U.S. patent application number 13/417008 was filed with the patent office on 2012-09-13 for fluorescence polarization herg assay.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Steve Duff, Stephen Hess, Thomas Livelli, David Piper, Mohammed Saleh Shekhani, Kurt Vogel, Zhong Zhong.
Application Number | 20120231541 13/417008 |
Document ID | / |
Family ID | 40756160 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120231541 |
Kind Code |
A1 |
Piper; David ; et
al. |
September 13, 2012 |
FLUORESCENCE POLARIZATION hERG ASSAY
Abstract
Disclosed are assays, methods, and kits for the screening of
test compounds for their capability to induce cardiotoxicity in a
subject. In particular, whether a test compound has the effect to
prolong the Q-T interval as measured by an electrocardiogram in a
human. The assays, methods, and kits disclosed herein make use of
the binding interaction between novel fluorescent tracers and the
hERG K.sup.+ channel, and the propensity of a test compound to
influence that binding interaction.
Inventors: |
Piper; David; (Madison,
WI) ; Hess; Stephen; (Verona, WI) ; Shekhani;
Mohammed Saleh; (Madison, WI) ; Livelli; Thomas;
(Madison, WI) ; Zhong; Zhong; (Shanghai, CN)
; Duff; Steve; (Middleton, WI) ; Vogel; Kurt;
(Madison, WI) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
40756160 |
Appl. No.: |
13/417008 |
Filed: |
March 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
12394605 |
Feb 27, 2009 |
8133695 |
|
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13417008 |
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61032809 |
Feb 29, 2008 |
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61032604 |
Feb 29, 2008 |
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61032390 |
Feb 28, 2008 |
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Current U.S.
Class: |
435/361 ;
435/358; 435/369 |
Current CPC
Class: |
Y10T 436/13 20150115;
G01N 33/582 20130101; G01N 33/6872 20130101; C07D 491/107
20130101 |
Class at
Publication: |
435/361 ;
435/358; 435/369 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/10 20060101 C12N005/10; C12N 5/073 20100101
C12N005/073 |
Claims
1-42. (canceled)
43. A hERG K.sup.+ channel-expressing cell population, wherein said
cell population expresses at least about 100 .mu.mol of hERG
K.sup.+ channel per mg of total membrane protein.
44. The hERG K.sup.+ channel-expressing cell population of claim
43, wherein cells thereof express a hERG K.sup.+ channel for which
the hERG current as determined by patch clamping with a fully
automated high throughput patch clamp system is in a range of about
1500 pA to about 2500 pA.
45. The hERG K.sup.+ channel-expressing cell population of claim
43, wherein cells thereof are HEK 293 cells or CHO cells.
46. The hERG K.sup.+ channel-expressing cell population of claim
43, wherein cells thereof have been transfected with an expression
vector selected from the group consisting of: i) an isolated and
purified nucleic acid comprising a nucleotide sequence which
encodes a hERG K.sup.+ channel having an amino acid sequence that
is at least 80% homologous to that of SEQ ID NO: 1 or a fragment
thereof; and ii) an isolated and purified nucleic acid comprising a
nucleotide sequence which encodes a hERG K.sup.+ channel having the
amino acid sequence of SEQ ID NO: 1 or a fragment thereof.
47. The hERG K.sup.+ channel-expressing cell population of claim
46, wherein the nucleic acid further comprises a nucleotide
sequence which encodes an internal ribosomal entry site protein and
a nucleotide sequence which encodes CD-8 plasma membrane
protein.
48. The hERG K.sup.+ channel-expressing cell population of claim
47, wherein the nucleotide sequences which encode the internal
ribosomal entry site protein and the CD-8 plasma membrane protein
are located successively downstream from the nucleotide sequence
which encodes the hERG K.sup.+ channel.
49. The hERG K.sup.+ channel-expressing cell population of claim
48, wherein expression of the hERG K.sup.+ channel is coupled to
expression of the CD-8 plasma protein by means of the nucleotide
sequence which encodes an internal ribosomal entry site
protein.
50. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/032,390,
filed Feb. 28, 2008, U.S. Provisional Patent Application No.
61/032,604, filed Feb. 29, 2008, and U.S. Provisional Patent
Application No. 61/032,809, filed Feb. 29, 2008, the disclosures of
which are hereby incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of cardiovascular
safety assays, in particular to assays, methods, and kits for the
screening of test compounds for their capability to induce
cardiotoxicity in a subject. The assays, methods, and kits
disclosed herein are based on the interaction of novel fluorescent
tracer compounds with the hERG K.sup.+ channel, which interaction
is exploited by means of membrane preparations from a cell line
engineered to have a high level of hERG K.sup.+ channel expression.
The assays, methods, and kits disclosed herein may be useful to
identify compounds with undesirable effects on cardiac
repolarization in man, in particular the propensity to prolong the
Q-T interval in an electrocardiogram.
BACKGROUND OF THE INVENTION
[0003] The human ether-a-go-go related gene (hERG) encodes the
rapidly delayed inward rectifying potassium channel (I.sub.Kr) that
profoundly effects the repolarization of the human ventricle (see,
Curran, M. E.; Splawski, I.; Timothy, K. W.; Vincent, G. M.; Green,
E. D.; Keating, M. T., A molecular basis for cardiac arrhythmia:
HERG mutations cause long QT syndrome. Cell 1995, 80, (5), 795-803;
Sanguinetti, M. C.; Jiang, C.; Curran, M. E.; Keating, M. T., A
mechanistic link between an inherited and an acquired cardiac
arrhythmia: HERG encodes the IKr potassium channel. Cell 1995, 81,
(2), 299-307; Trudeau, M. C.; Warmke, J. W.; Ganetzky, B.;
Robertson, G. A., HERG, a human inward rectifier in the
voltage-gated potassium channel family. Science 1995, 269, (5220),
92-5; and Warmke, J. W.; Ganetzky, B., A family of potassium
channel genes related to eag in Drosophila and mammals. Proc Natl
Acad Sci USA 1994, 91, (8), 3438-42). Block of I.sub.Kr
repolarizing current flowing through the channel in ventricular
muscle can result in prolongation of the Q-T interval, a
characteristic electrocardiogram pattern termed torsades de
pointes, and potentially lethal arrhythmia (see, Sanguinetti, M.
C.; Tristani-Firouzi, M., hERG potassium channels and cardiac
arrhythmia. Nature 2006, 440, (7083), 463-9; and Haverkamp, W.;
Breithardt, G.; Camm, A. J.; Janse, M. J.; Rosen, M. R.;
Antzelevitch, C.; Escande, D.; Franz, M.; Malik, M.; Moss, A.;
Shah, R., The potential for QT prolongation and proarrhythmia by
non-antiarrhythmic drugs: clinical and regulatory implications.
Report on a policy conference of the European Society of
Cardiology. Eur Heart J 2000, 21, (15), 1216-31). The promiscuous
nature of this channel, referred to herein as the hERG K.sup.+
channel, to bind a diverse set of chemical structures (see,
Cavalli, A.; Poluzzi, E.; De Ponti, F.; Recanatini, M., Toward a
pharmacophore for drugs inducing the long QT syndrome: insights
from a CoMFA study of HERG K(+) channel blockers. J Med Chem 2002,
45, (18), 3844-53), coupled with the potential fatal outcome that
may emerge from that interaction, have resulted in the
recommendation from the International Congress of Harmonization and
the U.S. Food and Drug Administration that all new drug candidates
undergo testing in a functional patch-clamp assay using the human
hERG protein, either in native form or expressed in recombinant
form (see, Bode, G.; Olejniczak, K., ICH topic: the draft ICH S7B
step 2: note for guidance on safety pharmacology studies for human
pharmaceuticals. Fundam Clin Pharmacol 2002, 16, (2), 105-18).
Although automated, high-throughput patch-clamp methods have been
recently developed, such systems require specialized operators,
live cells, and a substantial capital investment (see,
Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.;
Henthorn, D. C.; Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C.
G.; Abi-Gerges, N.; Persson, F.; Jacobson, I.; Sullivan, M.;
Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin, J. P.;
Pollard, C. E., Optimisation and validation of a medium-throughput
electrophysiology-based hERG assay using IonWorks HT. J Pharmacol
Toxicol Methods 2006, 54, (2), 189-99; and Dubin, A. E.; Nasser,
N.; Rohrbacher, J.; Hermans, A. N.; Marrannes, R.; Grantham, C.;
Van Rossem, K.; Cik, M.; Chaplan, S. R.; Gallacher, D.; Xu, J.;
Guia, A.; Byrne, N. G.; Mathes, C., Identifying modulators of hERG
channel activity using the PatchXpress planar patch clamp. J Biomol
Screen 2005, 10, (2), 168-81). Further, since patch-clamp testing
is costly, and because numerous, chemically-diverse scaffolds block
the hERG K.sup.+ channel, strategies to mitigate potential cardiac
liability during early-stage drug development typically employ a
binding assay to predict the ability of a compound to block hERG
current in the functional patch-clamp assay (see, Whitebread, S.;
Hamon, J.; Bojanic, D.; Urban, L., Keynote review: in vitro safety
pharmacology profiling: an essential tool for successful drug
development. Drug Discov Today 2005, 10, (21), 1421-33; and Diaz,
G. J.; Daniell, K.; Leitza, S. T.; Martin, R. L.; Su, Z.;
McDermott, J. S.; Cox, B. F.; Gintant, G. A., The [3H]dofetilide
binding assay is a predictive screening tool for hERG blockade and
proarrhythmia: Comparison of intact cell and membrane preparations
and effects of altering [K+]o. J Pharmacol Toxicol Methods 2004,
50, (3), 187-99).
[0004] Radioligand binding assays that use [.sup.3H]-dofetilide
(see, Diaz, G. J.; Daniell, K.; Leitza, S. T.; Martin, R. L.; Su,
Z.; McDermott, J. S.; Cox, B. F.; Gintant, G. A., The
[3H]dofetilide binding assay is a predictive screening tool for
hERG blockade and proarrhythmia: Comparison of intact cell and
membrane preparations and effects of altering [K+]o. J Pharmacol
Toxicol Methods 2004, 50, (3), 187-99; and Finlayson, K.; Turnbull,
L.; January, C. T.; Sharkey, J.; Kelly, J. S., [3H]dofetilide
binding to HERG transfected membranes: a potential high throughput
preclinical screen. Eur J Pharmacol 2001, 430, (1), 147-8),
[.sup.3H]-astemizole (see, Chiu, P. J.; Marcoe, K. F.; Bounds, S.
E.; Lin, C. H.; Feng, J. J.; Lin, A.; Cheng, F. C.; Crumb, W. J.;
Mitchell, R., Validation of a [3H]astemizole binding assay in
HEK293 cells expressing HERG K.sup.+ channels. J Pharmacol Sci
2004, 95, (3), 311-9), or [.sup.35S]-MK499 (see, Wang, J.; Della
Penna, K.; Wang, H.; Karczewski, J.; Connolly, T. M.; Koblan, K.
S.; Bennett, P. B.; Salata, J. J., Functional and pharmacological
properties of canine ERG potassium channels. Am J Physiol Heart
Circ Physiol 2003, 284, (1), H256-67) have been shown to be
predictive of hERG K.sup.+ channel block. However, the preparation,
storage, and disposal of the radioligands adds time and cost to the
assay procedure. Additionally, the radiometric assays that have
been described to assess compound binding to the hERG K.sup.+
channel are heterogeneous filter binding assays, and require a
separation of free from bound radioligand by capturing
radioligand-bound membrane protein on filter paper using a vacuum
manifold. This procedure makes the assay difficult to automate for
large-scale screening or routine compound profiling, thereby
limiting its practical utility. Additionally, over the past decade,
there has been a strong push within both industry and academia to
develop non-radioactive methods to replace such assays.
[0005] Fluorescence polarization (FP) assays provide a fully
homogenous, mix-and-read format to characterize the affinity of a
ligand for a receptor, and in many cases can be used to replace
many radiometric binding assays (see, Burke, T. J.; Loniello, K.
R.; Beebe, J. A.; Ervin, K. M., Development and application of
fluorescence polarization assays in drug discovery. Comb Chem High
Throughput Screen 2003, 6, (3), 183-94). The technique is based on
the ability of a compound to displace a fluorescent probe (a
"tracer") from a receptor, which is detected by a change in an
optical signal. In such an assay, the tracer typically consists of
a known, high-affinity ligand for the receptor that has been
chemically attached to a fluorescent molecule, without
substantially disrupting the affinity of the receptor-ligand
interaction (see, Huang, X., Fluorescence polarization competition
assay: the range of resolvable inhibitor potency is limited by the
affinity of the fluorescent ligand. J Biomol Screen 2003, 8, (1),
34-8). When a tracer molecule is excited with plane-polarized light
in an FP assay, the polarization of the emitted light is retained
if the fluorophore maintains its orientation during the time
(typically nanoseconds) between photon excitation and emission. In
solution, this orientation is largely maintained when the tracer is
bound to a larger molecule, such as a protein, because the
protein-tracer complex rotates more slowly than the free tracer
itself. When the tracer is displaced from the receptor by a ligand
that binds to the receptor, emission of light from the tracer is
depolarized relative to the excitation source.
[0006] An important practical distinction between a traditional
radioligand binding assay and an FP assay is that, in contrast to a
radioligand binding assay, FP assays are optimally configured using
a limiting amount of tracer, and a concentration of receptor that
is at or above the K.sub.d value for the receptor-tracer
interaction. This is because the optical signal that is measured is
dependant on the signal from all of the tracer that is
present--both free and bound, which is unlike that in a radioligand
binding assay in which (after separation) the only signal measured
is due to bound ligand, and free ligand does not contribute to the
signal. Thus, in an FP assay, any unbound tracer contributes to the
amount of depolarized light present, thereby lowering the
polarization signal that is measured, and lowering the assay
window. Typically, FP assays are configured such that between 50
and 70% of the total tracer is bound in the absence of competing
ligand in order to strike a balance between the assay window
(maximal-minimal polarization values that are measured) and the
assay sensitivity (ability of IC.sub.50 values to approach true
K.sub.i values) (see, Huang, X., Fluorescence polarization
competition assay: the range of resolvable inhibitor potency is
limited by the affinity of the fluorescent ligand. J Biomol Screen
2003, 8, (1), 34-8). When developing FP assays using purified,
soluble, recombinant proteins, this is typically not an issue
because many such proteins are readily prepared in quantities
sufficient for such assays. However, this requirement can pose a
challenge when developing assays for membrane-associated proteins,
such as hERG, which in most cases have not been purified in
functional form from their membrane components, which include both
insoluble lipid components as well as other proteins. Moreover, the
presence of large amounts of membrane components can interfere with
the assay by scattering light (see, Banks, P.; Gosselin, M.;
Prystay, L., Impact of a red-shifted dye label for high throughput
fluorescence polarization assays of G protein-coupled receptors. J
Biomol Screen 2000, 5, (5), 329-34) or by leading to increased
non-specific binding of the tracer (which often contains a
lipophilic fluorophore) with the membrane itself.
