U.S. patent application number 11/991275 was filed with the patent office on 2009-05-28 for class iia histone deacetylase (hdac) substrate.
This patent application is currently assigned to ISTITUTO DI RICERCHE DI BIOLOGIA MOLECOLARE P. ANG. Invention is credited to Philip Jones, Christian Steinkuhler.
Application Number | 20090136979 11/991275 |
Document ID | / |
Family ID | 35221039 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090136979 |
Kind Code |
A1 |
Jones; Philip ; et
al. |
May 28, 2009 |
Class IIA Histone Deacetylase (HDAC) Substrate
Abstract
The present invention relates to the use of trifluoroacetylated
lysine (Boc-L-Lys(.epsilon.- trifluoroacetyl)-MCA) as a substrate
for histone deacetylases, specific for the class Ha subtype.
##STR00001##
Inventors: |
Jones; Philip; (Rome,
IT) ; Steinkuhler; Christian; (Rome, IT) |
Correspondence
Address: |
MERCK AND CO., INC
P O BOX 2000
RAHWAY
NJ
07065-0907
US
|
Assignee: |
ISTITUTO DI RICERCHE DI BIOLOGIA
MOLECOLARE P. ANG
Pomezia (Rome)
IT
|
Family ID: |
35221039 |
Appl. No.: |
11/991275 |
Filed: |
September 5, 2006 |
PCT Filed: |
September 5, 2006 |
PCT NO: |
PCT/GB2006/050276 |
371 Date: |
February 29, 2008 |
Current U.S.
Class: |
435/19 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
17/00 20180101; A61P 11/00 20180101; C07D 487/04 20130101; C07D
487/08 20130101; A61P 25/00 20180101; A61P 1/16 20180101; C07D
417/12 20130101; A61P 25/16 20180101; A61P 31/04 20180101; A61P
17/06 20180101; C07D 417/14 20130101; A61P 19/02 20180101; A61P
25/18 20180101; A61P 1/12 20180101; A61P 7/06 20180101; A61P 35/00
20180101; C07D 333/38 20130101; A61P 27/02 20180101; A61P 43/00
20180101; A61P 9/10 20180101; A61P 17/02 20180101; A61P 29/00
20180101; C07D 409/12 20130101; A61P 33/00 20180101; C07D 471/04
20130101; A61P 5/14 20180101; A61P 31/12 20180101; A61P 9/00
20180101; A61P 31/18 20180101; A61P 37/02 20180101; C07D 409/14
20130101; A61P 19/08 20180101; C07D 333/34 20130101; C12Q 1/34
20130101; A61P 1/04 20180101; A61P 35/02 20180101; A61P 7/04
20180101; A61P 9/08 20180101; A61P 9/04 20180101; A61P 25/28
20180101; C07D 413/12 20130101; A61P 21/04 20180101; A61P 37/06
20180101; C07D 413/14 20130101; C07D 495/04 20130101; A61P 13/12
20180101; C07D 409/08 20130101; A61P 37/08 20180101; C07D 491/10
20130101; C07D 277/56 20130101 |
Class at
Publication: |
435/19 |
International
Class: |
C12Q 1/44 20060101
C12Q001/44 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 7, 2005 |
GB |
0518237.3 |
Claims
1. (canceled)
2. (canceled)
3. An assay for determining the concentration of a Class IIa HDAC
in a sample which comprises: (i) incubating said sample suspected
of containing said Class IIa HDAC with trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA); (ii) adding a suitable
enzyme capable of cleaving trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) developer and (iii)
measuring the concentration of said enzyme as a determinant of the
concentration of said Class IIa HDAC in said sample.
4. The assay for detecting Class IIa HDAC in a test sample,
comprising the steps of: (i) incubating said test sample
potentially containing Class IIa HDAC with suitable amounts of
trifluoroacetylated lysine (Boc-L-Lys
(.epsilon.-trifluoroacetyl)-MCA); and (ii) detecting the presence
of MCA by its fluorescence.
