Discovery of CHD1 Antagonists for PTEN-Deficient Prostate Cancer

Abstract

CHD1 is a chromodomain-helicase DNA-binding protein that preferentially recognizes di- and trimethylated lysine 4 on histone H3 (H3K4me2/3). Genetic studies have established CHD1 as a synthetic lethal target in phosphatase and tensin homologue (PTEN)-deficient cancers. Despite this attractive therapeutic link, no inhibitors or antagonists of CHD1 have been reported to date. Herein, we report the discovery of UNC10142, a first-in-class small molecule antagonist of the tandem chromodomains of CHD1 that binds with an IC₅₀ of 1.7 ± 0.2 μM. A cocrystal structure revealed a unique binding mode and competition pull-down experiments in cell lysates confirmed endogenous target engagement. Treatment of PTEN-deficient prostate cancer cells with UNC10142 led to a dose-dependent reduction in viability while PTEN-intact prostate cancer cells were unaffected, phenocopying genetic loss of CHD1. Overall, this study demonstrates the ligandability of the CHD1 chromodomains and suggests more potent and selective antagonists could translate to compounds of therapeutic value in PTEN-deficient cancers.

Figure 1.

CHD1 antagonist hit discovery and validation. (A) Schematic of CHD1 protein domains. (B) PTEN-deficient cancer cells are dependent on CHD1 and its downstream targets for survival. Thus, a CHD1 antagonist offers an avenue to selectively target PTEN-deficient cancer cells while leaving PTEN-intact cells unaffected. (C) CHD1 degradation occurs in normal, PTEN-intact cells, whereas loss of PTEN results in enhanced CHD1 stability. (D) Heat map of small molecule TR-FRET screen. Color gradient indicates average percent inhibition normalized to high (DMSO) and low (100 μM competitor) controls. Each box represents an individual compound screened at 10 μM. A threshold of ≥40% inhibition was used to triage compounds for follow up screening in a dose response format. (E) Structure of UNC622 with numbering of relevant carbon numbering highlighted in red (1). (F) UNC622 binds CHD1 with an IC₅₀ of 6.4 ± 0.2 μM (N = 3) by TR-FRET. (G) Representative ITC curve confirming binding of UNC622 (1) to CHD1 (K_d = 10 ± 1 μM, N = 2).

Figure 2.

Co-crystal structure of UNC10142 bound to the tandem chromodomains of CHD1. (A) UNC10142 binds in a shallow pocket at the intersection of the tandem chromodomains of CHD1. (B) The quinazoline core engages in π–π stacking interactions with W67. (C) The 9-membered ring binds in a shallow hydrophobic pocket. (D) A potential electrostatic interaction exists between the N-terminus and piperidine ring at the C7 position. PDB: 8UMG, Chain B. Polar and aromatic interactions are rendered as yellow lines and distance measurements are rendered as black lines.

Figure 3.

Selectivity and ITC validation of UNC10142. (A) Evaluation of the selectivity of UNC622 (1), UNC10142 (44), and less active control compound UNC10892 (55) against a panel of Kme reader domains by TR-FRET. Values are represented as mean ± SE for N = 2–3. (B) Representative ITC curve of UNC10142 binding to the tandem chromodomains of CHD1 (K_d = 4.3 ± 0.4 μM, N = 4).

Figure 4.

UNC10142 engages endogenous CHD1 and leads to loss of viability in PTEN-deficient cells. (A) Representative immunoblot analysis of CHD1 pulled down by a biotinylated H3K4me2 peptide and competed off with UNC10142 in HEK293T lysates (left). Bands are quantified in the bar graph relative to DMSO treatment (average ± SEM for N = 2) (right). (B) Schematic of expected effects on viability in PTEN-intact (DU 145) and PTEN deficient (PC-3 and LNCaP) cells treated with UNC10142 and negative control compounds UNC10892 and UNC9461. (C–E) Structures and cytotoxicity of UNC10142 (C), UNC10892 (D), and UNC9461 (E) in dose response as assessed by Cell Titer-Glo in DU 145, PC-3, and LNCaP cells (N = 3).

Scheme 1.. Synthetic Route for Quinazoline Derivative Conditions^a

^a (A) Substituted amine, DIPEA, DMF, RT, 2 h; (B) TFA, RT, 2 h; (C) K₂CO₃, chloro-iodo alkane, ACN, reflux, 2 h followed by NaI, TBAI, secondary amine, 60°C overnight; (D) DIPEA, substituted amine, isopropanol, 160°C, 2 h.