[0007] Accordingly, the development of a homogenous, FP-based assay
to identify and characterize the affinity of small molecules for
the hERG K.sup.+ channel, and demonstrate tight correlation with
data obtained from either radioligand binding or patch-clamp
assays, has heretofore not been realized.
SUMMARY OF THE INVENTION
[0008] In order to avoid the issues associated with radiometric
assays to assess hERG K.sup.+ channel binding, a homogenous,
FP-based substitute for these assays has been developed. A
traditional radioligand displacement assay was used initially to
identify candidate high-affinity fluorescent tracers. However,
because the level of hERG K.sup.+ channel expression (B.sub.max) in
the initial cell line was insufficient to configure an FP assay, a
strategy was developed to increase hERG K.sup.+ channel expression
levels by coupling the expression of a cell-surface marker (CD8) to
the expression of hERG K.sup.+ channel, using a bicistronic
expression vector that encoded both proteins. This strategy allowed
for the clonal isolation of high-expressing hERG K.sup.+ channel
cell lines using flow cytometry, and enabled the development of the
FP assay, including further tracer development and assay
optimization. The resulting FP assay is predictive of hERG K.sup.+
channel binding, is simple to perform using standard plate readers,
and is well suited to replace traditional radiometric binding
assays as a means of triaging compounds for hERG K.sup.+ channel
liability.
[0009] Described herein are FP assays, methods, and kits for the
screening of small molecules, i.e., test compounds, to characterize
their affinity for the hERG K.sup.+ channel, and their capability
to induce cardiotoxicity in a subject. In addition, described
herein are processes for preparing novel fluorescent tracer
compounds and membrane preparations having a high level of hERG
K.sup.+ channel expression for use in the disclosed assays,
methods, and kits.
[0010] One aspect of the present invention provides a novel
fluorescent tracer compound having the general structural formula
(I):
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein: Ar is an
aromatic ring selected from the group consisting of benzo, thieno,
furo, and pyrido; R.sup.1 and R.sup.2 are independently selected
from the group consisting of:
[0011] 1) hydrogen,
[0012] 2) C.sub.1-6 alkyl, either unsubstituted or substituted with
[0013] a) --NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5 are
independently hydrogen or C.sub.1-6 alkyl, [0014] b)
--N(R.sup.5)COC.sub.1-6 alkyl, [0015] c) --NHSO.sub.2(C.sub.1-6
alkyl), [0016] d) --CONR.sup.6R.sup.7, wherein R.sup.6 and R.sup.7
are independently [0017] i) hydrogen, [0018] ii) C.sub.1-6 alkyl,
or [0019] iii) R.sup.6 and R.sup.7 taken together with the nitrogen
atom to which they are attached represent a 5- or 6-membered
saturated heterocyclic ring, which may contain an additional
heteroatom selected from N, S(O).sub.n, or O, selected from the
group consisting of pyrrolidine, morpholine, piperidine,
piperazine, and N-methylpiperazine, [0020] e) --CO(C.sub.1-6
alkyl), [0021] f) --OH, [0022] g) --O(C.sub.1-6 alkyl), [0023] h)
--O(C.sub.1-6 alkyl)--O--(C.sub.1-3 alkyl), [0024] i)
--S(O).sub.n(C.sub.1-6 alkyl), [0025] j) imidazole, [0026] k)
2-imidazolidinone, [0027] l) 2-pyrrolidinone, [0028] m)
--NH--C(NHR.sup.5).dbd.N--CN, or [0029] n)
--NH--C(SR.sup.5).dbd.N--CN,
[0030] 3) --OH,
[0031] 4) C.sub.1-3 alkoxy, either unsubstituted or substituted
with C.sub.1-3 alkoxy,
[0032] 5) --N(R.sup.5)SO.sub.2(C.sub.1-6 alkyl),
[0033] 6) --N(R.sup.5)SO.sub.2(CH.sub.2).sub.gCO.sub.2H, wherein g
is 1-5,
[0034] 7) --N(R.sup.5)SO.sub.2(CH.sub.2).sub.gCO.sub.2C.sub.1-6
alkyl,
[0035] 8) --NO.sub.2,
[0036] 9) --N(R.sup.5)COC.sub.1-6 alkyl,
[0037] 10) --N(R.sup.5)SO.sub.2--C.sub.6H.sub.4--R.sup.4,
[0038] 11) --N(R.sup.5)CO--C.sub.6H.sub.4--R.sup.4,
[0039] 12) --NR.sup.4R.sup.5,
[0040] 13) halo,
[0041] 14) --CO--C.sub.1-6 alkyl,
[0042] 15) --CONR.sup.6R.sup.7,
[0043] 16) --CN,
[0044] 17) --CO.sub.2R.sup.5,
[0045] 18) --C(R.sup.5).dbd.N--OR.sup.8,
[0046] 19) benzoyl, either unsubstituted or substituted with
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halo, or hydroxy,
[0047] 20) --N(R.sup.5)COO(C.sub.1-6 alkyl),
[0048] 21) --N(R.sup.5)COO-phenyl, either unsubstituted or
substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy or
halo,
[0049] 22) --N(R.sup.5)CONR.sup.4R.sup.5,
[0050] 23) --S(O).sub.nC.sub.1-6 alkyl,
[0051] 24) --S(O).sub.n--C.sub.6H.sub.4--R.sup.4,
[0052] 25) --CF.sub.3,
[0053] 26) phenyl, either unsubstituted or substituted with
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halo or hydroxy,
[0054] 27) imidazolyl,
[0055] 28) --SO.sub.2NR.sup.6R.sup.7,
[0056] 29) --N[S(O).sub.2C.sub.1-6 alkyl][(CH.sub.2).sub.pCN],
wherein p is 2-5,
[0057] 30) --N(R.sup.5)--C(NR.sup.4R.sup.5).dbd.N--CN, and
[0058] 31) --N(R.sup.5)--C(SR.sup.5).dbd.N--CN;
the ring system comprising W, X, and Y is a 5-, 6-, or 7-membered
ring system wherein W, X, and Y are independently --O--, C.dbd.O,
--(CR.sup.4R.sup.5).sub.n--, C.dbd.NOR.sup.8, CHOR.sup.9,
--NR.sup.9--, CHNR.sup.10R.sup.11, --S(O).sub.n--, .dbd.CH--,
.dbd.N--, or a bond; wherein:
[0059] R.sup.4 and R.sup.5 are as defined above,
[0060] R.sup.8 is
[0061] a) hydrogen, or
[0062] b) C.sub.1-6 alkyl, unsubstituted or substituted with
--COOR.sup.5;
[0063] R.sup.9 is
[0064] a) hydrogen,
[0065] b) C.sub.1-6 alkyl,
[0066] c) (CH.sub.2).sub.n--C.sub.6H.sub.4--R.sup.12, wherein
R.sup.12 is [0067] i) --NO.sub.2, [0068] ii) C.sub.1-3 alkyl,
[0069] iii) --O--C.sub.1-3 alkyl, [0070] iv) halo, [0071] v)
--CF.sub.3, or [0072] vi)hydrogen,
[0073] d) --CO--C.sub.1-6 alkyl,
[0074] e) --CO--C.sub.6H.sub.4--R.sup.12,
[0075] f) --COO--C.sub.1-6 alkyl, or
[0076] g) --CONR.sup.4, R.sup.5;
[0077] R.sup.10 and R.sup.11 are independently
[0078] a) hydrogen,
[0079] b) C.sub.1-6 alkyl, unsubstituted or substituted with
--(CR.sup.4R.sup.5).sub.n--(CR.sup.4R.sup.5).sub.g--R.sup.13,
wherein g is 1-5, and R.sup.13 is [0080] i) hydrogen, [0081] ii)
--OH, or [0082] iii) --OC.sub.1-6 alkyl,
[0083] c) --CO--C.sub.1-6 alkyl, unsubstituted or substituted with
[0084] i) --OH, [0085] ii) --N(R.sup.4R.sup.5), [0086] iii)
--OC.sub.1-6 alkyl, or [0087] iv) --CO.sub.2R.sup.5,
[0088] d) --CO--C.sub.6H.sub.4--R.sup.13, or
[0089] e) R.sup.10 and R.sup.11 taken together with the nitrogen
atom to which they are attached represent a 5- or 6-membered
saturated heterocyclic ring, unsubstituted or substituted with
oxygen or hydroxy, which may contain an additional heteroatom
selected from N, S(O).sub.n or O, selected from the group
consisting of pyrrolidine, morpholine, piperidine, pyrrolidinone,
piperidinone, piperazine and N-methylpiperazine;
[0090] n is 0, 1, or 2;
B is a 5- to 7-membered N-containing ring; L is
--(CR.sup.4R.sup.5).sub.m-Q-(CR.sup.4R.sup.5).sub.q--NH--[CZ--(CR.sup.4R.-
sup.5).sub.u-(D).sub.w].sub.z--, wherein
[0091] R.sup.4 and R.sup.5 are as defined above,
[0092] m and q are independently 1 to about 5,
[0093] u is 0 to about 7,
[0094] w is 0 or 1,
[0095] z is 1 or 2,
[0096] Q is a bond, --O--, C.dbd.O, CHOH, --NR.sup.5-- or
--S(O).sub.n--,
[0097] Z is .dbd.O or .dbd.S, and
[0098] D is --O--, --S(O).sub.n--, --NR.sup.5--, or
--NR.sup.5SO.sub.2--; and
R.sup.3 is a fluorescent dye.
[0099] Another aspect of the present invention provides an assay
for screening test compounds, wherein the assay is a binding assay
using a fluorescent tracer described herein binding to a source of
the hERG K.sup.+ channel or fragment thereof.
[0100] Another aspect of the present invention provides a method
for characterizing the activity of a test compound as a hERG
K.sup.+ channel blocker, the method comprising the steps of:
[0101] a) contacting the test compound with a membrane preparation
containing a hERG K.sup.+ channel having the amino acid sequence of
SEQ ID NO: 1, the membrane preparation derived from cells
transfected with a nucleic acid expression vector including a
nucleotide sequence which encodes the hERG K.sup.+ channel, in an
assay buffer in the presence of a fluorescent tracer described
herein;
[0102] b) monitoring whether the test compound influences the
binding of the fluorescent tracer to the membrane preparation
containing the hERG K.sup.+ channel; and
[0103] c) determining the hERG K.sup.+ channel blocker activity of
the test compound.
[0104] Another aspect of the present invention provides a kit for
screening test compounds, the kit comprising:
[0105] a) a fluorescent tracer described herein;
[0106] b) a source of the hERG K.sup.+ channel or fragment thereof;
and
[0107] c) an assay buffer.
[0108] Another aspect of the present invention provides a hERG
K.sup.+ channel-expressing cell population, wherein the cell
population expresses at least about 100 pmol of hERG K.sup.+
channel per mg of total membrane protein.
[0109] Another aspect of the present invention provides a process
for preparing a fluorescent tracer compound of structural formula
(I)
##STR00002##
the process comprising:
[0110] a) reacting a compound of structural formula (II)
##STR00003##
in dimethylformamide/diisopropylethyl amine at room temperature
with a compound of structural formula (III)
[R.sup.14O].sub.k--[CZ--(CR.sup.4R.sup.5).sub.u-(D).sub.w].sub.x--R.sup.-
3 (III)
wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, Ar, B, D, L,
Q, W, X Y, Z, m, q, u, w, and z are as defined above; k is 0 or 1;
and R.sup.14 is a component of an active ester; provided that if Z
is .dbd.O, then k is 1, and provided that if Z is .dbd.S, then k is
0, u is 0, w is 1, z is 1, and D is .dbd.N.
[0111] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0112] FIG. 1 shows six candidate fluorescent tracers (identified
by their internal compound identification numbers) initially
evaluated for hERG K.sup.+ channel affinity as determined by a
radioligand displacement assay that displaced [.sup.3H]-astemizole
with an IC.sub.50 value of less than 30 nM.
[0113] FIG. 2 shows clonal isolation of high-expressing hERG
K.sup.+ channel cell line: (A) The top 10% of CD8+ cells were
isolated by FACS. (B) 192 single cells were expanded and analyzed
by immunocytochemistry for CD8 expression. (C) Six clones were
analyzed by manual patch clamp, and peak tail current recorded.
"T-REx" refers to the original inducible hERG cell line analyzed
and "Pool" refers to the original sort of high-CD8-expressing
cells. (D) A membrane preparation from clone D was analyzed by
radioligand binding. (.smallcircle.) total bound ligand (.cndot.)
specific bound ligand, (.times.) non-specific bound ligand.
[0114] FIG. 3 shows observed polarization values of Predictor.TM.
hERG Tracer Red to hERG-CD8 membranes in the presence
(.smallcircle.) or absence (.cndot.) of 30 .mu.M E-4031.
[0115] FIG. 4 shows displacement of Predictor.TM. hERG Tracer Red
from hERG-CD8 membranes. (A) Displacement by E-4031 (.quadrature.)
or astemizole (.smallcircle.). (B) Displacement by astemizole in
the absence (.smallcircle.) or presence (.times.) of 30 .mu.M
E-4031. Corrected data (.cndot.) accounts for the non-specific
displacement seen in the presence of E-4031.
[0116] FIG. 5 shows displacement of Predictor.TM. hERG Tracer Red
from CD8-hERG membranes by known hERG K.sup.+ channel blockers that
span a range of affinities for the hERG K.sup.+ channel. Raw data
are shown by respective symbols. Solid lines represent displacement
curves that have been corrected as described herein.