5. The assay for identifying a potential Class IIa HDAC inhibitor,
which comprises: (i) incubating a potential Class IIa HDAC
inhibitor with nucelci acid molecule under conditions favoring
expression a protein comprising a catalytic domain of Class IIa
HDAC; (ii) adding suitable amounts of trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) and an enzyme under
conditions favoring cleavage of said trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA); and (iii) determining
concentration of a cleavage product as a determinant of an HDAC
inhibitor relative to a control sample that does not contain said
inhibitor.
6. The assay according to claim 5 wherein said enzyme is selected
from the group consisting of BIOMOL Fluor De Lys, Trypsin and
Lys-C.
7. The assay according to claim 5 wherein said Class IIa HDAC
enzyme is present at a concentration of 0.05-50 nM.
8. The assay according to claim 7 wherein the assay is performed at
a pH value between 6.0 and 8.5.
9. The assay according to claim 8 further comprising monovalent
and/or divalent cations.
10. The assay according to claim 9 further comprising bovine serum
albumin.
11. The assay according to claim 10 wherein the substrate
trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) is added at a
concentration range between 1 .mu.M and 100 .mu.M.
12. The assay according to claim 11 wherein the protein which
expresses the catalytic domain of Class IIa HDAC is E coli.
13. The assay according to claim 12 wherein the Class IIa HDACs is
HDAC4.
Description
[0001] The present invention relates to a substrate for histone
deacetylases, specific for the class IIa subtype that is suitable
for use in an assay for screening for HDAC subtype-selective
inhibitors.
[0002] In eukaryotic cells the orderly packaging of DNA in the
nucleus plays an important role in the regulation of gene
transcription. Nuclear DNA is ordered in a compact complex called
chromatin. The core of the complex is an octamer of highly
conserved basic proteins called histones. Two each of histones H2A,
H2B, H3 and H4 associate and DNA winds around the basic amino acids
of the histones interacting with the negatively charged phosphate
groups of the DNA. One molecule of histone H1 is associated with
each wound core which accommodates approximately 146 bp of DNA. The
cores are, in turn, packaged into a compact regular structure with
about 200 bp of DNA between each core.
[0003] The amino-terminal tails of the histones are subject to
post-translational modification, in particular by acetylation of
lysine. Histone deacetylases (HDACs) and histone acetyl
transferases (HATs) determine the pattern of histone acetylation,
which together with other dynamic sequential post-translational
modifications might represent a `code` that can be recognised by
non-histone proteins forming complexes involved in the regulation
of gene expression. This and the ability of histone deacetylases
(HDACs) to also modify non-histonic substrates and participate in
multi-protein complexes contributes to the regulation of gene
transcription, cell cycle progression and differentiation, genome
stability and stress responses.
[0004] Eleven members of the Zn-dependent HDAC family have been
identified in humans, which share a conserved catalytic domain and
are grouped into two classes: class I (1,2,3,8), homologous to
yeast Rpd3; class IIa (4, 5, 7, 9) and IIb (6, 10), homologous to
yeast Hda1. HDAC11 shares homologies with both classes, but is at
the same time distinct from all the other ten subtypes. Interest in
these enzymes is growing because HDAC inhibitors (HDACi) are
promising therapeutic agents against cancer and other diseases. The
first generation of HDACi were discovered from cell-based
functional assays and only later identified as HDAC class I/II
inhibitors. Present HDAC inhibitors are pan-specific or poorly
selective. Those that entered clinical trials all show similar
adverse effects, mainly fatigue, anorexia, hematologic and
GI-toxicity, that becomes dose-limiting in clinical trials. It is
not at all clear whether the antitumor properties of HDAC
inhibitors are due to their lack of specificity or are the
consequence of hitting one or few "crucial" subtypes. This question
is of considerable interest because it may open the way for the
development of novel, more sensitive compounds with possibly
enhanced efficacy and/or tolerability. More recent studies were
therefore directed to better define the biological function of
different class members and to devise subtype-selective enzymatic
assays to assist in the development of improved cancer
chemotherapies.
