Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design

Bioorganic and Medicinal Chemistry Letters

Volumn 25, Issue 14, 2015, Pages 2818-2823

  Duffy, Bryan C ^a   Liu, Shuang ^a   Martin, Gregory S ^a   Wang, Ruifang ^a   Hsia, Ming Min ^a   Zhao, He ^a   Guo, Cheng ^a   Ellis, Michael ^a   Quinn, John F ^b   Kharenko, Olesya A ^c   Norek, Karen ^c   Gesner, Emily M ^c   Young, Peter R ^c   McLure, Kevin G ^c   Wagner, Gregory S ^c   Lakshminarasimhan, Damodharan ^d   White, Andre ^d   Suto, Robert K ^d   Hansen, Henrik C ^c   Kitchen, Douglas B ^a  

^a ALBANY MOLECULAR RESEARCH INC   (United States)

^b JFQuinn Consulting   (United States)

^c Zenith Epigenetics Corp   (Canada)

^d Xtal BioStructures Inc   (United States)

Author keywords

BRD4(1); Bromodomain inhibitors; Hit triage; Protein ligand docking; Virtual screening

Indexed keywords

BROMODOMAIN CONTAINING 4 INHIBITOR; BROMODOMAIN INHIBITOR; LIGAND; PROTEIN; UNCLASSIFIED DRUG; BRD4 PROTEIN, HUMAN; NUCLEAR PROTEIN; PROTEIN BINDING; QUINOXALINE DERIVATIVE; TRANSCRIPTION FACTOR;

ARTICLE; BINDING AFFINITY; CRYSTALLOGRAPHY; DRUG DESIGN; DRUG INHIBITION; DRUG SCREENING; DRUG STRUCTURE; DRUG SYNTHESIS; FLUORESCENCE RESONANCE ENERGY TRANSFER; GEOMETRY; MOLECULAR DOCKING; STRUCTURE ACTIVITY RELATION; X RAY CRYSTALLOGRAPHY; ANTAGONISTS AND INHIBITORS; BINDING SITE; CHEMISTRY; HUMAN; METABOLISM; PRECLINICAL STUDY; PROTEIN TERTIARY STRUCTURE;

BINDING SITES; CRYSTALLOGRAPHY, X-RAY; DRUG DESIGN; DRUG EVALUATION, PRECLINICAL; HUMANS; LIGANDS; MOLECULAR DOCKING SIMULATION; NUCLEAR PROTEINS; PROTEIN BINDING; PROTEIN STRUCTURE, TERTIARY; QUINOXALINES; STRUCTURE-ACTIVITY RELATIONSHIP; TRANSCRIPTION FACTORS;

EID: 84930274651     PISSN: 0960894X     EISSN: 14643405     Source Type: Journal    
DOI: 10.1016/j.bmcl.2015.04.107     Document Type: Article

References (24)