[0117] FIG. 6(A) shows a time course study to determine signal
stability (polarization shift and IC.sub.50 value) and assay
robustness (Z' value) over time. The assay plate was read
(.box-solid.) 30 minutes, (.quadrature.) 1 hour, (.cndot.) 2 hours,
(.smallcircle.) 4 hours, (.tangle-solidup.) 6 hours, (.times.) 24
hours after addition of tracer and membrane to a dilution series of
E-4031. Each datapoint represents the average of duplicate
measurements, except the 30 .mu.M E-4031 datapoint which contains
28 replicates in order to calculate Z' values (Table 2). Error bars
are shown but in general are smaller than the symbol used to mark
the datapoint. In FIG. 6(B), the assay was repeated in the presence
of various concentrations of DMSO (.quadrature.), methanol
(.cndot.), or ethanol (.DELTA.) and read after 2 hours, IC.sub.50
values are connected by solid lines, Z' values are connected by
dashed lines.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0118] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It should be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise.
[0119] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein:
[0120] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. This term includes, by way of example, linear and
branched hydrocarbyl groups such as methyl (CH.sub.3--), ethyl
(CH.sub.3CH.sub.2--), n-propyl (CH.sub.3CH.sub.2CH.sub.2--),
isopropyl ((CH.sub.3).sub.2CH--), n-butyl
(CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0121] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0122] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 5 to 14 carbon atoms having a single ring (e.g.,
benzo) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom.
[0123] "Amino" refers to the group --NH.sub.2.
[0124] "Alkenyl" refers to alkenyl groups having from 2 to 6 carbon
atoms and preferably 2 to 4 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkenyl unsaturation. Such groups
are exemplified, for example, by vinyl, allyl, and
but-3-en-1-yl.
[0125] "Carboxyl" or "carboxy" refers to --COOH or salts
thereof.
[0126] "H" indicates hydrogen.
[0127] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0128] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0129] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties.
[0130] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or unsaturated group having a
single ring or multiple condensed rings, including fused bridged
and spiro ring systems, from 1 to 10 carbon atoms and from 1 to 4
hetero atoms selected from the group consisting of nitrogen, sulfur
or oxygen within the ring wherein, in fused ring systems, one or
more the rings can be cycloalkyl, aryl or heteroaryl provided that
the point of attachment is through the non-aromatic ring. In one
embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic
group are optionally oxidized to provide for the N-oxide, sulfinyl,
sulfonyl moieties.
[0131] "Spirocyclyl" or "spiro" refers to divalent saturated cyclic
group from 3 to 10 carbon atoms having a cycloalkyl or heterocyclyl
ring with a spiro union (the union formed by a single atom which is
the only common member of the rings).
[0132] "Salt" refers to acceptable salts of a compound, which salts
are derived from a variety of organic and inorganic counter ions
well known in the art and include, by way of example only, sodium,
potassium, calcium, magnesium, ammonium, and tetraalkylammonium;
and when the molecule contains a basic functionality, salts of
organic or inorganic acids, such as hydrochloride, hydrobromide,
tartrate, mesylate, acetate, maleate, and oxalate.
[0133] The term "dye" as used herein refers to a compound that
emits light to produce an observable detectable signal.
[0134] The term "fluorophore" or "fluorogenic" as used herein
refers to a composition that demonstrates a change in fluorescence
upon binding to a biological compound or analyte interest.
Preferred fluorophores of the present invention include fluorescent
dyes having a high quantum yield in aqueous media. Exemplary
fluorophores include xanthene, indole, borapolyazaindacene, furan,
and benzofuran, among others. The fluorophores of the present
invention may be substituted to alter the solubility, spectral
properties or physical properties of the fluorophore.
[0135] The term "linker" as used herein, refers to a series of
stable covalent bonds incorporating atoms selected from the group
consisting of C, N, O, and S that covalently attach the fluorogenic
or fluorescent compounds to another moiety such as a chemically
reactive group or a biological and non-biological component.
Exemplary linking members include a moiety that includes
--C(O)NH--, --C(O)O, NH--, --S--, --O--, and the like.
[0136] The term "BSA" as used herein refers to bovine serum
albumin.
[0137] The term "CMV" as used herein refers to cytomegalovirus.
[0138] The term "D-MEM" as used herein refers to Dulbecco's
Modified Eagle Medium.
[0139] The term "DMSO" as used herein refers to dimethyl
sulfoxide.
[0140] The term "EDTA" as used herein refers to ethylenediamine
tetraacetic acid.
[0141] The term "FACS" as used herein refers to fluorescence
automated cell sorting.
[0142] The term "FBS" as used herein refers to fetal bovine
serum.
[0143] The term "FP" as used herein refers to fluorescence
polarization.
[0144] The term "HEPES" as used herein refers to
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
[0145] The term "hERG" as used herein refers to the human
ether-a-go-go related gene.
[0146] The term "LQTS" as used herein refers to long Q-T
syndrome.
[0147] The term "MEM NEAA" as used herein refers to minimal
essential medium with non-essential amino acids.
[0148] The term "PBS" as used herein refers to phosphate buffered
saline.
[0149] The term "TdP" as used herein refers to Torsades de
Pointe.
Particular Aspects of the Invention:
[0150] Development of an FP assay to assess hERG K.sup.+ channel
binding required that a series of fluorescent tracer compounds be
synthesized with varying scaffolds, substituents, linkers and
fluorophores (see, Singleton, D. H.; Boyd, H.; Steidl-Nichols, J.
V.; Deacon, M.; Groot, M. J.; Price, D.; Nettleton, D. O.; Wallace,
N. K.; Troutman, M. D.; Williams, C.; Boyd, J. G., Fluorescently
Labeled Analogues of Dofetilide as High-Affinity Fluorescence
Polarization Ligands for the Human Ether-a-go-go-Related Gene
(hERG) Channel. J Med Chem 2007, 50, (13), 2931-2941). Accordingly,
one aspect of the present invention provides a novel fluorescent
tracer compound having the general structural formula (I):
##STR00004##
or a pharmaceutically acceptable salt thereof, wherein: Ar is an
aromatic ring selected from the group consisting of benzo, thieno,
furo, and pyrido; R.sup.1 and R.sup.2 are independently selected
from the group consisting of:
[0151] 1) hydrogen,
[0152] 2) C.sub.1-6 alkyl, either unsubstituted or substituted with
[0153] a) --NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5 are
independently hydrogen or C.sub.1-6 alkyl, [0154] b)
--N(R.sup.5)COC.sub.1-6 alkyl, [0155] c) --NHSO.sub.2(C.sub.1-6
alkyl), [0156] d) --CONR.sup.6R.sup.7, wherein R.sup.6 and R.sup.7
are independently [0157] i) hydrogen, [0158] ii) C.sub.1-6 alkyl,
or [0159] iii) R.sup.6 and R.sup.7 taken together with the nitrogen
atom to which they are attached represent a 5- or 6-membered
saturated heterocyclic ring, which may contain an additional
heteroatom selected from N, S(O).sub.n, or O, selected from the
group consisting of pyrrolidine, morpholine, piperidine,
piperazine, and N-methylpiperazine, [0160] e) --CO(C.sub.1-6
alkyl), [0161] f) --OH, [0162] g) --O(C.sub.1-6 alkyl), [0163] h)
--O(C.sub.1-6 alkyl)--O--(C.sub.1-3 alkyl), [0164] i)
--S(O).sub.n(C.sub.1-6 alkyl), [0165] j) imidazole, [0166] k)
2-imidazolidinone, [0167] l) 2-pyrrolidinone, [0168] m)
--NH--C(NHR.sup.5).dbd.N--CN, or [0169] n)
--NH--C(SR.sup.5).dbd.N--CN,
[0170] 3) --OH,
[0171] 4) C.sub.1-3 alkoxy, either unsubstituted or substituted
with C.sub.1-3 alkoxy,
[0172] 5) --N(R.sup.5)SO.sub.2(C.sub.1-6 alkyl),
[0173] 6) --N(R.sup.5)SO.sub.2(CH.sub.2).sub.gCO.sub.2H, wherein g
is 1-5,
[0174] 7) --N(R.sup.5)SO.sub.2(CH.sub.2).sub.gCO.sub.2C.sub.1-6
alkyl,
[0175] 8) --NO.sub.2,
[0176] 9) --N(R.sup.5)COC.sub.1-6 alkyl,
[0177] 10) --N(R.sup.5)SO.sub.2--C.sub.6H.sub.4--R.sup.4,
[0178] 11) --N(R.sup.5)CO--C.sub.6H.sub.4--R.sup.4,
[0179] 12) --NR.sup.4R.sup.5,
[0180] 13) halo,
[0181] 14) --CO--C.sub.1-6 alkyl,
[0182] 15) --CONR.sup.6R.sup.7,
[0183] 16) --CN,
[0184] 17) --CO.sub.2R.sup.5,
[0185] 18) --C(R.sup.5).dbd.N--OR.sup.8,
[0186] 19) benzoyl, either unsubstituted or substituted with
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halo, or hydroxy,
[0187] 20) --N(R.sup.5)COO(C.sub.1-6 alkyl),
[0188] 21) --N(R.sup.5)COO-phenyl, either unsubstituted or
substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy or
halo,
[0189] 22) --N(R.sup.5)CONR.sup.4R.sup.5,
[0190] 23) --S(O).sub.nC.sub.1-6 alkyl,
[0191] 24) --S(O).sub.n--C.sub.6H.sub.4--R.sup.4,
[0192] 25) --CF.sub.3,
[0193] 26) phenyl, either unsubstituted or substituted with
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halo or hydroxy,
[0194] 27) imidazolyl,
[0195] 28) --SO.sub.2NR.sup.6R.sup.7,
[0196] 29) --N[S(O).sub.2C.sub.1-6 alkyl][(CH.sub.2).sub.pCN],
wherein p is 2-5,
[0197] 30) --N(R.sup.5)--C(NR.sup.4R.sup.5).dbd.N--CN, and
[0198] 31) --N(R.sup.5)--C(SR.sup.5).dbd.N--CN;
the ring system comprising W, X, and Y is a 5-, 6-, or 7-membered
ring system wherein W, X, and Y are independently --O--, C.dbd.O,
--(CR.sup.4R.sup.5).sub.n--, C.dbd.NOR.sup.8, CHOR.sup.9,
--NR.sup.9--, CHNR.sup.10R.sup.11, --S(O).sub.n, .dbd.CH--,
.dbd.N--, or a bond; wherein:
[0199] R.sup.4 and R.sup.5 are as defined above,
[0200] R.sup.8 is
[0201] a) hydrogen, or
[0202] b) C.sub.1-6 alkyl, unsubstituted or substituted with
--COOR.sup.5;
[0203] R.sup.9 is
[0204] a) hydrogen,
[0205] b) C.sub.1-6 alkyl,
[0206] c) (CH.sub.2).sub.n--C.sub.6H.sub.4--R.sup.12, wherein
R.sup.12 is [0207] i) --NO.sub.2, [0208] ii) C.sub.1-3 alkyl,
[0209] iii) --O--C.sub.1-3 alkyl, [0210] iv) halo, [0211] v)
--CF.sub.3, or [0212] vi) hydrogen,
[0213] d) --CO--C.sub.1-6 alkyl,
[0214] e) --CO--C.sub.6H.sub.4--R.sup.12,
[0215] f) --COO--C.sub.1-6 alkyl, or
[0216] g) --CONR.sup.4R.sup.5;
[0217] R.sup.10 and R.sup.11 are independently
[0218] a) hydrogen,
[0219] b) C.sub.1-6 alkyl, unsubstituted or substituted with
--(CR.sup.4R.sup.5).sub.n--(CR.sup.4R.sup.5).sub.g--R.sup.13,
wherein g is 1-5, and R.sup.13 is [0220] i) hydrogen, [0221] ii)
--OH, or [0222] iii) --OC.sub.1-6 alkyl,
[0223] c) --CO--C.sub.1-6 alkyl, unsubstituted or substituted with
[0224] i) --OH, [0225] ii) --N(R.sup.4R.sup.5), [0226] iii)
--OC.sub.1-6 alkyl, or [0227] iv) --CO.sub.2R.sup.5,
[0228] d) --CO--C.sub.6H.sub.4--R.sup.13, or
[0229] e) R.sup.10 and R.sup.11 taken together with the nitrogen
atom to which they are attached represent a 5- or 6-membered
saturated heterocyclic ring, unsubstituted or substituted with
oxygen or hydroxy, which may contain an additional heteroatom
selected from N, S(O).sub.n or O, selected from the group
consisting of pyrrolidine, morpholine, piperidine, pyrrolidinone,
piperidinone, piperazine and N-methylpiperazine;
[0230] n is 0, 1, or 2;
B is a 5- to 7-membered N-containing ring; L is
--(CR.sup.4R.sup.5).sub.m-Q-(CR.sup.4R.sup.5).sub.q--NH--[CZ--(CR.sup.4R.-
sup.5).sub.u-(D).sub.w].sub.z--, wherein
[0231] R.sup.4 and R.sup.5 are as defined above,
[0232] m and q are independently 1 to about 5,
[0233] u is 0 to about 7,
[0234] w is 0 or 1,
[0235] z is 1 or 2,
[0236] Q is a bond, --O--, C.dbd.O, CHOH, --NR.sup.5-- or
--S(O).sub.n--,
[0237] Z is .dbd.O or .dbd.S, and
[0238] D is --O--, --S(O).sub.n--, --NR.sup.5--, or
--NR.sup.5SO.sub.2--; and
R.sup.3 is a fluorescent dye.
[0239] Despite recognition that a subset of the aforementioned
fluorescent tracers exhibited high-affinity binding for the hERG
K.sup.+ channel (FIG. 1), a finding suggesting that at least one
such fluorescent tracer might prove useful for assay development,
standard hERG K.sup.+ channel-containing membranes were
insufficient to enable a robust FP assay. Specifically, the highest
affinity fluorescent tracers were examined for their utility in an
FP assay using membrane preparations derived from the
hERG-T-REx.TM. 293 cell line. These initial experiments failed to
exhibit a measurable difference in fluorescence polarization in the
presence or absence of known hERG K.sup.+ channel blockers such as
E-4031 or dofetilide. These results were not surprising as a robust
FP assay requires both a high affinity tracer and protein
concentrations sufficient to bind .about.50% or more of the tracer
in the absence of displacing compounds (see, Huang, X.,
Fluorescence polarization competition assay: the range of
resolvable inhibitor potency is limited by the affinity of the
fluorescent ligand. J Biomol Screen 2003, 8, (1), 34-8).
