Discovery of a PCAF Bromodomain Chemical Probe

Abstract

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Keywords: bromodomains; chemical probes; epigenetics; medicinal chemistry; structure-based design.

Figure 1

Reported PCAF bromodomain inhibitors.

Scheme 1

Synthesis of [1,2,4]triazolo[4,3‐a]phthalazine derivatives. Reagents and conditions: a) Acetohydrazide, DMF 120 °C 16 h, 62 %; b) N₂H₄ ⋅H₂O, EtOH, 120 °C, 10 min, quant.; c) TFA, 100 °C, 2 h, 43 %; d) R′₂NH (1.5–2.0 equiv) KI (0.1 equiv), HCl (0.05 equiv), EtOH or iPrOH, reflux, 3 days 8–94 %.

Scheme 2

Synthesis of threo‐substituted derivatives 39–45. Reagents and conditions: a) NH₄OAc (0.2 equiv), EtNO₂, reflux, 1:1 E/Z, quant.; b) Me₂NH (5 equiv), THF, RT, 16 h, d.r. 4.6:1–33:1; c) H₂ (1 atm), Pd/C (10 %), MeOH, RT, 16 h, 11–15 % over two steps, single diastereomer; d) H₂ (1 atm), Ra/Ni (0.3 equiv), MeOH, RT, 16 h, 25–28 %, over two steps, single diastereomer; e) 5 (0.8 equiv) KI (0.1 equiv), HCl (0.05 equiv), EtOH or iPrOH, reflux, 3 days 16–79 %.

Figure 2

A) Profile of L ‐45. B) L ‐45 is selective in a DSF assay panel of 48 Brds (black text). C) Displacement of PCAF‐Brd from H3.3‐nanoLuc in live HEK‐293 cells using the nanoBRET assay. [a] clogD was calculated using ChemAxon.18 [b] Ligand efficiency.19

Figure 3

Co‐crystal structure of L ‐45 with PfGCN5 (PDB ID 5TPX). A) L ‐45 (pale sticks) binds in the KAc‐binding pocket of PfGCN (blue ribbon and sticks) and makes H‐bonds (dotted lines) through the triazole to N1436 and the first of a network of four water molecules (red spheres). The dimethylamino group forms a salt bridge with E1389. Blue mesh: 2 F _o  F _c omitted map contoured at 2.5 σ. B) Surface view of complex of PfGCN5 (surface, blue sticks) and L ‐45 (pale sticks). The phenyl group of L ‐45 lies in a hydrophobic groove between W1379 and the alkyl linker of K1383. The structure of PCAF (orange sticks, PDB ID 5FTZ) is superimposed to show key residue similarities (black text PfGCN5/PCAF) and differences (blue text PfGCN5, red text PCAF).

Scheme 3

Asymmetric synthesis of L ‐45. Reagents and conditions: a) Boc₂O, DIPEA, CH₂Cl₂, RT, 16 h, 51 % b) SOCl₂, Pyridine, MeCN, 2 h, −40 °C to 0 °C; c) NaIO₄ (1.5 equiv), RuCl₃⋅3 H₂O (0.05 equiv), MeCN, 1 h, 0 °C, 48 % (over two steps); d) Me₂NH (3 equiv), THF, RT, 16 h, 63 %; e) TFA, CH₂Cl₂, quant.; f) 5 (0.8 equiv) KI (0.1 equiv), HCl (0.05 equiv), iPrOH, reflux, 3 days, 30 %.