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21. The diverse set of 230,000 compounds was docked into each independent BRD4(1) docking models using Schrödinger's Glide Program in the high throughput virtual screen (HTVS) mode. 20,21 Poses of compounds with a Glide score less than -7.5 in either model were retained. This diverse set of 1475 compounds was used to select compounds from the virtual library with 2D atom pair similarity scores ≥0.80 Dice similarity level. These similarity calculations resulted in 17,774 compounds and the compounds were filtered using the same property values and this filtering resulted in a total of 3394 molecules. The HTVS selection set of compounds was re-docked at the Glide Standard Precision (SP) precision level for more accurate scoring. The top scoring 1229 compounds from both grids contained 82 top-level HierS scaffolds.13
22. Bromodomain expression: BRD4(1) and BRD2(2) bromodomain constructs were based on (Filippakopoulos et al., 2010, Nicodeme et al., 2010) with an added N-terminal His-tag. These constructs were cloned, expressed, and purified by nickel affinity and size exclusion chromatography by either Genscript or Xtal BioStructures, frozen at -80°C for competition experiments. Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay: 200 nM N-terminally His-tagged bromodomains, BRD4(1) or BRD2(2) and 25-50 nM biotinylated tetra-acetylated histone H4 peptide (Millipore) were incubated in the presence of Europium Cryptate-labeled streptavidin (Cisbio Cat. #610SAKLB) and XL665-labeled monoclonal anti-His antibody (Cisbio Cat.#61HISXLB) in a white 96 well microtiter plate (Greiner). For inhibition assays, serially diluted compound was added to these reactions in a 0.2% final concentration of DMSO. Final buffer concentrations were 30 mM HEPES pH 7.4, 30 mM NaCl, 0.3 mM CHAPS, 20 mM PO4 pH 7.0, 320 mM KF, 0.08% BSA). After 2 h incubation at room temperature, the fluorescence by FRET was measured at 665 and 620 nm by a SynergyH4 plate reader (Biotek). Duplicate measurements were obtained at each concentration. IC50 values were determined from a dose response curve.
23. X-ray Crystallography: The recombinant human BRD4(1)10 protein at 1 mg/mL was mixed with 0.6 mM 16a from 100 mM DMSO stock. The complex was incubated at room temperature for 30 min and then concentrated to 4.0 mg/mL using 3000MWCO concentrators. Protein co-crystals were obtained by vapor diffusion with sitting drops against a reservoir solution containing 200 mM Potassium thiocyanate and 20% (W/V) PEG 3350. Selected monocrystals were briefly treated with a cryo-protectant solution containing the well solution supplemented with 20% 2-methyl-2,4-pentanediol and flash-frozen in liquid nitrogen. Apo-BRD4(1) crystals were obtained by vapor diffusion using sitting drop against a reservoir solution containing 10% (W/V) PEG 3350, 100 mM HEPES pH 7.5 and 200 mM l-proline. Well-formed crystals were soaked for 3 h at room temperature with the same reservoir solution supplemented with 20 mM 19d. Soaked crystals were briefly treated with the soaked solution where the PEG3350 concentration was increased to 35% (W/V) and flash-frozen in liquid nitrogen. Single-crystal X-ray diffraction was obtained at Beam line X29 of the National Synchrotron Light Source, Brookhaven National Laboratory, using an automated sample mount system. The X-ray diffraction data were reduced using HKL200022 The BRD4(1)-16a and BRD4(1)-19d crystals both belonged to the space group P212121 and they diffracted to 1.33 Å and 1.60 Å resolution, respectively, (Table S1). The protein structures were solved by molecular replacement and refined using REFMAC523 as previously done.10 Model rebuilding was pursued using COOT24. In each structure, a single compound conformation was observed and refined. The BRD4(1)-19d co-structure was refined to an R/Rfree of 16.0%/19.9% with good stereochemistry. Given the high resolution of the BRD4(1)-16a co-structure, individual anisotropic temperature factors were also refined to an R/Rfree of 11.0/14.8% with good stereochemistry. The final crystallographic data reduction statistics are summarized in Table S2. The structures have been deposited in the PDB with the following codes: 4yh3 and 4yh4.
24. The synthesis of these analogues is exemplified by the preparation of 16e as described below: Step 1: A mixture of 4-methoxybenzaldehyde (1.36 g, 10.0 mmol), 2-(4-methoxyphenyl)acetic acid (1.66 g, 10.0 mmol), acetic anhydride (2.04 g, 20.0 mmol), and triethylamine (1.0 mL, 7.0 mmol) was heated at 140°C under nitrogen for 16 h. The reaction mixture was cooled to room temperature and diluted with ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was recrystallized in ethyl acetate and hexanes to afford (E)-2,3-bis(4-methoxyphenyl)acrylic acid (0.65 g, 23%) as a yellow solid: 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H), 7.17 (dd, J = 6.7, 2.1 Hz, 2H), 7.05 (dd, J = 7.0, 1.9 Hz, 2H), 6.93 (dd, J = 6.7, 2.1 Hz, 2H), 6.71 (dd, J = 7.0, 2.0 Hz, 2H), 3.85 (s, 3H), 3.77 (s, 3H). Step 2&3: To a solution of (E)-2,3-bis(4-methoxyphenyl)acrylic acid (142 mg, 0.5 mmol) in dichloromethane (3 mL) was added oxalyl chloride (0.17 mL, 2.0 mmol). The reaction mixture was stirred at room temperature for 1 h and then concentrated. The residue was dissolved in tetrahydrofuran (2 mL) and was added into an ice cold solution of 3,4-dihydroquinoxalin-2(1H)-one (67 mg, 0.45 mmol) and triethylamine (50 mg, 0.5 mmol) in tetrahydrofuran (7 mL). The mixture was warm to room temperature, stirred for 16 h. The mixture was diluted with ethyl acetate and saturated sodium carbonate (200/50 mL). The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by chromatography (silica gel, 0-40% ethyl acetate/hexanes) to give Example 16e (170 mg, 82%) as an off-white solid: 1H NMR (500 MHz, DMSO-d6) δ 10.57 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.06 (td, J = 7.7, 1.3 Hz, 1H), 7.01-6.99 (m, 4H), 6.94 (td, J = 7.9, 1.3 Hz, 1H), 6.86 (dd, J = 7.9, 1.2 Hz, 1H), 6.81-6.76 (m, 5H), 4.34 (s, 2H), 3.71 (s, 3H), 3.70 (s, 3H); ESI m/z 415 [M+H]+