[0240] Accordingly, another aspect of the present invention
provides for increasing the specific activity (B.sub.max) of hERG
K.sup.+ channel membrane preparations. Since the B.sub.max levels
required to configure an FP assay are well above those typically
described for cell lines used in radioligand binding and
patch-clamp assays, increasing the specific activity (B.sub.max) of
hERG K.sup.+ channel membrane preparations was no less important
than identifying fluorescent tracer candidates with sufficient
affinity. To accomplish the former objective, an expression vector
(SEQ ID NO: 2) was constructed using a CMV promoter to drive
transcription of a bicistronic element composed of nucleotide
sequences encoding the hERG K.sup.+ channel and the CD8 cell
surface marker, wherein translation of the two proteins was linked
by an internal ribosomal entry site sequence (IRES). A
puromycin-resistance marker was included on the expression vector
to provide a means of selecting cells wherein stable, genomic
incorporation of the expression cassette had occurred. In one
illustrative variation, the expression vector includes a nucleotide
sequence encoding a hERG K.sup.+ channel having the amino acid
sequence of SEQ ID NO: 1. In another illustrative variation, the
expression vector may include a nucleotide sequence encoding a hERG
K.sup.+ channel having an amino acid sequence that is at least 80%
homologous to that of SEQ ID NO: 1.
[0241] Following transfection and isolation of high-expressing
cells by two successive rounds of FACS, single cell clone
expansion, and immunocytochemical staining, a hERG K.sup.+
channel-expressing cell population with a B.sub.max of greater than
450 pmol of hERG K.sup.+ channel per mg of total membrane protein
was obtained. Further, the underlying methodology enables the
production of hERG K.sup.+ channel-expressing cell populations with
B.sub.max values over a broad range, i.e., preferably at least
about 100 pmol to greater than 450 pmol of hERG K.sup.+ channel per
mg of total membrane protein, more preferably about 200 pmol to
greater than about 450 pmol of hERG K.sup.+ channel per mg of total
membrane protein, even more preferably about 300 pmol to greater
than about 450 pmol of hERG K.sup.+ channel per mg of total
membrane protein, and most preferably a B.sub.max of greater than
450 pmol of hERG K.sup.+ channel per mg of total membrane
protein.
[0242] Another aspect of the present invention provides a process
for preparing a fluorescent tracer compound of structural formula
(I)
##STR00005##
the process comprising:
[0243] a) reacting a compound of structural formula (II)
##STR00006##
in dimethylformamide/diisopropylethyl amine at room temperature
with a compound of structural formula (III)
[R.sup.14O].sub.k--[CZ--(CR.sup.4R.sup.5).sub.u-(D).sub.w].sub.z--R.sup.-
3 (III)
wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, Ar, B, D, L,
Q, W, X Y, Z, m, q, u, w, and z are as defined above; k is 0 or 1;
and R.sup.14 is a component of an active ester; provided that if Z
is .dbd.O, then k is 1, and provided that if Z is .dbd.S, then k is
0, u is 0, w is 1, z is 1, and D is .dbd.N.
[0244] In one illustrative embodiment, compound (III) is a
carboxylic acid succinimidyl ester, such that R.sup.14 is
succinimidyl. In another illustrative embodiment, compound (III) is
a carboxylic acid 4-sulfo-2,3,5,6-tetrafluorophenyl ester, such
that R.sup.14 is 4-sulfo-2,3,5,6-tetrafluorophenyl. In yet another
illustrative embodiment, compound (III) is an isothiocyanate.
[0245] Another aspect of the present invention provides an assay
for screening test compounds, wherein the assay is a binding assay
using a fluorescent tracer described herein binding to a source of
the hERG K.sup.+ channel or fragment thereof. Illustratively, the
assay includes the steps of: [0246] a) incubating the fluorescent
tracer or salt thereof with the source of the hERG K.sup.+ channel
or fragment thereof in an assay buffer in the presence or absence
of different amounts of a test compound or a mixture of test
compounds; and [0247] b) measuring an effect of the test compound
or the mixture of test compounds on the amount of the fluorescent
tracer bound to the hERG K.sup.+ channel or fragment thereof. In an
illustrative embodiment, the assay buffer comprises 15 mM to 50 mM
HEPES, 5 mM to 20 mM KCl, 0.5 mM to 2 mM MgCl.sub.2, and 0.02% to
about 0.1% PLURONIC F-127, and the source of the hERG K.sup.+
channel or fragment thereof is selected from the group consisting
of: [0248] i) membrane preparations derived from cells expressing
on the surface thereof the hERG K.sup.+ channel of fragment
thereof; [0249] ii) cells expressing on the surface thereof the
hERG K.sup.+ channel of fragment thereof; and [0250] iii) membrane
preparations derived from tissue expressing on the surface thereof
the hERG K.sup.+ channel of fragment thereof. In a preferred
embodiment, the source of the hERG K.sup.+ channel or fragment
thereof are membrane preparations derived from cells expressing on
the surface thereof the hERG K.sup.+ channel or fragment thereof,
and the assay buffer comprises 25 mM HEPES, 15 mM KCl, 1 mM
MgCl.sub.2, and 0.05% PLURONIC F-127, wherein the pH of the assay
buffer is between pH 7.2 and pH 7.6 at room temperature. In a most
preferred embodiment, the cells express greater than about 450
.mu.mol of hERG K.sup.+ channel per mg of total membrane protein,
and the assay buffer is at pH 7.4.
[0251] Another aspect of the present invention provides a method
for characterizing the activity of a test compound as a hERG
K.sup.+ channel blocker. Illustratively, the method includes the
steps of:
[0252] a) contacting the test compound with a membrane preparation
containing a hERG K.sup.+ channel having the amino acid sequence of
SEQ ID NO: 1, the membrane preparation derived from cells
transfected with a nucleic acid expression vector including a
nucleotide sequence which encodes the hERG K.sup.+ channel, in an
assay buffer in the presence of a fluorescent tracer described
herein;
[0253] b) monitoring whether the test compound influences the
binding of the fluorescent tracer to the membrane preparation
containing the hERG K.sup.+ channel; and
[0254] c) determining the hERG K.sup.+ channel blocker activity of
the test compound.
In an illustrative embodiment, the nucleic acid expression vector
further includes a nucleotide sequence which encodes an internal
ribosomal entry site protein and a nucleotide sequence which
encodes CD-8 plasma membrane protein, wherein the nucleotide
sequences which encode the internal ribosomal entry site protein
and the CD-8 plasma membrane protein are located successively
downstream from the nucleotide sequence which encodes the hERG
K.sup.+ channel. In another illustrative embodiment, the nucleic
acid expression vector has the nucleotide sequence of SEQ ID NO: 2,
and monitoring whether the test compound influences the binding of
the fluorescent tracer to the membrane preparation containing the
hERG K.sup.+ channel is measured by fluorescence polarization. In a
preferred embodiment, the assay buffer is at pH 7.4 and comprises
25 mM HEPES, 15 mM KCl, 1 mM MgCl.sub.2, and 0.05% PLURONIC F-127,
and expression of the hERG K.sup.+ channel is coupled to expression
of the CD-8 plasma protein by means of the nucleotide sequence
which encodes an internal ribosomal entry site protein.
[0255] Another aspect of the present invention provides a kit for
screening test compounds. Illustratively, the kit includes:
[0256] a) a fluorescent tracer described herein;
[0257] b) a source of the hERG K.sup.+ channel or fragment thereof;
and
[0258] c) an assay buffer.
In a preferred embodiment, the source of the hERG K.sup.+ channel
or fragment thereof are membrane preparations derived from cells
expressing on the surface thereof the hERG K.sup.+ channel or
fragment thereof, wherein the cells express at least about 100 pmol
of hERG K.sup.+ channel per mg of total membrane protein, and the
assay buffer includes 25 mM HEPES, 15 mM KCl, 1 mM MgCl.sub.2, and
0.05% PLURONIC F-127 at pH 7.4.
[0259] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLES
Chemical Syntheses
Preparation of Linker (4)
##STR00007##
[0260] 2,2'-Oxybis(ethane-2,1-diyl)dimethanesulfonate (2)
##STR00008##
[0262] Diethylene glycol (1, 5.0 mL, 53 mmol) and triethylamine
(Et.sub.3N, 16.2 mL, 116 mmol) were dissolved in 40 mL of
dichloromethane in a 100 mL 3-neck round bottom flask equipped with
a 10 mL addition funnel, a thermometer, an argon inlet, and a
magnetic stir bar. The solution was cooled to 0-5.degree. C. in an
ice bath. Methanesulfonyl chloride (MsCl, 8.4 mL, 108 mmol) was
added dropwise, via the addition funnel, at a rate so as to keep
the reaction solution below 15.degree. C. The ice bath was removed
and the reaction was stirred at ambient temperature overnight
(.about.16 hours). Water (25 mL) was added and the mixture was
stirred until the solids dissolved. The layers were separated and
the organic (lower) layer was washed successively with two 20 mL
portions of ice cold 3M hydrochloric acid, 25 mL of 5% aqueous
sodium carbonate, and 25 mL of saturated aqueous sodium chloride.
The organic phase (lower) was dried over anhydrous sodium sulfate,
filtered, and evaporated to dryness on a rotary evaporator to
provide 13 g of orange solid.
[0263] This material was purified by flash chromatography on 150 g
of Silica Gel 60 (230-400 mesh), eluting with 1:1 ethyl
acetate-toluene and collecting .about.125 mL fractions. Based on
TLC (silica gel, 4:1 ethyl acetate-toluene, ceric ammonium
molybdate visualization; R.sub.f (1)=0.05-0.3, R.sub.f
(2)=0.46-0.64), fractions were combined and concentrated to a
slurry by rotary evaporation under reduced pressure. The slurry was
cooled in an ice bath and the solid was collected by vacuum
filtration, washed with ice-cold toluene, and dried in vacuo at
25.degree. C. to afford
2,2'-oxybis(ethane-2,1-diyl)dimethanesulfonate (2) as a white solid
(11.79 g, 85% yield) that was homogeneous by TLC. .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 3.0 (s, 6H), .delta. 3.8 (m, 4H), and
.delta. 4.4 (m, 4H).
2-(2-(bis(tert-Butoxycarbonyl)amino)ethoxy)ethyl methanesulfonate
(3)
##STR00009##
[0265] 2,2'-Oxybis(ethane-2,1-diyl)dimethanesulfonate (2, 10 g, 38
mmol), potassium carbonate powder (5.3 g, 38 mmol), and
di-tert-butyl iminodicarboxylate (9.1 g, 42 mmol) were dissolved in
25 mL of anhydrous DMF in a 100 mL round bottom flask. This mixture
was stirred at 60-65.degree. C. for .about.3 hours. An additional
10 mL of anhydrous DMF was added and stifling at 60-65.degree. C.
was continued. After .about.24 h, TLC (silica gel, 1:1 ethyl
acetate-toluene, ceric ammonium molybdate visualization; R.sub.f
(2)=0.3-0.4, R.sub.f (3)=0.6-0.7) still showed starting material
(2), so stifling at 60-65.degree. C. was continued. After an
additional .about.24 hours, TLC showed no further change, so the
reaction was cooled to room temperature. Water (30 mL) and ethyl
acetate (60 mL) were added and the mixture was transferred to a
separatory funnel. The layers were separated and the aqueous
(lower) layer was extracted with ethyl acetate. The ethyl acetate
extracts were combined and washed successively with 25 mL of 1 M
aqueous hydrochloric acid, two 25 mL portions of water, and 25 mL
of saturated aqueous sodium chloride. The ethyl acetate solution
was dried over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure on a rotary evaporator followed by high
vacuum to afford 13.72 g of very pale yellow oil.
[0266] This material was purified by flash chromatography on 260 g
of Silica Gel 60 (230-400 mesh), eluting successively with 15:85
ethyl acetate-hexanes, 25:75 ethyl acetate-hexanes, and 30:70 ethyl
acetate-hexanes, collecting .about.125 mL fractions. Based on TLC
(silica gel, 15:85 ethyl acetate-hexanes, ceric ammonium molybdate
visualization), fractions were combined and concentrated by rotary
evaporation under reduced pressure to give 7.21 g of clear,
colorless oil. This material was still not homogeneous by TLC
(silica gel, 1:1 ethyl acetate-hexanes, ceric ammonium molybdate
visualization; R.sub.f (2)=0.08-0.2, R.sub.f (3)=0.65-0.75 with
minor impurities at R.sup.f=0.45-0.5 and R.sub.f=0.55-0.65) so it
was purified again by flash chromatography on 150 g of Silica Gel
60 (230-400 mesh), eluting with 20:80 ethyl acetate-hexanes and
collecting .about.125 mL fractions. Based on TLC (silica gel, 1:1
ethyl acetate-hexanes, ceric ammonium molybdate visualization),
fractions were combined and concentrated under reduced pressure on
a rotary evaporator followed by high vacuum to afford
2-(2-(bis(tert-butoxycarbonyl)amino)ethoxy)ethyl methanesulfonate
(3) as a clear, colorless oil (4.58 g, 31% yield). TLC (silica gel,
1:1 ethyl acetate-hexanes, ceric ammonium molybdate visualization)
shows 3 at R.sub.f=0.65-0.75 with a trace impurity at
R.sub.f=0.45-0.5. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 1.5
(s, 18H), .delta. 3.1 (s, 3H), and .delta. 3.5-3.9 (m)+.delta. 4.3
(m)=8H.
tert-Butyl 2-(2-bromoethoxy)ethylcarbamate (4)
##STR00010##
[0268] Lithium bromide (7.6 g, 87 mmol) was added to a solution of
compound 3 (3.35 g, 8.74 mmol) in 33 mL of acetone and heated in an
oil bath at 55-60.degree. C. After .about.3 hours TLC (1:1 ethyl
acetate-hexanes, ceric ammonium molybdate visualization) showed
complete disappearance of starting material (3, R.sub.f=0.7-0.8)
and appearance of a major new product (4, R.sub.f=0.8-0.9). The
reaction mixture was cooled to room temperature and water
(.about.15 mL) was added. The resulting solution was extracted
twice with 25 mL portions of ethyl acetate. The combined ethyl
acetate extract was washed with water (15 mL), dried over anhydrous
sodium sulfate, filtered, and concentrated by rotary evaporation
under reduced pressure to give a clear, colorless oil (2.05 g).