[0005] The class IIa HDACs contain a highly conserved C-terminal
catalytic domain (.about.420 amino acids) homologous to yHDA1 and
an extended N-terminal domain that is able to interact with
several, different proteins, including transcription factors,
transcriptional co-repressors and 14-3-3 proteins. The activity of
the class IIa HDACs is regulated at several levels, including
tissue-specific gene expression, recruitment of distinct cofactors
and nucleocytoplasmic shuttling. Whereas most class I HDACs are
ubiquitously expressed, class IIa HDACs are expressed in a
restricted number of cell types.
[0006] Riester et al., Biochem. Biophys. Res. Comm., (2004)
324:1116-1123 and Wegener et al, Chem. Biol. (2003) 10:61-68,
describe the substrate specificities of different HDACs (rat liver
HDAC, HDAC8) towards using short fluorogenic substrates. HDAC from
rat liver (which according to immunochemical analysis is a mixture
of HDAC1, 2 and 3) was shown to have a very low sequence
specificity and was able to work also on a substrate composed only
of an acetylated lysine (Boc-L-Lys(.epsilon.-acetyl)-MCA;
K.sub.M=3.7 .mu.M). For (recombinant) human HDAC8, no good
substrates were reported at the time of the paper. This was true
for an H4-derived peptide, as well as for other synthetic
substrates. With Boc-L-Lys(.epsilon.-acetyl)-MCA or
Ac-Arg-Gly-L-Lys(.epsilon.-acetyl)-MCA, for example, a
K.sub.m.gtoreq.500 .mu.M was published.
[0007] In Riester et al. various modifications of .epsilon.-acetyl
moieties of L-Lysine were also studied. In particular,
Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA proved to be a superior
substrate for HDAC8 with K.sub.M value (K.sub.M=247 .mu.M)
significantly improved as compared to
Boc-L-Lys(.epsilon.-acetyl)-MCA. Rat liver HDAC did not convert
Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA to a significant
degree.
[0008] It has now surprisingly been found that trifluoroacetylated
lysine (Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) is a superior
substrate for ClassIIa HDACs.
[0009] Thus, in a first aspect of the invention there is provided
the use of trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) as a substrate for class
IIa histone deacetylases.
[0010] In another aspect there is provided a complex of
trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) bound to a Class IIa
HDAC.
[0011] In another aspect there is provided an assay for determining
the concentration of a Class IIa HDAC in a sample which comprises:
[0012] (i) incubating a sample containing a Class IIa HDAC with the
substrate trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA); [0013] (ii) adding a
developer and measuring the concentration by the fluorescence (ex
360 nM, em 460 nM).
[0014] Preferably, the developer is a commercially available kit
component (BIOMOL Fluor De Lys) or a proteolytic enzyme such as
Trypsin or Lys-C that will cleave the bond between the deacetylated
lysine and the MCA fluorophore, thereby liberating a fluorescent
compound. This assay is capable of determining the sub- nanomolar
concentration of the enzyme.
[0015] In another aspect is provided an assay for detecting Class
IIa HDAC which comprises: [0016] (i) incubating a sample
potentially containing Class IIa HDAC with the substrate
trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA); and [0017] (ii)
detecting the presence of MCA by its fluorescence.
[0018] In an embodiment this assay can be used to specifically
detect HDAC 4.
[0019] In another aspect there is provided an assay specific for
Class IIa HDAC inhibitors, which comprises: [0020] (i) incubating a
potential Class IIa HDAC inhibitor with a protein which expresses
the catalytic domain of Class IIa HDAC, in a suitable assay buffer;
[0021] (ii) adding the substrate trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) and a developer and
incubating; and [0022] (iii) stopping the incubation and
determining the effect the putative Class IIa HDAC inhibitor has on
enzyme activity by comparison with a standard.
[0023] Preferred Class IIa HDACs are HDAC4, HDAC5 and HDAC7,
particularly HDAC4.
[0024] Preferably, the protein which expresses the catalytic domain
of Class IIa HDAC is E coli, particularly strain BL21.
[0025] Preferable, the concentration of the Class IIa HDAC enzyme
in the assay for screening for inhibitors is 0.05-50 nM preferably
0.5 nM.
[0026] Preferably, the assays of the present invention can be
performed at a pH value between 6.0 and 8.5, most preferably at pH
7.5. A preferred buffer used for assuring pH stability during the
assay is Hepes at a concentration of 20 mM.
