Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor

Abstract

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.

Figure 1

A. Three validated p300 HAT small molecule inhibitors C646, C375, and C146 and their inhibitory constants measured as described in B and Supplementary Figure 1. B. Inhibitory characteristics of C646 toward p300. A. Plot of 1/v vs. 1/[acetyl-CoA] at fixed H4-15 peptide substrate (100 μM) and three concentrations of C646 shows competitive inhibition. C646 K_i=400±60 nM, apparent acetyl-CoA K_m=8.5±1.4 μM, apparent k_cat=18±1 min^-1. C. Plot of 1/v vs. 1/[peptide substrate (H4-15)] at fixed acetyl-CoA (10 μM) and three concentrations of C646 shows noncompetitive inhibition. C646 K_i=530±40 nM, apparent H4-15 K_m=155±19 μM, apparent k_cat=40±2 min^-1.

Figure 2

Interactions of C646 with p300. A. In silico model of C646 bound to p300 HAT active site (upper) shows overlapping interactions with the X-ray crystal structure of p300 HAT complexed with Lys-CoA (lower). B. Kinetic analysis of selected active site mutants

Figure 3

Synthetic analogs of C646 and their relative IC₅₀s for p300 inhibition referenced to C646.

Figure 4

C646 treatment reduces histone H3 and H4 acetylation levels and abrogates TSA-induced acetylation in cells. Quiescent C3H 10T1/2 mouse fibroblasts were pretreated with C646 (25 uM, lanes 2-4, 6-8) for the indicated times (1, 2 or 3h) or the control compound, C37 (25 uM, 3h, lanes 9, 10), and TSA (33 nM) added where indicated (lanes 6-8, 10) for the final 30 min of incubation. Histones were extracted and analysed by western blotting to visualize total H3 and H3K9ac using SDS (Panel A) or H3, H3K9ac, and H4K12ac using acid-urea (Panel B) gel electrophoresis. Acid-urea gels separate histones on the basis of charge. Each acetylation event incrementally retards migration to produce a ‘ladder’ of bands, numbers on either side of the blot indicating extent of modification. (lane 1, untreated control cells; lane 5, TSA-treated cells).

Figure 5

(A) C646 has a more potent effect on cell growth than Lys-CoA-Tat does. Cells were treated for 24 h and proliferation was measured via ³H-thymidine incorporation pre- and post-treatment. Data for each cell line were normalized to ³H counts measured pre-treatment. C37 is an inactive derivative of C646. DMSO serves as a vehicle control for C37 and C646. Ac-DDDD-Tat is a control peptide for Lys-CoA-Tat. WM35, WM983A, and 1205Lu are melanoma cell lines representing three stages of cancer progression: radial, vertical, and metastatic, respectively. (B) Two out of three non-small cell lung adenocarcinoma cell lines demonstrate significant growth inhibition after treating with C646 and Lys-CoA-Tat for 24 h. (C) C646 does not have a significant inhibitory effect on NIH3T3 cell proliferation.

Figure 6

(A) C646 inhibits WM983A melanoma cell proliferation but C37 does not. Cells were treated with each compound for 24 h. Proliferation was measured via ³H-thymidine incorporation. (B) C646 blocks H3 acetylation in WM983A cells. Cells were treated for 6 h with increasing concentrations of C646. Nuclear lysates were subjected to western blot analysis for acetylated H3 (Upstate 06-599). Total H3 (Abcam ab1791) was blotted as a loading control. (C) C646 causes growth arrest in melanoma cell line WM35 and WM983A, indicated by a decrease in %S phase. Cells were treated for 24 h with 20 μM C646 or DMSO, then collected and stained with propidium iodide, followed by FACS analysis.

Scheme 1