[0269] This material was purified by flash chromatography on 60 g
of Silica Gel 60 (230-400 mesh) eluting with 15:85 ethyl
acetate-hexanes and collecting .about.50 mL fractions. Based on TLC
(silica gel, 50:50 ethyl acetate-hexanes, ceric ammonium molybdate
visualization), fractions were combined and concentrated under
reduced pressure on a rotary evaporator followed by high vacuum to
afford tert-butyl 2-(2-bromoethoxy)ethylcarbamate (4) as a clear,
colorless oil (1.92 g, 82% yield) that was homogenous by TLC. Mass
spec: m/z=268.4 (100%), 270.2 (93%) [M+H].sup.+1H NMR (300 MHz,
CDCl.sub.3): .delta. 1.4 (s, 9H), .delta. 3.2-3.8 (m, 8H), and
.delta. 4.9 (br s, 1H).
Preparation of Spiropiperidine Ketone Linked to Dye (13)
##STR00011##
[0270] 1'-Benzoylspiro[chroman-2,4'-piperidin]-4-one (7)
##STR00012##
[0272] A solution of 2'-hydroxyacetophenone (5, 5.88 mL, 48.85
mmol), 1-benzoyl-4-piperidone (6, 10 g, 49 mmol), and pyrrolidine
(4.1 mL, 49 mmol) in 55 mL of methanol was heated in an oil bath at
65.degree. C. After .about.18 hours the oil bath was removed and
the solution was allowed to cool to room temperature. Additional
1-benzoyl-4-piperidone (6, 100 mg, 0.5 mmol) was added and heating
at 65.degree. C. was resumed. After an additional .about.2.5 hours
the oil bath was removed and the solution was allowed to cool to
room temperature. This mixture was concentrated under reduced
pressure on a rotary evaporator followed by high vacuum to give a
viscous, orange oil. This oil was triturated with diethyl ether to
provide a solid that was collected by vacuum filtration, washed
with diethyl ether, and dried in vacuo at room temperature to
afford 1'-benzoylspiro[chroman-2,4'-piperidin]-4-one (7) as an
off-white solid (14.72 g, 93.8% yield). The filtrate was evaporated
to dryness, triturated with diethyl ether, filtered, washed with
diethyl ether, and dried in vacuo at room temperature to afford
additional compound 7 as a pale tan solid (0.54 g, 3.7% yield).
Both samples were homogeneous by TLC (silica gel, 1.5% methanol in
dichloromethane, UV visualization; R.sub.f (7)=0.15-0.2).
1'-Benzoyl-6-nitrospiro[chroman-2,4'-piperidin]-4-one (8)
##STR00013##
[0274] 1'-Benzoylspiro[chroman-2,4'-piperidin]-4-one (7, 12.6 g,
39.2 mmol) was suspended in 120 mL of acetic anhydride under argon
with vigorous stirring. The mixture was cooled to 0-5.degree. C. in
an ice bath and fuming nitric acid (15 mL) was added dropwise. The
suspended solid dissolved as the nitric acid was added. After the
addition was completed, the reaction was stirred at ice bath
temperature for 5 minutes and then allowed to warm to room
temperature. After .about.45 minutes at room temperature, the
reaction rapidly became exothermic and was cooled again in an ice
bath. After .about.1 hour total reaction time, TLC (silica gel,
50:50 ethyl acetate-hexanes, iodine and UV visualization) of a
small aliquot (quenched into saturated aqueous sodium carbonate and
extracted into ethyl acetate) showed multiple products but complete
consumption of starting compound 7 [R.sub.f (7)=0.3-0.4]. After
.about.1.5 hours total reaction time the reaction mixture was
poured into 300 mL of ice-cold saturated aqueous sodium carbonate.
After the mixture stirred for a few minutes, solid sodium carbonate
was added until the solution reached pH 6-7. This solution was
extracted with three 200 mL portions of ethyl acetate. The organic
extracts were combined, dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure on a rotary
evaporator to afford an orange-brown foam (16.6 g).
[0275] This material was combined with 0.62 g of material from a
previous, smaller-scale reaction, and preabsorbed onto silica gel
by dissolving in ethyl acetate, adding Silica Gel 60 (70-230 mesh,
50 g), and removing solvent by rotary evaporation under reduced
pressure. The resulting powder was applied to the top of a
slurry-packed (with 10:90 ethyl acetate-hexanes) 100 g Silica Gel
60 (70-230 mesh) flash chromatography column and eluted
successively with 10:90 ethyl acetate-hexanes, 20:80 ethyl
acetate-hexanes, 30:70 ethyl acetate-hexanes, 40:60 ethyl
acetate-hexanes, and 50:50 ethyl acetate-hexanes, collecting
.about.125 mL fractions. Based on TLC (silica gel, 50:50 ethyl
acetate-hexanes), fractions were combined and concentrated under
reduced pressure on a rotary evaporator to provide 8.33 g of
slightly impure (by TLC as above) yellow solid.
[0276] This material was again preabsorbed onto silica gel by
dissolving in dichloromethane, adding Silica Gel 60 (70-230 mesh,
25 g), and removing solvent by rotary evaporation under reduced
pressure. The resulting powder was applied to the top of a
slurry-packed (with 50:50 ethyl acetate-hexanes) 335 g Silica Gel
60 (70-230 mesh) flash chromatography column and eluted
successively with 50:50 ethyl acetate-hexanes and 60:40 ethyl
acetate-hexanes, collecting .about.125 mL fractions. Based on TLC
(silica gel, 1:1 ethyl acetate-hexanes), fractions were combined
and concentrated under reduced pressure on a rotary evaporator to
provide 6.59 g of yellow foamy solid that was still slightly impure
by TLC (silica gel, 50:50 ethyl acetate-hexanes or 3% methanol in
dichloromethane, UV visualization).
[0277] This material was repurified by flash chromatography on 264
g of Silica Gel 60 (70-230 mesh), eluting successively with
dichloromethane, 1% methanol in dichloromethane, and 2% methanol in
dichloromethane, collecting .about.125 mL fractions. Based on TLC
(silica gel, 3% methanol in dichloromethane, UV visualization),
fractions were combined and concentrated under reduced pressure on
a rotary evaporator to afford
1'-benzoyl-6-nitrospiro[chroman-2,4'-piperidin]-4-one (8, 6.5 g,
45% yield) as a pale yellow foamy solid. By TLC (silica gel, 3%
methanol in dichloromethane, UV visualization), this material
contained one major component (R.sub.f=0.2-0.3) plus a minor
contaminant (R.sub.f=0.14-0.17).
6-Amino-1'-benzoylspiro[chroman-2,4'-piperidin]-4-one (9)
##STR00014##
[0279] A solution of tin(II) chloride dihydrate (25.4 g, 112 mmol)
in 152 mL of concentrated hydrochloric acid was added dropwise,
over .about.50 minutes, to a solution of
1'-benzoyl-6-nitrospiro[chroman-2,4'-piperidin]-4-one (8, 5.9 g, 16
mmol) in 152 mL of tetrahydrofuran under an argon atmosphere. The
reaction mixture was then stirred at room temperature. After
.about.2 hours total, TLC (silica gel, 10% methanol in
dichloromethane, UV visualization, R.sub.f (8)=0.75-0.85) of a
small aliquot (quenched into excess aqueous sodium hydroxide and
extracted into ethyl acetate) showed that the reaction was
complete. The reaction mixture was cooled to 0-5.degree. C. in an
ice bath, and 40% aqueous sodium hydroxide was added dropwise until
the solution reached pH 11-13. The resulting solution was extracted
with three 150 mL portions of ethyl acetate, and the combined ethyl
acetate extracts were washed successively with 50 mL portions of
water and saturated aqueous sodium chloride. The organic extract
was dried over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure on a rotary evaporator followed by high
vacuum to give 8.2 g of viscous, yellow oil that showed several
spots (one major) on TLC [silica gel, 10% methanol in
dichloromethane (R.sub.f 0.4-0.65) or 3% methanol in
dichloromethane (R.sub.f 0.2-0.35), UV visualization].
[0280] This material was purified by flash chromatography on 220 g
of Silica Gel 60 (230-400 mesh) eluting successively with
dichloromethane and 2% methanol in dichloromethane, collecting
.about.200 mL fractions. Based on TLC (silica gel, 10% methanol in
dichloromethane, UV visualization), fractions were combined and
concentrated under reduced pressure on a rotary evaporator followed
by high vacuum to provide 4.2 g of orange residue that showed
several spots by TLC.
[0281] This material was repurified by flash chromatography on 200
g of Silica Gel 60 (230-400 mesh) eluting successively with 40:60
ethyl acetate-toluene, 50:50 ethyl acetate-toluene, and 70:30 ethyl
acetate-toluene, collecting .about.125 mL fractions. Based on TLC
(silica gel, 1:1 ethyl acetate-toluene, UV visualization),
fractions were combined and concentrated under reduced pressure on
a rotary evaporator followed by high vacuum to provide 2.15 g (39%
yield) of 6-amino-1'-benzoylspiro-[chroman-2,4'-piperidin]-4-one
(9) as a yellow solid that was nearly homogeneous by TLC [R.sub.f
(9)=0.25-0.3, silica gel, 1:1 ethyl acetate-toluene, UV
visualization].
N-(1'-Benzoyl-4-oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfonamide
(10)
##STR00015##
[0283] Methanesulfonyl chloride (0.51 mL, 6.6 mmol) was added to a
solution of 6-amino-1'-benzoylspiro-[chroman-2,4'-piperidin]-4-one
(9, 1.85 g, 5.50 mmol) in 18.5 mL of pyridine. The reaction mixture
was stirred at room temperature. After .about.1.5 hours, TLC
(silica gel, 1:1 ethyl acetate-toluene, UV visualization) of a
small aliquot (quenched into ice-cold 3 M aqueous hydrochloric acid
and extracted into ethyl acetate) showed that the reaction was
complete [R.sub.f (9)=0.25-0.3, R.sub.f (10)=0.15-0.25]. After
.about.2 hours total the reaction mixture was poured into 75 mL of
ice cold 3 M aqueous hydrochloric acid and stirred for ten minutes.
The solid was collected by vacuum filtration, washed with water,
and dried in vacuo at ambient temperature to give 2.17 g of pink
solid. This material was suspended in a mixture of methanol (50 mL)
and ethanol (50 mL) and heated to boiling. Additional methanol was
added until dissolution was complete and the hot solution was
filtered (gravity). The resulting solution was allowed to cool to
room temperature (some precipitation) and then cooled in the
refrigerator at 0-5.degree. C. The solid that precipitated was
collected by vacuum filtration, washed with ice-cold 1:1
methanol-ethanol, and dried in vacuo at ambient temperature to
afford 0.86 g (38% yield) of
N-(1'-benzoyl-4-oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfonamide
(10) as a light pink solid. This material was homogeneous by TLC
(silica gel, 5% methanol in dichloromethane, UV visualization,
R.sub.f (10)=0.3-0.35).
[0284] An additional 0.42 g (18% yield) of
N-(1'-benzoyl-4-oxospiro[chroman-2,4'-piperidine]-6-yl)-methanesulfonamid-
e (10) as a light pink solid was obtained by evaporating the mother
liquor to dryness under reduced pressure by rotary evaporation
followed by recrystallization of the residue from 2:1
methanol-ethanol. This material was homogeneous by TLC (silica gel,
5% methanol in dichloromethane, UV visualization, R.sub.f
(10)=0.3-0.35).
N-(4-Oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfonamide
hydrochloride (11)
##STR00016##
[0286]
N-(1'-Benzoyl-4-oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfo-
namide (10, 0.81 g, 1.95 mmol) was suspended in a mixture of
absolute ethanol (10 mL) and 6 M aqueous hydrochloric acid (10 mL)
and stirred at 85-90.degree. C. in an oil bath. After 1 hour the
oil bath temperature was increased to 95.degree. C. After
.about.3.5 hours at 95.degree. C. the reaction was allowed to cool
to room temperature. TLC (silica gel, 10% methanol in
dichloromethane, UV visualization) showed no remaining starting
material [R.sub.f (10)=0.65-0.7]. Solvent was removed under reduced
pressure on a rotary evaporator. Ethanol was added to the residue
and then evaporated to dryness under reduced pressure on a rotary
evaporator. The ethanol addition and evaporation to dryness was
repeated two more times. The residue was dried in vacuo to afford
0.77 g (114% yield) of
N-(4-oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfonamide
hydrochloride (11) as a light yellow solid that was homogeneous by
TLC [silica gel, 10% methanol in dichloromethane containing a small
amount of concentrated aqueous ammonium hydroxide, UV
visualization, R.sub.f (11)=0.12-0.19]. Mass spec: m/z=311.2
[M+H].sup.+
tert-Butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-
-1'-yl)ethoxy)ethylcarbamate (12)
##STR00017##
[0288] A solution of N,N-diisopropylethylamine (1.3 mL, 7.5 mmol),
tert-butyl 2-(2-bromoethoxy)ethyl-carbamate (4, 0.59 g, 2.2 mmol),
and N-(4-oxospiro[chroman-2,4'-piperidine]-6-yl)methanesulfon-amide
hydrochloride (11, 0.5 g, 1.4 mmol) in 10 mL of anhydrous DMF was
stirred at 60-65.degree. C. After .about.24 hours TLC (silica gel,
10% methanol in dichloromethane, UV visualization) showed almost
complete disappearance of starting amine 11 (R.sub.f=0.02-0.06) and
appearance of one major new product (R.sub.f=0.35-0.45). The
reaction was allowed to cool to room temperature and 10 mL of water
was added. The resulting mixture was transferred to a separatory
funnel with the aid of ethyl acetate and the layers were separated.
The aqueous layer was extracted with 20 mL of ethyl acetate. The
organic extracts were combined, washed successively with 10 mL
portions of water and saturated aqueous sodium chloride, dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure on a rotary evaporator followed by high vacuum to provide
0.94 g of orange-brown oil.