[0027] In an embodiment, monovalent and/or divalent cations can be
added to the assays of the present invention, preferable at
millimolar concentrations. Preferred cations are Na, K and Mg.
[0028] Preferably, the cations are present in a concentration of
0.5 mM to 200 mM.
[0029] Preferred salts which provide the cations are NaCl, KCl and
MgCl.sub.2.
[0030] In an embodiment NaCl is present at a concentration of
100-150, particularly 137 mM.
[0031] In another embodiment KCl is present at a concentration of
0.5-10, particularly 2.7 mM.
[0032] In another embodiment MgCl.sub.2 is present in a
concentration of 0.5-5, particularly 1 mM.
[0033] In an embodiment, a protein such as bovine serum albumin is
added to the assay buffer. Preferable, the bovine serum albumin is
present at a concentration of 0.01 and 10 mg/ml, particularly at
0.1 mg/ml.
[0034] The substrate trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) can be added in a
concentration range between 1 .mu.M and 100 .mu.M, preferably at 20
or 40 .mu.M
[0035] The assays of the present invention can be carried out at a
temperature of about 20 to 37.degree. C., preferably about
37.degree. C.
[0036] In a preferred embodiment is an assay specific for Class IIa
HDAC inhibitors, which comprises: [0037] (i) incubating a potential
Class IIa HDAC inhibitor with a protein which expresses the
catalytic domain of Class IIa HDAC, in a suitable assay buffer to
maintain a pH of 6.0 to 8.5; [0038] (ii) adding the substrate
trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA) to produce a
concentration of 1 .mu.M and 100 .mu.M in the sample; BIOMOL Fluor
De Lys, Trypsin or Lys-C; and a developer and incubating; and
[0039] (iii) stopping the incubation and determining the effect the
putative Class IIa HDAC inhibitor has on enzyme activity by
comparison with a standard.
EXAMPLE 1
The Deacetylating and Enzymatic Activity of HDAC4
[0040] The deacetylating activity of HDAC4, a class IIa histone
deacetylase, was analysed by incubating different concentrations of
C-terminally flagged HDAC4 (HDAC4-FLAG; 3-24 nM) with a histone
substrate (histone H4-derived octamer peptide substrate containing
a single acetylated lysine). FIG. 1 shows the activity at different
concentrations as a percentage conversion of the acetylated
substrate. The higher the concentration of the HDAC4-FLAG, the
higher the deacetylating activity. The enzymatic activity of
HDAC4-FLAG was measured in a conventional assay kit (Biomol) and
with histone substrates by measuring its inhibition by Apicidin,
MS27-275 and by LAQ824 (FIG. 2).
[0041] A cross-linking assay to analyze the binding of HDAC4-FLAG
to the inhibitors was performed. The only compound able to
significantly and specifically bind HDAC4 was LAQ824. Apicidin and
MS25-275 were unable to bind the deacetylase, whilst showing good
inhibition constants (in particular Apicidin with a low nanomolar
IC.sub.50) (FIG. 2). A possible explanation of this result could be
that HDAC4-FLAG does not significantly contribute to the observed
enzymatic activity, which rather reflects the involvement of one or
more active endogenous co-purified HDACs. Infact, histone
deacetylases expressed in mammalian cells interact with several
endogenous HDACs.
[0042] To further analyze this point, the catalytic domain of HDAC4
(6xHis tagged) was expressed in E. coli. The HDAC4 C-terminal
region from T-653 to L-1084, containing therefore the entire
catalytic domain (HDAC4-CD), was produced, purified to homogeneity
and the enzymatic activity analyzed with the histone-based (FIG.
3A) and the commercial Biomol HDAC assay (FIG. 3B; RFU,
Fluorescence Units).
