Communications
DOI: 10.1002/anie.200703198
Microarrays
Fluorous-Based Small-Molecule Microarrays for the Discovery of
Histone Deacetylase Inhibitors**
Arturo J. Vegas, James E. Bradner, Weiping Tang, Olivia M. McPherson, Edward F. Greenberg,
Angela N. Koehler, and Stuart L. Schreiber*
Reversible acetylation plays a key role in defining chromatin
states and in regulating transcription from genomic DNA
differentially across distinct tissues.[1–3] Histone deacetylases
(HDACs) function in this process by catalyzing the hydrolysis
of N-acetyl groups on lysine residues found in the N-terminal
tails of histone proteins.[4] This process mediates cell differ-
entiation, correlates with epigenetic inheritance, and is
deregulated in human disease, among others.[1–3,5]
Identifying novel HDAC inhibitors is an increasingly
active area of research.[6–10] Trapoxin, which is a naturally
occurring HDAC inhibitor, was instrumental in the original
discovery of HDAC1.[4] Suberoylaniline hydroxamic acid
(SAHA/vorinostat), which inhibits multiple members of the
HDAC family of enzymes, has been approved recently for the
treatment of cutaneous T-cell lymphoma.[11–13] Tubacin, which
is the first homologue-selective inhibitor (having selectivity
for HDAC6), has illuminated the function of HDAC6 and
tubulin acetylation.[14–19]
reported uses of SMMs to identify new HDAC inhibitors,
including ones having selectivity for specific members of the
HDAC family. Traditional SMMs use various chemistries to
attach compounds covalently.[20–25] Many of these approaches
either take advantage of latent functionalities that result in
heterogeneous molecular display on the surface, or require
synthetic modification of compounds to obtain homogeneous
display. Fluorous tags are versatile tagging groups for
chemical-library synthesis and can facilitate noncovalent
immobilization on fluorinated glass surfaces.[26–31,37] A pre-
vious report from Pohl and co-workers demonstrated fluorous
microarrays as a powerful screening tool for carbohydrate-
binding proteins.[26] Herein, we demonstrate that fluorous-
based SMMs enable screening for HDAC inhibitors by
allowing controlled molecular display of inhibitory function-
ality, low uniform background signals, and excellent signal-to-
noise ratios.
SMMs were evaluated as a tool for identifying HDAC
Biochemical, enzyme-activity assays involving fluorescent
readouts are frequently used to identify new HDAC inhib-
itors. However, this approach requires expensive reagents and
equipment, and can be difficult to perform in a high-
throughput manner. Small-molecule microarrays (SMMs)
provide an attractive alternative for high-throughput iden-
tification of HDAC inhibitors. Currently, there are no
binders or inhibitors by using a three-part validation
(Figure 1). Quantitative fluorescence data were collected
from probed arrays and used to generate a list of positives.
Non-fluorous tagged equivalents of the compounds were then
tested in a fluorescence-based biochemical activity assay with
the same set of enzymes to determine enzymatic inhibition.
Furthermore, thermodynamic and kinetic binding data were
collected for non-fluorous-tagged compounds binding to one
of the HDACs by using surface plasmon resonance (SPR)
[*] A. J. Vegas, Dr. J. E. Bradner, O. M. McPherson, E. F. Greenberg,
Dr. A. N. Koehler, Prof. Dr. S. L. Schreiber
Howard HughesMedical Institute
Chemistry and Chemical Biology
Harvard University
Broad Institute of Harvard and MIT
7 Cambridge Center
Cambridge, MA 02142 (USA)
Fax: (+1)617-324-9601
E-mail: stuart_schreiber@harvard.edu
Prof. Dr. W. Tang
School of Pharmacy
University of Wisconsin
777 Highland Avenue
Madison, WI 53705 (USA)
[**] We would like to thank Dr. Kara Herlihy, Dr. Ralph Mazitschek, Dr.
Carlos Tassa, Jason Fuller, Dr. Steve Haggarty, Dr. Jianping Cui, Dr.
Letian Kuai, Dr. Marvin Yu, and Dr. Philip Yeske (Fluorous
Technologies) for reagents or comments. Work described herein has
been funded in whole or in part with Federal fundsfrom the
National Cancer Institute’s Initiative for Chemical Genetics,
National Institutes of Health, under contract no. N01-CO-12400.
Supporting information for thisarticle isavailable on the WWW
Figure 1. Experimental approach to validating the use of fluorous-based
SMMsfor HDAC inhibitor discovery.
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 7960 –7964