[0289] This material was combined with 94 mg from a previous,
smaller reaction and purified by flash chromatography on 43 g of
Silica Gel 60 (230-400 mesh), eluting with 5% methanol in
dichloromethane and collecting .about.40 mL fractions. Based on TLC
(silica gel, 10% methanol in dichloromethane, UV visualization)
fractions were combined and concentrated under reduced pressure on
a rotary evaporator to afford 0.51 g (59% combined yield) of
tert-butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-1'-yl)eth-
oxy)ethylcarbamate (12) as a yellow foam that was homogeneous by
TLC (R.sub.f=0.4-0.5). Mass spec: m/z=498.18 [MH].sup.+ 1H NMR (300
MHz, CDCl.sub.3): .delta. 1.4 (s, 9H), .delta. 1.9-2.6 (m, 8H),
.delta. 2.65 (s, 2H), .delta. 2.9 (s, 3H), .delta. 3.1-3.6 (m, 8H),
.delta. 5.1 (br s, 1 H), .delta. 6.9-7.6 (m, 4H).
Fluorescent Tracer (13)
##STR00018##
[0291] A solution of tert-butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-1'-yl)eth-
oxy)ethylcarbamate (12, 5.0 mg, 0.010 mmol) in a mixture of
dichloromethane (0.9 mL) and trifluoroacetic acid (TFA, 0.1 mL) was
stirred at room temperature for .about.3 hours. This solution was
evaporated to dryness under reduced pressure on a rotary evaporator
at <35.degree. C. Toluene (.about.1 mL) was added and the
solution was evaporated to dryness under reduced pressure on a
rotary evaporator at <35.degree. C. The toluene
addition/evaporation sequence was repeated one or two more
times.
[0292] The residue was dissolved in 4.0 mL of anhydrous DMF and 1.0
mL aliquots of the solution were transferred to 5 mL round bottom
flasks containing .about.1 mg of "amine-reactive" [isothiocyanate,
carboxylic acid succinimidyl ester, or carboxylic acid STP
(4-sulfo-2,3,5,6-tetrafluorophenyl)ester] fluorescent dye.
Anhydrous diisopropylethylamine (0.2 mL) was added to each flask.
The flasks were wrapped with aluminum foil to block the light and
the reactions were stirred at room temperature, under argon,
overnight (16-20 hours). Methanol (0.5 mL) was added to each flask
and the solution was stirred at room temperature for 1-3 hours.
Solvent was removed under reduced pressure on a rotary evaporator
at <35.degree. C. Toluene (.about.1 mL) was added and the
solution was evaporated to dryness under reduced pressure on a
rotary evaporator at <35.degree. C. The toluene
addition/evaporation sequence was repeated one or two more times.
The resulting material was purified by preparative HPLC. (The same
general procedure was employed to prepare additional fluorescent
tracers of general formula 13, from intermediate compound 14;
fluorescent tracers of general formula 20, from intermediate
compound 17; and fluorescent tracers of general formula 21, from
intermediate 19, all of which tracers are listed in Table 1).
[0293] Representative HPLC purification conditions:
[0294] Column: Zorbax RX, C-8, 5 microns, 4.6 mm.times.25 cm
[0295] Buffer A: 0.1% TFA, 10% acetonitrile
[0296] Buffer B: 0.085% TFA, 90% acetonitrile
[0297] Gradient: 2-25 min 10-50% B; 35-45 min 50-100% B
[0298] Flow rate: 1.0 mL/min
[0299] Injection: 100 .mu.L of 1.4 mM in Buffer A
N-(1'-(2-(2-Aminoethoxy)ethyl)-4-oxospiro[chroman-2,4'-piperidine]-6-yl)me-
thanesulfon-amide dihydrochloride (14)
##STR00019##
[0301] Ten drops of concentrated hydrochloric acid was added to a
solution of tert-butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-1'-yl)eth-
oxy)ethylcarbamate (12, 0.51 g, 1.02 mmol) in 5 mL of glacial
acetic acid and the resulting solution was stirred at room
temperature. After .about.1.5 hours, TLC (silica gel, 10% methanol
in dichloromethane, UV visualization) shows complete disappearance
of starting compound 12 (R.sub.f=0.2-0.3) and a new spot at the
origin. Volatile components were removed under reduced pressure on
the rotary evaporator. Toluene (.about.10 mL) was added and
evaporated under reduced pressure on the rotary evaporator. The
toluene addition and evaporation was repeated two more times and
the residue was dried under high vacuum. The resulting residue was
triturated with 3 mL of diethyl ether, resulting in formation of a
tan solid. The diethyl ether was removed under reduced pressure on
the rotary evaporator followed by high vacuum to give a tan
solid.
(R)-tert-Butyl
2-(2-(4-hydroxy-6-(methylsulfonamido)spiro[chroman-2,4'-piperidine]-1'-yl-
)ethoxy)ethylcarbamate (16)
##STR00020##
[0303] tert-Butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-1'-yl)eth-
oxy)ethyl-carbamate (12, 0.25 g, 0.50 mmol) was dissolved in 5 mL
of dichloromethane containing 0.038 mL (0.5 mmol) of 2-propanol and
the solution was cooled to -20.degree. C. Borane dimethyl sulfide
complex (Me.sub.2S.BH.sub.3, 0.126 mL, .about.1.26 mmol) was added
dropwise and the solution was stirred at -20.degree. C. for 1 hour.
(S)-Tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo-[1,2-c][1,3,2]oxazabor-
ole-borane complex (15, 15 mg, 0.05 mmol; prepared from
(S)-tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaboro-
le as described in Xavier, L. C.; et al. Org. Syn. 1998, Coll. Vol.
9, 676) was added in a single portion and the mixture was stirred
at -20.degree. C. for 30 minutes. The reaction mixture was allowed
to warm slowly (over .about.30 minutes) to 0.degree. C. and then
stirred at 0.degree. C. for 2.5-3 hours. TLC (silica gel, 10%
methanol in dichloromethane, UV visualization) show complete
disappearance of starting ketone 12 (R.sub.f=0.4-0.5). Methanol
(4.5 mL) was added and the reaction was allowed to warm to room
temperature. The reaction flask was fitted with a short path
distillation head, placed in an oil bath, and heated to remove
volatile components until the distillate temperature reached
62.degree. C. An additional 5 mL of methanol was added and the
flask was heated in an oil bath at .about.75.degree. C. for 30
minutes as about half of the methanol was removed by distillation.
The flask was cooled to room temperature, acetonitrile (5 mL) was
added, and the mixture was evaporated to dryness under reduced
pressure on the rotary evaporator followed by high vacuum at
ambient temperature, affording a yellow solid (crude 16) that
showed multiple components by TLC .about.silica gel, 10% methanol
in dichloromethane or 10% methanol in dichloromethane containing a
small amount of aqueous ammonium hydroxide, iodine visualization;
R.sub.f (12)=0.35-0.4, R.sub.f (major component)=0.03-0.17 in 10%
MeOH/CH.sub.2Cl.sub.2; R.sub.f (12)=0.47-0.53, R.sub.f (major
component)=0.25-0.35 in 10% MeOH/CH.sub.2Cl.sub.2 containing
NH.sub.4OH].
[0304] This material (crude 16) was purified by flash
chromatography on a 10 g Silica Gel 60 (230-400 mesh) column,
eluting with 7% methanol in dichloromethane containing 0.2% aqueous
ammonium hydroxide and collecting .about.10 mL fractions. Based on
TLC (silica gel, 10% methanol in dichloromethane, iodine
visualization), fractions were combined and concentrated under
reduced pressure on a rotary evaporator to afford 195 mg (77%
yield) of (R)-tert-butyl
2-(2-(4-hydroxy-6-(methylsulfonamido)spiro[chroman-2,4'-piperidine]-1'-yl-
)ethoxy)ethylcarbamate (16) as a foamy, white solid. .sup.1H NMR:
consistent with the desired product (16). Mass spec: m/z=500.1551
(expected for [M+H].sup.+=500.2425)
N-(1'-(2-(2-aminoethoxy)ethyl)spiro[chromene-2,4'-piperidine]-6-yl)methane-
sulfonamide dihydrochloride (17)
##STR00021##
[0306] (R)-tert-butyl
2-(2-(4-hydroxy-6-(methylsulfonamido)spiro[chroman-2,4'-piperidine]-1'-yl-
)ethoxy)-ethylcarbamate (16, 193 mg, 0.386 mmol) was dissolved in 5
mL of glacial acetic acid. The solution was cooled in a cool water
bath and 8 drops of concentrated hydrochloric acid were added. The
water bath was removed and the reaction was allowed to warm to room
temperature. After 3 hours, TLC (silica gel, 10% methanol in
dichloromethane containing a small amount of aqueous ammonium
hydroxide, iodine visualization) showed complete disappearance of
starting compound 16 (R.sub.f=0.3-0.35) and appearance of a single
new compound (17, R.sub.f=0.18-0.25). The reaction mixture was
concentrated to dryness under reduced pressure on a rotary
evaporator. Toluene was added and evaporated to dryness under
reduced pressure on a rotary evaporator. This toluene addition and
evaporation was repeated two more times and the residue was dried
under high vacuum at ambient temperature, giving a viscous yellow
residue. This material was triturated with diethyl ether to give
210 mg (120% yield) of
N-(1'-(2-(2-aminoethoxy)ethyl)spiro[chromene-2,4'-piperidine]-6-yl)methan-
esulfonamide dihydrochloride (17) as a pale yellow solid. Mass
spec: m/z=382.13 [M+H].sup.+
tert-Butyl
2-(2-(4-(hydroxyimino)-6-(methylsulfonamido)spiro[chroman-2,4'--
piperidine]-1'-yl)ethoxy)ethylcarbamate (18)
##STR00022##
[0308] Anhydrous pyridine (40 .mu.L, 0.5 mmol) and hydroxylamine
hydrochloride (8 mg, 0.11 mmol) were added to a solution of
tert-butyl
2-(2-(6-(methylsulfonamido)-4-oxospiro[chroman-2,4'-piperidine]-1'-yl)eth-
oxy)ethylcarbamate (12, 50 mg, 0.10 mmol) in 5 mL of anhydrous
methanol. This solution was stirred at room temperature for
.about.1 hour and then at .about.60.degree. C. for .about.1 hour.
Additional hydroxylamine hydrochloride (6.2 mg, 0.09 mmol) and
anhydrous pyridine (40 .mu.L, 0.5 mmol) were added and stifling was
continued at 60-65.degree. C. for .about.1 hour. More hydroxylamine
hydrochloride (13.9 mg, 0.20 mmol) was added and stifling was
continued at 60-65.degree. C. for .about.80 minutes. Hydroxylamine
hydrochloride (7 mg, 0.10 mmol) was added and stirring was
continued at 60-65.degree. C. for .about.2 hours. TLC (silica gel,
10% methanol in dichloromethane, UV visualization) showed complete
disappearance of starting ketone 12 (R.sub.f=0.35-0.4) and a single
new spot (18, R.sub.f=0.18-0.27). The reaction mixture was
concentrated to dryness under reduced pressure on a rotary
evaporator. The residue was dissolved in 5 mL of ethyl acetate and
extracted successively with 10 mL of 0.5 M aqueous hydrochloric
acid, mL of water, and 5 mL of saturated aqueous sodium chloride.
TLC (as above) showed the product (18) in the combined aqueous
extracts, which were adjusted to pH .about.13 by addition of sodium
hydroxide pellets and then to pH .about.4 by addition of 3 M
aqueous hydrochloric acid. The aqueous solution was extracted with
three 20 mL portions of ethyl acetate. The combined ethyl acetate
extract was dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness under reduced pressure on a rotary evaporator
followed by high vacuum overnight to afford 50.3 mg (98% yield)
tert-butyl
2-(2-(4-(hydroxyimino)-6-(methylsulfonamido)-spiro[chroman-2,4'-piperidin-
e]-1'-yl)ethoxy)ethylcarbamate (18).
N-(1'-(2-(2-Aminoethoxy)ethyl)-4-(hydroxyimino)spiro[chroman-2,4'-piperidi-
ne]-6-yl)methanesulfonamide dihydrochloride (19)
##STR00023##
[0310] tert-Butyl
2-(2-(4-(hydroxyimino)-6-(methylsulfonamido)-spiro[chroman-2,4'-piperidin-
e]-1'-yl)ethoxy)ethylcarbamate (18, 50.3 mg, 0.10 mmol) was
dissolved in 1 mL of glacial acetic acid. The solution was cooled
in a cool water bath and 2 drops of concentrated hydrochloric acid
was added. The water bath was removed and the solution was allowed
to warm to room temperature. After 45 minutes, TLC (silica gel, 10%
methanol in dichloromethane, UV visualization) showed complete
disappearance of starting compound (R.sub.f (18)=0.15-0.25) and
appearance of a single new product with R.sub.f=0. Toluene (5 mL)
was added and then evaporated under reduced pressure on a rotary
evaporator. This toluene addition and evaporation was repeated two
more times and the residue was dried under high vacuum for .about.1
hour to afford 39.2 mg (82% yield) of
N-(1'-(2-(2-amino-ethoxy)ethyl)-4-(hydroxyimino)spiro[chroman-2,4'-piperi-
dine]-6-yl)methanesulfonamide dihydrochloride (19).