[0043] The conclusions which can be drawn from these experiments
are that: [0044] (a) HDAC4-CD enzymatic activity was about 100-fold
lower (micromolar amounts of enzyme) than that measured with
flagged-HDAC4 in transfected mammalian cells, thus suggesting that
the pure enzyme is a very weak deacetylase in the absence of
interacting endogenous enzymes; and [0045] (b) HDAC4-CD was
resistant to the inhibitory effect of Apicidin, in contrast with
the sensitivity shown by flagged-HDAC4-associated activity and in
agreement with its incapability of binding this compound
EXAMPLE 2
The Enzymatic Activity of HDACs 1, 3 and 8
[0046] A panel of HDAC inhibitors on class I HDACs 1, 3 and 8 was
tested in an analogous manner to the HDAC4 (see Vannini et al.,
(2004) Proc. Natl. Acad. Sci. U.S.A., 101:15064-15069). The results
showed that HDAC8 has a distinct inhibition pattern, which differs
from that of HDAC1 and HDAC3. Whereas HDACs 1 and 3 were inhibited
by all of the inhibitors, HDAC8 was inhibited not appreciably by
Apicidin or MS27-275 (see FIG. 4). Thus, HDAC8 shows a more
restricted inhibition pattern than other class I enzymes.
EXAMPLE 3
Preparation of tert-Butyl
[(1S)-5-(trifluoroacetylamino)-1-[[(4-methyl-2-oxo-2H-1-benzopyran-6-yl)
amino]carbonyl]pentyl]-carbamic Acid Ester (Trifluoroacetylated
Lysine (Boc-L-Lys (.epsilon.-trifluoroacetyl)-MCA))
##STR00002##
[0048] To a solution of Boc-Lys-AMC (1 equivalent, BACHEM: I-1880)
in DCM and Et.sub.3N (2 equivalents) at RT was added dropwise
trifluoroacetic anhydride (1.2 equivalents). The resulting mixture
was stirred at RT for 2 hours and was then diluted with DCM, washed
with saturated aqueous NaHCO.sub.3 solution and brine. The solution
was dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure whilst dry loading onto silica. The mixture was columned
on silica eluting with 70-80% EtOAc/petroleum ether to yield the
desired amide. .sup.1H NMR (300 MHz, d6-DMSO) .delta.: 10.40 (1H,
br. S), 9.39 (1H, Br. S), 7.76 (1H, d, J=1.6 Hz), 7.72 (1H, d,
J=8.4 Hz), 7.48 (1H, d, J=8.4 Hz), 7.10 (1H, d, J=7.6 Hz), 6.62
(1H, s), 4.10-4.00 (1H, m), 3.21-3.10 (2H, m), 2.38 (3H, s),
1.70-1.55 (2H, m), 1.54-1.43 (2H, m), 1.40-1.25 (2H, m), 1.37 (9H,
s). MS (ES) C.sub.23H.sub.28F.sub.3N.sub.3O.sub.6 requires: 499,
found: 500 (M+H).sup.+.
EXAMPLE 4
Comparing Activities on Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA
with Activities on the Biomol Standard Substrate
[0049] The activities measured on
Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA were compared with
activities on the Biomol standard substrate (not shown). It was
found that HDAC4-CD and HDAC8 work better on the Lys-Trifluoro
substrate, HDAC3 works with the same efficiency and HDAC1 and HDAC6
did not work on this substrate (see FIG. 5).
[0050] An enzyme titration was performed on the HDAC4-CD enzyme,
with a concentration of 0.5 nM being sufficient for the assay on
the new substrate (data not shown) and which was about a thousand
fold less than the amount needed for the commercial Biomol assay.
The activity of HDAC4-CD (0.5 nM) on Lys-Trifluoro substrate is
inhibited by LAQ824 and resistant to Apicidin, as shown in FIG.
6.
[0051] The results obtained on the Biomol substrate (HDAC4-CD=500
nM) are also shown in FIG. 6.
[0052] The experiments supra described with the Lys-Trifluoro
substrate were done with the HDAC4catalytic domain (HDAC4-CD). To
test the behavior of the complete protein, the full length
HDAC4protein (HDAC4-FLAG) was also analysed. Other class IIa HDACs,
namely HDAC 5 and 7, were also tested, together with HDAC1, HDAC3
and HDAC8 as controls. After transfection in mammalian cells, the
proteins were purified and analyzed. In these conditions associated
HDACs did not significantly contribute to the activity, since very
low (nanomolar) amounts of enzyme were used in the assay. Results
are shown in FIG. 7 and FIG. 8.
