phase synthesis were purchased from Novabiochem (Darmstadt, Germany) and used without additional purification. All amino acids used here were of l-configuration. Peptides were synthesized in Protein Technologies (Rainin) PS3 peptide synthesizer and purified using a Varian ProStar HPLC equipped with C18 reverse phase column. Amino acid analysis was performed in the Harvard microchemistry facility or the W.M. Keck facility at Yale University. All buffering and

Results

We designed and synthesized a set of three analogs of H4-CoA-20 (1) in which the acetyl bridge between Lys and CoA is replaced by a propionyl, a hexanoyl, and an octanoyl lipid linker (Fig. 2, 24). Two additional analogs incorporate a protected lysyl linker between either the natural Lys at position 8 of the H4-tail (5) or an amino-alanine at position 8 of the H4-tail (6) followed by CoA attachment. One final analog (7) incorporates two consecutive lysyl linkers attached to the natural Lys-8.

Discussion

Bisubstrate analog inhibitors for enzymes that catalyze reactions involving two substrates can be useful structural and functional tools [22], [23], [24], [25], [26]. Among the major challenges in bisubstrate analog inhibitor design is constructing the appropriate linker that can allow for optimal engagement of the two substrate binding sites while limiting entropic loss associated with forming a noncovalent tripartite complex. Here, we have addressed the paradoxical observation that a

Acknowledgments

We thank NIH for funding this study. Also, we thank Dr. Ling Wang for her assistance with the purification of p300 HAT and HAT assays.

References (26)

  • V.V. Ogryzko et al.

    Cell

    (1996)
  • H.S. Mellert et al.

    Trends Biochem. Sci.

    (2009)
  • L. Wang et al.

    Curr. Opin. Struct. Biol.

    (2008)
  • W. Gu et al.

    Cell

    (1997)
  • M. Cebrat et al.

    Bioorg. Med. Chem.

    (2003)
  • E.M. Bowers et al.

    Chem. Biol.

    (2010)
  • P.R. Thompson et al.

    J. Biol. Chem.

    (2001)
  • K.G. Tanner et al.

    J. Biol. Chem.

    (2000)
  • O.D. Lau et al.

    Mol. Cell

    (2000)
  • J. Wu et al.

    Bioorg. Med. Chem.

    (2009)
  • A.J. Bannister et al.

    Nature

    (1996)
  • P.R. Thompson et al.

    Nat. Struct. Mol. Biol.

    (2004)
  • R.H.S. Goodman

    Genes Dev.

    (2000)
  • Cited by (9)

    • Targeted degradation of the enhancer lysine acetyltransferases CBP and p300

      2021, Cell Chemical Biology
      Citation Excerpt :

      Over the last 2 decades, many efforts have focused on producing pharmacologic ligands of p300/CBP. Early studies demonstrated the KAT-inhibitory effects of bisubstrate analogues that link acetyllysine to the p300/CBP cofactor coenzyme A (Cebrat et al., 2003; Karukurichi and Cole, 2011; Lau et al., 2000; Sagar et al., 2004). The chemical probe C646 was discovered by virtual screening methods and has been used by many groups to study p300/CBP KAT activity in cellular model systems, yet its utility is limited by apparent off-target and pan-assay inhibitory effects (Bowers et al., 2010; Dahlin et al., 2017; Shrimp et al., 2015).

    • Histone Acetyltransferases: Enzymes, Assays, and Inhibitors

      2015, Epigenetic Technological Applications
    View all citing articles on Scopus
    View full text