TABLE-US-00001 TABLE 1 13 ##STR00024## 20 ##STR00025## 21
##STR00026## Fluorescent Tracers of General Formulae 13, 20, and 21
HPLC Mass Spec Compound Scaffold Fluorescent Dye Retention Time
Purity Exact Mass Observed m/z 20a Alkene 5-FAM 16.3 min 100%
739.22 739.22 20b Alkene FITC 19.2 min 100% 786.24 771.19 20c
Alkene Rhodamine Red-X 25.2 min ND 1034.40 1034.34 20d Alkene Texas
Red-X 31.2 min ND 1082.40 1082.34 20e Alkene BODIPY .RTM. TR 30.6
min ND 787.25 788.23 20f Alkene BODIPY .RTM. FL 30.8 min 100%
655.28 656.27 20g Alkene BODIPY .RTM. TR-X 30.9 min ND 900.33
901.34 20h Alkene DDAO 28.6 min ND 770.23 771.21 20m Alkene Alexa
Fluor .RTM. 546 21.2-23.4 min ND 1322.28 1322.27 13a Ketone 5-FAM
14.4 min 100% 755.21 756.19 13b Ketone FITC 17.9 min 100% 786.20
786.20 13c Ketone Rhodamine Red-X 28.1 min 100% 1050.39 1050.38 13d
Ketone Texas Red-X 30.0 min 100% 1098.39 1098.39 13e Ketone BODIPY
.RTM. TR 20.3 min 100% 803.24 804.22 13f Ketone BODIPY .RTM. FL
27.5 min 100% 671.28 672.18 13g Ketone BODIPY .RTM. TR-X 35.0 min
100% 916.33 917.31 13h Ketone DDAO 26.8 min 100% 786.23 787.19 13i
Ketone 5-TAMRA 26.7 min 95% 809.31 810.0 13j Ketone 6-TAMRA 24.0
min 98% 809.31 810.0 13k Ketone BODIPY .RTM. TMR 35.3 min 100%
777.32 778.0 13l Ketone BODIPY .RTM. TMR-X 35.4 min 100% 890.84
891.2 21b Oxime FITC 20.0 min 100% 801.21 802.27 21e Oxime BODIPY
.RTM. TR 16.0 min 100% 818.25 819.33 21g Oxime BODIPY .RTM. TR-X
19.6 min 100% 931.34 932.35
Materials and Methods
[0311] Membrane Preparations
[0312] The hERG-T-REx.TM. 293 cell line (Invitrogen, Carlsbad,
Calif.) was used to generate membrane preparations for testing the
affinity of fluorescent tracer molecules. Cells were maintained
following the manufacturers recommended protocol at 37.degree. C.
in 5% CO.sub.2 atmosphere, and were induced to express the hERG
channel by the addition of 1 .mu.g/mL doxycycline (MP biomedicals,
Solon, Ohio). Following a 24 hr of induction, cells were washed
with divalent ion free PBS (Invitrogen), harvested and washed once
with Versene (Invitrogen) and then spun down at .about.500 g for 5
minutes. The cell pellet was kept on ice and resuspended with
ice-cold homogenization buffer containing 20 mM HEPES (pH 7.4), 5
mM KCl, 1 mM EDTA, 1 mM PMSF, 0.01 mM E-64, and 10 .mu.g/ml
leupeptin. The cells were then homogenized with a Bio Polytron hand
held homogenizer (Brinkmann, Westbury, N.Y.), spun down at 40,000 g
for 10 min at 4.degree. C., and the supernatant was discarded. The
membrane pellet was resuspended in ice-cold homogenization buffer,
then homogenized and centrifuged again. The supernatant was
discarded and the membrane pellet was resuspended in storage buffer
containing 20 mM HEPES (pH 7.4), 5 mM KCl, 1 mM MgCl.sub.2, and 1
mM EGTA. The membrane pellet was broken up by pipetting, and
sonicated until a uniform suspension was achieved. The resulting
membrane preparation was aliquotted and stored at -80.degree. C.
During the process of assay optimization, the homogenization and
storage buffers were replaced with the experimentally-determined
assay buffer containing 25 mM HEPES (pH 7.5), 15 mM KCl, 1 mM
MgCl.sub.2, and 0.05% Pluronic F-127.
[0313] Radioligand Binding Assays
[0314] For saturation binding assays, hERG membranes were diluted
into assay buffer containing 60 mM KCl, 71.5 mM NaCl, 1 mM
CaCl.sub.2, 2 mM MgCl.sub.2,0.1% BSA, and 10 mM HEPES, pH 7.4.
Next, 80 .mu.L of the membrane/buffer mixture was added to each
well of a 96-well deep-well assay block (Corning, Lowell, Mass.)
containing either 5 .mu.L of 20.times. unlabeled astemizole (to
determine non-specific binding) or empty wells (total binding).
Labeled [.sup.3H]-astemizole was added as 20 .mu.L of a 5.times.
stock of the appropriate concentration. Non-specific binding was
determined in parallel for each concentration of label assayed. All
assays were performed using triplicate wells, and the final
concentration of membrane protein in the assay was 20
.mu.g/well.
[0315] To determine IC.sub.50 values of test compounds, hERG
membranes were diluted into assay buffer and 80 .mu.L of
membrane/buffer mixture was added per well to a 96 well deep well
assay block containing either 5 .mu.L of 20.times. unlabeled
astemizole (non-specific binding), 5 .mu.L of 20.times. reference
compounds or empty wells (total binding). [.sup.3H]-astemizole was
added as 20 .mu.L of a 5.times. stock. Test compounds were
typically tested using eight concentrations in duplicate wells. The
final concentration of membrane protein in the assay was 10
.mu.g/well and the final concentration of [.sup.3H]-astemizole was
1.5 nM.
[0316] After gentle vortexing to mix, the assay blocks were covered
with parafilm and incubated at room temperature for two hours. The
reaction was terminated by filtration through GF/B Unifilters
(PerkinElmer, Waltham, Mass.) that had been presoaked for two hours
in a 0.3% polyethylenimine solution (Sigma-Aldrich, St. Louis,
Mo.). The filter plates were then washed with 6-8 volumes of cold
(4.degree. C.) wash buffer containing 131.5 mM NaCl, 1 mM
CaCl.sub.2, 2 mM MgCl.sub.2, 10 mM HEPES and 0.1% BSA and then
dried on a hot block at 85.degree. C. for 1-2 hours. The bottom of
the plates were sealed and 50 .mu.L of Microscint 20 (PerkinElmer)
was added to each well. The top of the plate was sealed with
TopSeal (PerkinElmer) and the plates were analyzed after a minimum
of 2 hours on a TopCount scintillation counter (PerkinElmer).
[0317] Patch-Clamp Recording
[0318] Cells expressing hERG were plated on 55 mm round coverslips
and allowed to adhere in an incubator overnight. Coverslips were
placed on a microscope stage in a bath chamber and perfused with
PBS or equivalent at 1 mL/min. After obtaining a G .OMEGA. seal,
currents were recorded using the whole-cell recording mode (see,
Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.,
Improved patch-clamp techniques for high-resolution current
recording from cells and cell-free membrane patches. Pflugers Arch
1981, 391, (2), 85-100). Cells were held at -90 mV, and the
currents were filtered at 667 Hz and sampled at 2.0 kHz using an
EPC10/2 amplifier (HEKA, Oberkochen, Germany). Voltage-dependent
activation curves were obtained by stepping the command potential
to -70 mV for 50 ms, then stepping the command potential through
the range of -70 to +40 mV in 10 mV increments for a duration of 2
s, returning the command potential to -70 mV for 2 s and then
returning to the holding potential of -90 mV, every 5 s.
[0319] Cell Engineering
[0320] An expression vector was constructed using a CMV promoter to
drive transcription of a bicistronic element composed of nucleotide
sequences encoding the hERG channel and the CD8 cell surface
marker. Translation of the two proteins was linked by an internal
ribosomal entry site sequence (IRES). A puromycin-resistance marker
was included on the expression vector to provide a means of
selecting cells wherein stable, genomic incorporation of the
expression cassette had occurred. A culture of 293 cells was
maintained in 293 growth medium composed of high glucose
D-MEM+Na-pyruvate+GlutaMAX.TM. supplemented with FBS (10%),
MEM-NEAA, HEPES, and P/S at 37.degree. C. in 5% CO.sub.2. The day
before transfection, the cells were plated at .about.80% confluency
into 6-well dishes (Corning). Cells were transfected with plasmid
DNA using Lipofectamine.TM. LTX and Plus.TM. reagents (Invitrogen)
according to the manufacturer's protocol. The next day, the cells
were harvested, expanded into a T175 flask (Corning), and put under
selection by the addition of puromycin (Sigma) to the medium to a
final concentration of 0.3 .mu.g/mL. Cells were maintained and
split under selection for .about.3 weeks to generate a stable pool
of hERG-CD8-expressing 293 cells.
Immunocytochemistry and FACS
[0321] For single-cell FACS, cells were washed with PBS and
harvested with trypsin-EDTA (Invitrogen), which was then
inactivated with at least an equal volume of 293 growth medium.
Cells were counted, spun down at 1000.times.g for 5 min and
resuspended in PBS supplemented with 0.1% BSA at a density of
10.times.10.sup.6 cells/mL. Following the manufacturer's suggested
protocol, 10 .mu.L of mouse anti-human CD8 Alexa Fluor.RTM. 488
monoclonal antibody (Invitrogen) was added per 2.times.10.sup.6
cells (in 200 .mu.L) and allowed to incubate for 30-60 min at room
temperature. Cells were then repeatedly (3 times) spun down at
1000.times.g for 5 minutes and washed in PBS+0.1% BSA, before being
resuspended in 2 mL PBS+0.1% BSA and filtered to achieve a disperse
single cell suspension at .about.1.times.10.sup.6 cells/mL for
sorting. Cell suspensions were then run on a FACSVantage (BD
Biosciences, San Jose, Calif.) using the 488 nm laser line and
collecting with an emission filter centered at 530 nm. Single cells
from the top 10% of the stained population were isolated into
96-well microplates and were expanded for .about.3 weeks.
[0322] Immunocytochemistry was performed in 96-well microplates
(Corning) by washing cells with PBS and fixing in 4%
paraformaldehyde in PBS for 10 min. Cells were permeabilized with
0.25% TritonX-100 in PBS for 3 min, washed three times with PBS and
blocked with 1% BSA in PBS for 30 min. Cells were then stained with
primary mouse anti-human CD8 monoclonal antibody (2 .mu.g/mL) in
PBS for 60 min at room temperature. Primary antibody was washed off
three times with 1% BSA in PBS; cells were stained with secondary
goat anti-mouse Alexa Fluor.RTM. 488 (1:500) for 30 min at room
temperature, and then washed three times with 1% BSA in PBS and
once with PBS. The immunofluorescence was then measured on a Tecan
Safire.sup.2 plate reader (Tecan Instruments, Raleigh-Durham, N.C.)
using 488 nm excitation and 520 nm emission (10 nm bandwidth).
[0323] Fluorescence Polarization Assays
[0324] Tracer evaluation was conducted by incubating diluted
membrane preparations and fluorescent tracer in the presence or
absence of 10 .mu.M dofetilide (Sequioa Research Products,
Pangbourne, UK) in order to assess the degree of hERG-specific (and
displaceable) tracer binding. Experiments were performed in a
variety of buffers (data not shown), and the optimal FP assay
buffer composition was experimentally determined to consist of 25
mM HEPES (pH 7.5), 15 mM KCl, 1 mM MgCl.sub.2, and 0.05% Pluronic
F-127. Compound-displacement assays were performed by first
dispensing 10 .mu.L of assay buffer with or without test compounds
to wells of a 384-well untreated polystyrene assay plate (Corning
#3677), and then adding 10 .mu.L of a mixture of membrane
preparation and tracer at twice the final assay concentration.
Reactions were incubated for 2 to 4 hours and then read on a Tecan
InfiniTE F500 or Tecan Safire.sup.2 microplate reader using
polarized excitation and emission filters or monochromator settings
that were appropriate to the tracer being evaluated. Optimal
conditions for FP assays were determined by titrating a matrix of
membrane protein against varying concentrations of fluorescent
tracer in the presence and absence of 30 .mu.M E-4031 (Tocris
Bioscience, Ellisville, Mo.). These experimentally-determined
concentrations of total membrane protein and fluorescent tracer
were then used to perform competition assays against a dilution
series of compounds known to block the hERG channel. Using the
final optimized tracer (Predictor.TM. hERG Tracer Red), final
optimized assay conditions contained 1 nM tracer and 85 .mu.g/mL
membrane protein (B.sub.max of membrane preparation .about.450
.mu.mol/mg) in a 20 .mu.L final assay volume. Assay wells were
excited at 530 nm and emission was measured at 585 nm (20 nm
bandwidth) using a Tecan Safire.sup.2 microplate reader. In
experiments designed to measure the amount of non-hERG specific
tracer that could be displaced, the assay also contained 30 .mu.M
E-4031.
[0325] Data Analysis
[0326] Data were analyzed using Microsoft.RTM. Office Excel 2003
and Prism 4 for Windows (GraphPad Software Inc., San Diego,
Calif.).
[0327] Identification of a High Affinity Fluorescent Tracer
[0328] A series of candidate tracers were synthesized in order to
generate compounds which varied in their affinity for the hERG
K.sup.+ channel. Compound variation was accomplished by combining a
number of chemical scaffolds with various functional constituents,
linkers and fluorophores (see, Singleton, D. H.; Boyd, H.;
Steidl-Nichols, J. V.; Deacon, M.; Groot, M. J.; Price, D.;
Nettleton, D. O.; Wallace, N. K.; Troutman, M. D.; Williams, C.;
Boyd, J. G., Fluorescently Labeled Analogues of Dofetilide as
High-Affinity Fluorescence Polarization Ligands for the Human
Ether-a-go-go-Related Gene (hERG) Channel. J Med Chem 2007, 50,
(13), 2931-2941). Tracer affinity was initially evaluated using a
radioligand displacement assay to measure the affinity of the
tracer for the hERG K.sup.+ channel. A subset of these compounds
was determined to bind the hERG K.sup.+ channel with high affinity,
a finding that suggested one might prove useful as a fluorescent
tracer molecule for assay development (FIG. 1).
[0329] Generating Membrane Preparations with Higher Specific
Activity
[0330] The highest-affinity tracers shown in FIG. 1 were examined
for their performance in an FP assay using membrane preparations
derived from the hERG-T-REx.TM. 293 cell line. Membrane
preparations from this cell line had a specific activity (B.sub.max
value) of approximately 7 .mu.mol hERG protein/mg of total protein.
Initial FP experiments failed to produce a measurable difference in
polarization values in the presence or absence of known hERG
channel blockers such as E-4031 or dofetilide with any of the
candidate tracers, even when using total membrane concentrations as
high as 6001 mg/mL in the assay. These results were not surprising
given that a robust FP assay requires both a high affinity tracer
as well as protein concentrations that are sufficient to ideally
bind at least .about.50% or more of the tracer in the absence of
displacing compounds (see, Huang, X., Fluorescence polarization
competition assay: the range of resolvable inhibitor potency is
limited by the affinity of the fluorescent ligand. J Biomol Screen
2003, 8, (1), 34-8). High specific content of the protein of
interest is also desirable in order to minimize non-specific
interactions with the membranes or other membrane proteins.
