2
F. F. Wagner et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(86% and 95%, respectively). Binding kinetics represent key and
requisite parameters to consider when developing therapeutics8 to
understand and define the target engagement–pharmacodynamic
(PD) response relationships. At one extreme, prolonged target
engagement (slow-off or pseudo-irreversible inhibitors), as observed
for the HDAC1,2 inhibitor compound 609 (Supplemental Table S1),
may be desirable to affect a prolonged PD response to drive effi-
cacy. Alternatively, prolonged target engagement across multiple
HDAC isoforms may drive mechanism-based toxicity, such as
thrombocytopenia, and decrease the therapeutic window.10 By
developing a structure–kinetic relationship (SKR)11 for inhibitors
towards HDAC1 and HDAC2, the development of kinetically differ-
ent binding parameters for each isoform will define optimal target
engagement to maximize the PD response and therapeutic index.
compounds detected by SIR detection on a single quadrupole mass
spectrometer.
2.4. Microsomal stability
Microsomal stability was determined at 37 °C at 60 min in
mouse microsomes. Each compound was prepared in duplicate at
1 lM with 0.3 mg/mL microsomes in PBS pH 7.4 (1% DMSO). Com-
pounds were incubated at 37 °C for 60 min with a 350-rpm orbital
shake with time points taken at 0 min and 60 min. Samples were
analyzed by UPLC–MS (Waters, Milford, MA) with compounds
detected by SIR detection on
spectrometer.
a single quadrupole mass
2.5. Neuronal histone acetylation assays
2. Material and methods
2.1. Chemistry
Measurements of increases in neuronal histone acetylation in
mouse fore-brain primary neuronal cultures induced by HDAC
inhibitor compounds was performed exactly as described in Fass
et al.12 On the 13th day after generating the cultures, cells were
See SI for details. Representative procedure for the synthesis of
ortho-aminoanilide analogs is detailed in Supporting information.
All final compounds were confirmed to be of >95% purity based
on HPLC LCMS analysis (Alliance 2795, Waters, Milford, MA). Purity
was measured by UV absorbance at 210 nm. Identity was deter-
mined on a SQ mass spectrometer by positive and negative electro-
spray ionization. All reagents and solvents were purchased from
commercial vendors and used as received. 1H and 13C NMR spectra
were recorded on a Bruker 300 MHz or Varian UNITY INOVA
500 MHz spectrometer as indicated. Proton and carbon chemical
shifts (d) are reported in ppm relative to tetramethylsilane (d 0
for both 1H and 13C) and DMSO-d6 (1H d 2.50, 13C d 39.5). NMR data
were collected at 25 °C. Flash chromatography was performed
treated for 24 h with compounds at 10 lM. Cells were fixed with
formaldehyde, stained with antibodies to acetyl-histone H3, lysine
9 (AcH3K9), or acetyl-histone H4, lysine 12 (AcH4K12), and green
fluorescent secondary antibodies, and cellular fluorescence signals
were quantitated using laser-scanning cytometry (Acumen eX3,
TTP Laptech). To determine the efficacy of HDAC inhibitor com-
pounds, we calculated the percentage of compound-treated cells
with a fluorescence signal above a baseline threshold established
in vehicle (DMSO) treated cells.
2.6. Protein purification and crystallization
Protein was produced essentially as described in Bressi et al.13
Briefly, C-terminally His-tagged full-length protein was expressed
in insect cells und purified by affinity and size exclusion chro-
matography. The protein was C-terminally truncated by treatment
with trypsin for 1 h at 25 °C. The reaction was stopped by addition
of 1 mM PMSF and the protein finally purified by gel filtration
using 40–60 lm Silica Gel (60 Å mesh) on a Teledyne Isco Combi-
flash Rf system.
2.2. HDAC inhibition assays
All HDACs were purchased from BPS Bioscience. The substrates,
Broad Substrate A, and Broad Substrate B, were synthesized in
house.21 All the other reagents were purchased from Sigma. Caliper
EZ reader II system was used to collect all data. Purified HDACs
chromatography. For crystallization 0.5
at 12 mg/ml, containing the ligand at a concentration of 2 mM,
was mixed with 0.5 L of the crystallization buffer (34% PEG400,
lL of the protein solution
l
0.10 M NaKPO4 pH 6.50, 0.20 M KF). Crystals were harvested in a
cryo-loop and frozen directly in liquid nitrogen.
were incubated with
2 lM carboxyfluorescein (FAM)-labeled
acetylated or trifluoroacetylated peptide substrate (Broad Sub-
strate A and B respectively) and test compound for 60 min at room
temperature, in HDAC assay buffer that contained 50 mM HEPES
(pH 7.4), 100 mM KCl, 0.01% BSA and 0.001% Tween-20. Reactions
were terminated by the addition of the known pan HDAC inhibitor
2.7. Data collection, structure solution and refinement
Data collection was performed with synchrotron radiation
at the Swiss Light Source, Paul-Scherrer-Institute, Villingen,
Switzerland. Crystals belonged to space group P212121 with cell
LBH-589 (panobinostat) with a final concentration of 1.5 lM. Sub-
strate and product were separated electrophoretically and fluores-
cence intensity in the substrate and product peaks was determined
and analyzed by Labchip EZ Reader. The reactions were performed
in duplicate for each sample. IC50 values were automatically calcu-
lated by Origion8 using 4 Parameter Logistic Model. The percent
inhibition was plotted against the compound concentration, and
the IC50 value was determined from the logistic dose–response
curve fitting by Origin 8.0 software.
dimensions a = 92.30 Å, b = 98.01 Å and c = 139.25 Å,
a = b = c = 90°.
X-ray intensities and data reduction were evaluated by using
the XDS program package14 (see Table S3 for data collection
and processing statistics). Structures were solved and refined
with programs of the CCP4 suite (MOLREP and REFMAC5)15 (see
Table S4 for refinement statistics).
3. Results and discussion
2.3. Plasma stability
We previously reported the discovery of highly ligand efficient
carbamide (sp3-linked) HDAC inhibitors, such as BRD4884 (Fig. 1),
with excellent potency and moderate kinetic selectivity for
HDAC2.16 Following an initial phase (ꢀ1 h) of good kinetic selectiv-
ity for HDAC1, driven by faster on-rate, BRD4884 then displays a 7-
fold kinetic bias towards HDAC2 versus HDAC1 with residence
times of 142 min and 20 min, respectively. In order to obtain a
Plasma stability was determined at 37 °C at 5 h in mouse
plasma. Each compound was prepared in duplicate at 5 lM in
plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile,
0.05% DMSO). Compounds were incubated at 37 °C for 5 h with a
350-rpm orbital shake with time points taken at 0 h and 5 h.
Samples were analyzed by UPLC–MS (Waters, Milford, MA) with