Bioorganic & Medicinal Chemistry Letters 10 (2000) 559±562
Fluoresceinated FKBP12 Ligands for a High-Throughput
Fluorescence Polarization Assay
Gene M. Dubowchik,* Jonathan L. Ditta, John J. Herbst, Sagarika Bollini
and Alexander Vinitsky
Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, PO Box 5100, Wallingford, CT 06492-7660, USA
Received 19 November 1999; accepted 17 January 2000
AbstractÐSeveral ¯uoresceinated FKBP12 ligands have been prepared for a high-throughput ¯uorescence polarization assay. Kis
for FKBP12 rotamase inhibition by these ligands range from 1.3 mM to 32 nM, and their design is based on X-ray crystal structures
of FKBP12 complexed with known immunophilin ligands. # 2000 Elsevier Science Ltd. All rights reserved.
FKBP12 is the most prominent member of a growing
family of FK506 binding proteins (FKBPs) that are
known to be involved in many cellular processes as
chaperones and partners in multiprotein complexes.1
Most well known is the ability of FKBP12 to cause
immunosuppression by inhibition of calcineurin and
FRAP (also known as RAFT or mTOR) following
binding of the immunosuppressive drugs FK506 and
rapamycin, respectively.2 However, FKBP12 is also
found in complex with intracellular calcium channels
(RyR and IP3R)3 as well as members of the TGF-b
family of receptors4 where it most likely serves to mod-
ulate signaling. Recently, FKBP12 and related FKBPs
have been associated with recovery from neuronal
injury5 and implicated as targets for neuroregeneration
and neuroprotection.6±8
The trans isomer, but not the cis, is a substrate for chy-
motrypsin, which releases the ¯uorophore, whose rate
of appearance can be measured in the presence of an
FKBP inhibitor. The assay suers from high back-
ground signal due to the tendency of the peptide sub-
strate to be in the energetically more favorable trans
conformation, as well as a severe temperature sensitivity.
As a result, it is not suitable for high-throughput
screening of FKBP-binding compounds.
Recently, a scintillation proximity assay for FKBP12
binding has been described that utilizes radiolabeled
FK506.10 We sought an alternative screen that would
avoid the expensive, hydrophobic ligand, and chose to
develop a ¯uorescence polarization (FP) assay. This
method relies on the decrease of a ¯uorescence signal
generated by a small molecule ¯uorescent FKBP12
ligand bound to the protein measured in the presence of
inhibitor. For this assay, we desired a ligand whose
FKBP12 binding and solubility properties were compa-
tible with reasonable screening concentrations of both
protein and small molecule substrates.
Screening for compounds that bind to FKBP12 has
primarily depended on a fairly laborious assay that
measures inhibition of the cis-trans peptidyl-prolyl iso-
merase (rotamase) activity of FKBPs.9 In this assay, a
variable existing fraction (usually <50%) of the cis-
isomer of an AA-Pro-containing ¯uorogenic tetra-
peptide is converted to the trans isomer by the FKBP.
For convenience, we used ¯uorescein as the reporter
¯uorophore since its optical properties are well known
in this context.11 Ligand design was based on pre-
vious non-immunosuppressive FKBP12-binding com-
pounds.12±14 These take advantage of a small accessory
binding site that is connected to the primary pipecolate
binding pocket by a shallow hydrophobic groove to
increase anity by linking an aromatic ring to the
pipecolate/proline core via a short ester-alkyl chain
(Fig. 1). Since available SAR suggests that this second-
ary site is quite promiscuous, we decided to design our
*Corresponding author. Tel.: +1-203-677-7539; fax: +1-203-677-
7702; e-mail: gene.dubowchik@bms.com
Abbreviations: Boc, t-butyloxycarbonyl; DMAP, 4-dimethylamino-
pyridine; EDC, 3-ethyl-1-dimethylaminopropylcarbodiimide; FITC,
¯uorescein isothiocyante; FKBP, FK506 binding protein; FP, ¯uores-
cence polarization; FRAP, FKBP12-rapamycin-associated protein;
IP3R, inositol triphosphate receptor; MMT, monomethoxytrityl;
mTOR, mammalian target of rapamycin; Phe, l-phenylalanine; Pro,
l-proline; RAFT, rapamycin-associated FKBP12 target; RyR, ryano-
dine receptor; TFA, tri¯uoroacetic acid.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00045-7