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this assay avoids the use of radiolabeled reagents, the
artificial substrate may not reveal catalysis by many
HDACs and the requisite extraction step is not condu-
cive to HTS. The most widely used in vitro HDAC assay
for drug discovery employs an e-acetylated lysine based
substrate that is C-terminally coupled with a 4-methyl-
coumarin-7-amide residue.16,17 Once deacetylated, tryp-
sin recognizes the product as a substrate and releases 7-
amino-4-methylcoumarin (AMC). Free AMC, in con-
trast to acylated AMC, is highly fluorescent and easily
monitored with a fluorescence microtiter plate reader.18
Although the assay is readily adapted to HTS it does
have some drawbacks. Most importantly, the artificial
substrate is not recognized by all HDACs, and many
HDACs require prolonged incubation times due to
low catalytic turnover.19 Also, trypsin inhibitors are of-
ten identified as false positives in an HTS campaign with
this assay.25
Direct binding of 1 to purified Class I HDAC3/NcoR2,
Class II HDAC6 or crude HeLa nuclear extract was
measured by a change in FP. The fraction of bound 1
relative to protein concentration is plotted in Figure 1.
The binding isotherm data were analyzed by nonlinear
regression to calculate the dissociation equilibrium con-
stant (Kd).21,22 The binding affinity of 1 to HDACs with-
in the HeLa cell nuclear extract is a composite value
based upon both Class I and Class II HDACs. Since
the relative HDAC concentration and composition
within the HeLa nuclear extract is unknown, the equilib-
rium Kd could not be calculated; however, half maximal
binding was seen at 247.6 1.2 lg/mL. Compound 1
bound tightly to both HDAC3/NcoR2 and HDAC6
with a Kd = 2.0 0.2 and 4.6 0.2 nM, respectively.
A displacement assay was then used to assess the affinity
of various known HDAC inhibitors to HDACs within
HeLa nuclear extract as well as purified HDAC3/NcoR2
and HDAC6. Protein was diluted in assay buffer and 1
was added to 2 nM. Increasing concentrations of 2,
apicidin, TSA, FK228, or SAHA were added to the
HDAC/1 complex and the fluorescence anisotropy was
measured at equilibrium as described in the supporting
information. Half maximal inhibitory concentrations
(IC50) were determined by non-linear regression analysis
and the dissociation constant for inhibitor binding (Ki)
was calculated (Table 1).23 As expected, the Class III
specific HDAC inhibitor splitomicin did not displace
1. The binding affinity for SAHA, Apicidin, TSA, and
FK228 correlate well to the IC50 values determined by
the fluorogenic substrate assay.18
This report describes a single-step, fluorescence polari-
zation (FP) based displacement assay that is suitable
for HTS of compounds targeting HDACs. While the
HDAC assays described above measure the inhibition
of HDAC activity, this assay detects molecules that
compete for the substrate binding site. The assay mea-
sures ligand binding under equilibrium conditions and
does not suffer from product inhibition. The amount
of HDAC required per well is comparable to the amount
of HDAC used with the fluorogenic substrate. The Ki
values for various literature reported HDAC inhibitors
correlate well with the IC50 values derived from enzy-
matic turnover screens.
We have designed the FP-HDAC probe 1 based on the
unselective HDAC inhibitor M344,20 which has been
coupled through a short linker element to FITC. The
synthesis was accomplished in eight steps starting from
commercially available 7-amino heptanoic acid in 40%
overall yield (Scheme 1).
The adaptability of the FP based 1 displacement assay
for HTS against human HDAC3/NcoR2 and HDAC6
was assessed using a statistical measure described by
Zhang et al.24 A Z-factor between 0.5 and 1 is generally
favorable for a HTS assay. The minimum amount of
HDAC protein required to yield a statistically signifi-
O
O
O
O
a
b,c
H2N
H2N
OH
H2N
OMe
N
H
OMe
3
4
6
d
O
O
O
O
e
N
OMe
N
H
OMe
H
N
H
N
H
5
2
N
N
N
H2N
f
O
O
O
O
S
OH
g
OH
N
N
H
N
H
N
H
HN
O
N
H
N
N
H
N
H
N
H
N
N
N
N
OH
H2N
1
O
OH
O
Scheme 1. Synthesis of fluorescence polarization assay probe 1. Reagents and conditions: (a) methanol, SOCl2, rt, 5 h; (b) 4-nitrobenzoic acid,
isobutyl chloroformate, N-methylmorpholine, THF, 0 °C to room temperature; (c) MeOH, Pd/C, H2; (d) propargyl bromide, K2CO3, DMA, 16 h,
50 °C; (d) Cu(MeCN)4PF6, ligand, 3-azidopropyl amine, MeCN; (e) KCN, H2NOH, MeOH, THF; (f) FITC, DMF.