EXAMPLE 5
HDAC4 Assay
[0053] The following assay was used to screen for potential HDAC4
inhibitors and measure their activity.
HDAC 4 Expression and Affinity Purification
[0054] The His-tagged HDAC 4, wild-type catalytic domain, was
expressed in E. coli strain BL21 Star.TM. (DE3). The cells were
grown at 37.degree. C. in minimum medium supplemented with 1 g/l
(.sup.15NH.sub.4).sub.2SO.sub.4 and 5 g/l glucose, and 100 .mu.M of
ZnCl.sub.2 to an optical density of 0.8 at 600 nm and induced with
IPTG for 16 hr at 23.degree. C. At 23.degree. C. more than 80% of
the protein was soluble.
[0055] Bacterial pellets were resuspended in 25 mM Hepes pH 7.5,
200 mM KCl, 0.5% NP-40, 20% glycerol 1 mM DTT and supplemented with
Complete EDTA-free protease inhibitor. Subsequently bacterial
pellets were lysed by microfluidizer, and centrifuged at 15000 rpm
for 30 min.
[0056] The soluble fraction was diluted 1:1 with 25mM Hepes pH 7.5,
200 mM KCl, 1 mM DTT and was loaded directly on His Trap HP 5 ml
(Amersham Biosciences). The protein was eluted at 200 mM Imidazole.
The fractions with HDAC 4 were diluted 1:3 with 25 mM Hepes pH 7.5,
5% glycerol, 0.1% of NP-40, 1 mM DTT. Then the solution was loaded
on a Resource Q equilibrated with 25 mM Hepes pH 7.5, 10% glycerol,
50 mM KCl, 0.1% of NP-40, 1 mM DTT. HDAC 4 was eluted with a salt
gradient (0-250) mM of KCl. The product was fractionated by
preparative SEC (G-75, Superdex 75 26/60 Amersham Biosciences) (25
mM Hepes pH 7.5, 150 mM KCl, 0.1% of .beta.-octyl glucopiranoside,
1 mM DTT) to give the final product. Analytical SEC indicated that
this product was monomeric. The protein was concentrated at
.apprxeq.100 .mu.M.
HDAC 4 Assay
Working Reagents
[0057] TSA Stock: TSA is provided as a 10 mM solution in 100% DMSO.
[0058] Assay buffer: 25 mM Tris/HCl pH8, 137 mM NaCl, 2.7 mM KCl, 1
mM MgCl.sub.2, 0.1 mg/ml BSA [0059] Diluted substrate solution:
tert-butyl
{(1S)-1-{[(4-methyl-2-oxo-2H-chromen-7-yl)amino]carbonyl}-5-[(trifluoroac-
etyl) amino]pentyl} carbamate (trifluoroacetylated lysine
(Boc-L-Lys(.epsilon.-trifluoroacetyl)-MCA)) is diluted to 200 .mu.M
with Tris 1 mM pH 7.4 prior to each use. The final concentration in
the assay is 20 .mu.M. [0060] Diluted developer solution: The
commercial 20X developer concentrate (KI-105, BioMol Research
Laboratories) is diluted 1:167 into Tris 1 mM pH7.4. 2 .mu.M
[final] TSA to this solution increases its ability to stop the
reaction. [0061] Enzyme working solution: Enzyme is diluted in
1.25.times. assay buffer prior to each use from a fresh aliquot of
enzyme. The final concentration in the assay is 0.2 nM.
Experimental Design:
[0062] The reaction is performed in 96-well microplate in a final
volume of 50 .mu.l/well. Add 5 .mu.l of DMSO/compound solution, add
40 .mu.l of HDAC 4 enzyme in assay buffer and incubate 10' at RT.
Start the reaction by adding 5 .mu.l of the 200 .mu.M substrate
solution and incubate 1 hr at 37.degree. C. Stop the reaction by
adding 50 .mu.l of developer/4 .mu.M TSA solution and incubate 30
min at RT. Measure the fluorescence at ex.360 nM and em.460 nM.
* * * * *