Therefore, we sought to increase the B.sub.max of the hERG channel
membrane preparations by generating a stable pool of 293 cells
using a bicistronic vector that coupled expression of the hERG
channel to the CD8 receptor by virtue of an IRES element. In such
cells, high levels of the CD8 marker would be expected to correlate
with high levels of hERG channel. High-expressing cells were
isolated by FACS, and cells from the top 10% of the CD8+ population
(FIG. 2, panel A) were sorted and isolated as single cells into
96-well plates. Single cell clones were expanded, then stained to
identify individual clones with the highest CD8 expression level
(FIG. 2, panel B). Of the .about.192 clones thus examined, six were
isolated for further study and were examined by patch-clamp
recording to determine the degree of functional hERG channel
expression at the plasma membrane (FIG. 2, panel C). To ensure a
true clonal population and to ensure the best cellular substrate
for hERG channel containing membranes, one of these clones (clone
D) was expanded, and then subjected to a second round of FACS
isolation, clonal expansion, and immunocytochemical staining.
Membrane protein from the highest-expressing clone was prepared and
characterized by radioligand binding, in which a B.sub.max of
>450 pmol/mg was determined (FIG. 2, panel D). This is a
>50-fold increase as compared to the membrane preparations
derived from the hERG-T-REx.TM. 293 cell line.
[0331] Fluorescence Polarization Assay Optimization
[0332] Using the six candidate high-affinity tracers that were
originally identified in the radioligand displacement assay, a
membrane preparation from the hERG-CD8 293 cell line was evaluated
for use in an FP experiment by titrating a fixed amount of each
tracer (1 nM) with increasing concentrations of membrane
preparation. The assays were performed in the presence or absence
of 10 .mu.M dofetilide in order to discriminate non-specific from
specific binding. Of the six candidate tracers, only one (IM-0107)
provided an assay window of >100 mP between specific- and
non-specific binding at a concentration of membrane required to
elicit .about.70% bound tracer. Although further assay optimization
was possible using this tracer, the excitation and emission spectra
of the fluorophore used was similar to that of Texas Red, which
falls between those of common "red" (TAMRA-like) or "far-red"
(Cy5-like) fluorophores. Because of this, both non-standard filters
and a custom dichroic mirror were required in the plate reader
(Tecan InfiniTE F-500) for optimal performance. To allow the assay
to be easily performed on a variety of commercially-available plate
readers, another round of iterative tracer synthesis was
undertaken, based on the results of the initial evaluations. In
this second round of synthesis, tracer evaluation was facilitated
by characterizing tracer performance using the FP assay rather than
the more cumbersome radioligand displacement assay.
[0333] This second round of synthesis resulted in the
identification of Predictor.TM. hERG Tracer Red, a tracer with
TAMRA-like excitation and emission spectra that showed strong
specific binding to hERG-CD8 membranes with a large polarization
shift between bound and displaced tracer at a concentration of
membrane required for 75% specific binding of tracer (85 .mu.g/mL
total protein, FIG. 3). As with all of the tracers evaluated,
substantial non-specific binding of the tracer was observed, as
seen by the membrane-dependent increase in mP values in the
presence of saturating E-4031. This polarization signal was not an
artifact due to scattered light from the membranes, as the specific
signal from the tracer in the presence of membrane was >40-fold
that of membrane alone in both the parallel and perpendicular
emission channels for all measurements when using the Safire.sup.2
plate reader.
[0334] Initial tracer displacement assays using 1 nM Predictor.TM.
hERG Tracer Redand 85 .mu.g (total protein)/mL of CD8_hERG
membranes were performed using two well-characterized hERG binding
ligands, astemizole and E-4031, which have been shown to bind to
hERG with K.sub.i values in the low single- to low double-digit nM
range, respectively (see, Finlayson, K.; Turnbull, L.; January, C.
T.; Sharkey, J.; Kelly, J. S., [3H]dofetilide binding to HERG
transfected membranes: a potential high throughput preclinical
screen. Eur J Pharmacol 2001, 430, (1), 147-8; Chiu, P. J.; Marcoe,
K. F.; Bounds, S. E.; Lin, C. H.; Feng, J. J.; Lin, A.; Cheng, F.
C.; Crumb, W. J.; Mitchell, R., Validation of a [3H]astemizole
binding assay in HEK293 cells expressing HERG K.sup.+ channels. J
Pharmacol Sci 2004, 95, (3), 311-9; and Finlayson, K.; Pennington,
A. J.; Kelly, J. S., [3H]dofetilide binding in SHSY5Y and HEK293
cells expressing a HERG-like K.sup.+ channel? Eur J Pharmacol 2001,
412, (3), 203-12). Displacement with E-4031 produced data
consistent with a one-site competition model, and an IC.sub.50
value of 11 nM (FIG. 4). Displacement by astemizole, however
produced data that appeared to be consistent with a two-site
binding model, with binding of the tracer to a second site being
displaced only in the presence of high concentrations (>1 .mu.M)
of astemizole. When the experiment was repeated using control
membranes from the parental 293 cells which lacked overexpressed
hERG K.sup.+ channels, this same lower-affinity displacement was
also seen, suggesting that a non-hERG component in the membrane, or
the membrane itself, can bind the tracer, and that this interaction
can be displaced by certain compounds. To correct for this non-hERG
binding component, displacement of tracer by astemizole was
repeated in the presence or absence of 30 .mu.M E-4031, which is
expected to compete all hERG-specific binding of the tracer. When
the data were corrected by removing the non-hERG component of the
displacement curve (FIG. 4, panel B), an IC.sub.50 value of 2.7 nM
as obtained. As a simpler alternative to performing the astemizole
displacement assay in the presence or absence of E-4031,
displacement data that provided a polarization value of less than
that seen in a control well containing saturating E-4031 could be
discarded, and the astemizole data then fit to a curve with the
minimum mP value fixed to that seen in the control well contained
saturating E-4031.
[0335] The FP assay using Predictor.TM. hERG Tracer Red was then
validated against a series of compounds that are known to block the
hERG K.sup.+ channel across a wide range of affinities, with nM to
.mu.M K.sub.i or IC.sub.50 values reported in the literature (FIG.
5). Like astemizole, several compounds were able to displace the
non-hERG binding component of the tracer at high compound
concentrations, but this was easily corrected and there was
excellent correlation between the corrected IC.sub.50 values and
values that had been reported in the literature (Table 2). We then
evaluated signal stability (IC.sub.50 value) and assay robustness
(determination of Z' value; see, Zhang, J. H.; Chung, T. D.;
Oldenberg, K. R., A simple statistical parameter for use in
evaluation and validation of high throughput screening assays. J
Biomol Screen 1999, 4, 67-73) at different time points after the
addition of all assay components. As shown in FIG. 6 and Table 3,
the assay reports an IC.sub.50 value that varies by less than 25%
between 30 minutes and 6 hours, and then increases slightly
(approximately 2-fold) within 24 hours. Although the total
polarization shift continued to increase over the course of this
experiment, Z' values were excellent (.gtoreq.187) at all time
points examined. Additionally, when the assay was repeated in the
presence of increasing concentrations of DMSO, ethanol, or methanol
(to determine assay tolerance to solvents that are commonly used
for compound storage), negligible effect was seen on either Z' or
E-4031 IC.sub.50 values at up to 10% solvent (FIG. 6, panel B). In
separate experiments, the assay was seen to provide data of similar
quality using polypropylene plates (Matrical MP101-1-PP), but
performance was slightly compromised when using NBS-coated
polystyrene plates (Corning 3676).
TABLE-US-00002 TABLE 2 Comparison of IC.sub.50 values (in nM) as
reported by patch-clamp or radioligand displacement assays, and in
the FP assay described herein. Compound Patch clamp Radioligand FP
Astemizole 1 1-7 2.7 Pimozide 18 3-80 7.2 Dofetilide 12-15 6-40 11
E-4031 8-48 20-80 17 Terfenadine 16-204 30-110 33 Haloperidol
28-174 90-180 187 Bepridil 550 170-450 279 Thioridazine 36-1250
737-1710 655 Fluoxetine 990 1920-3040 2880 Amitriptyline 10,000
2440 8135 Literature values for radioligand displacement or
patch-clamp assays are found in Diaz, G. J.; Daniell, K.; Leitza,
S. T.; Martin, R. L.; Su, Z.; McDermott, J. S.; Cox, B. F.;
Gintant, G. A., The [3H]dofetilide binding assay is a predictive
screening tool for hERG blockade and proarrhythmia: Comparison of
intact cell and membrane preparations and effects of altering
[K+]o. J Pharmacol Toxicol Methods 2004, 50, (3), 187-99; Deacon,
M.; Singleton, D.; Szalkai, N.; Pasieczny, R.; Peacock, C.; Price,
D.; Boyd, J.; Boyd, H.; Steidl-Nichols, J. V.; Williams, C., Early
evaluation of compound QT prolongation effects: a predictive
384-well fluorescence polarization binding assay for measuring hERG
blockade. J Pharmacol Toxicol Methods 2007, 55, (3), 238-47; and
Wible, B. A.; Hawryluk, P.; Ficker, E.; Kuryshev, Y. A.; Kirsch,
G.; Brown, A. M., HERG-Lite: a novel comprehensive high-throughput
screen for drug-induced hERG risk. J Pharmacol Toxicol Methods
2005, 52, (1), 136-145.
TABLE-US-00003 TABLE 3 Assay signal stability and robustness over
time for the FP assay described herein. Time IC.sub.50 (nM)
Delta-mP Z' 30 Minutes 14 107 .87 1 hour 13 146 .90 2 hours 14 173
.91 4 hours 16 191 .92 6 hours 19 194 .93 24 hours 29 210 .94
IC.sub.50 value is for E-4031; Z' values were calculated from 28
replicate well containing either DMSO (control) or 30 .mu.M
E-4031.
[0336] The FP assay results using astemizole to displace the tracer
suggest the presence of a second, lower-affinity binding site in
membranes prepared from the high-expression hERG cell line
described herein and in non-transfected 293 cells. A second,
lower-affinity binding site for dofetilide (see, Finlayson, K.;
Turnbull, L.; January, C. T.; Sharkey, J.; Kelly, J. S.,
[3H]dofetilide binding to HERG transfected membranes: a potential
high throughput preclinical screen. Eur J Pharmacol 2001, 430, (1),
147-8) or astemizole (see, Chiu, P. J.; Marcoe, K. F.; Bounds, S.
E.; Lin, C. H.; Feng, J. J.; Lin, A.; Cheng, F. C.; Crumb, W. J.;
Mitchell, R., Validation of a [3H]astemizole binding assay in
HEK293 cells expressing HERG K.sup.+ channels. J Pharmacol Sci
2004, 95, (3), 311-9) has previously been identified in radioligand
binding studies. These sites remain uncharacterized in radioligand
studies yet do not prevent accurate determination of hERG affinity
for test compounds. Described herein for the hERG K.sup.+ channel
of the present invention are two straightforward procedures to
correct data from test compounds that show apparent inhibition
beyond that seen in the presence of 30 .mu.M E-4031.
[0337] Although the ultimate measure of predictability of any hERG
in vitro assay is often taken to be correlation with prolongation
of the Q-T interval in animal models (see, Lynch, J. J., Jr.;
Wallace, A. A.; Stupienski, R. F., 3rd; Baskin, E. P.; Beare, C.
M.; Appleby, S. D.; Salata, J. J.; Jurkiewicz, N. K.; Sanguinetti,
M. C.; Stein, R. B.; et al., Cardiac electrophysiologic and
antiarrhythmic actions of two long-acting spirobenzopyran
piperidine class III agents, L-702,958 and L-706,000 [MK-499]. J
Pharmacol Exp Ther 1994, 269, (2), 541-54; and Gintant, G. A.; Su,
Z.; Martin, R. L.; Cox, B. F., Utility of hERG assays as surrogate
markers of delayed cardiac repolarization and QT safety. Toxicol
Pathol 2006, 34, (1), 81-90), the hERG K.sup.+ channel assay
described herein achieves the penultimate result, namely, an
excellent correlation with literature data on patch-clamp IC.sub.50
values for compounds with a documented ability to block hERG
currents. The assay agrees with literature patch-clamp IC.sub.50
values with no more than a 3-fold discrepancy across the
patch-clamp data set, and when the FP results are compared across
both patch-clamp and radioligand binding data, the FP data falls
within the ranges reported by these techniques. The assay is fully
homogenous, uses a red-shifted tracer to lessen problems of
compound interference, and has a Z' value of >0.8 over at least
a 24-hour assay read window. Although we observed that some
compounds produce a greater displacement of the FP signal than does
the standard, E-4031, this signal is not hERG-dependent and can be
easily identified and corrected for during IC.sub.50 profiling.
Together, these features make the assay well suited to routine and
even automated compound profiling.
[0338] Each of the above-cited references, as well as U.S. Pat. No.
5,206,240 and all synthetic methods disclosed therein, are hereby
incorporated by reference as if set forth fully herein.
Sequence CWU 1
1
211159PRTHomo sapiens 1Met Pro Val Arg Arg Gly His Val Ala Pro Gln
Asn Thr Phe Leu Asp1 5 10 15Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser
Arg Lys Phe Ile Ile Ala 20 25 30Asn Ala Arg Val Glu Asn Cys Ala Val
Ile Tyr Cys Asn Asp Gly Phe 35 40 45Cys Glu Leu Cys Gly Tyr Ser Arg
Ala Glu Val Met Gln Arg Pro Cys 50 55 60Thr Cys Asp Phe Leu His Gly
Pro Arg Thr Gln Arg Arg Ala Ala Ala65 70 75 80Gln Ile Ala Gln Ala
Leu Leu Gly Ala Glu Glu Arg Lys Val Glu Ile 85 90 95Ala Phe Tyr Arg
Lys Asp Gly Ser Cys Phe Leu Cys Leu Val Asp Val 100 105 110Val Pro
Val Lys Asn Glu Asp Gly Ala Val Ile Met Phe Ile Leu Asn 115 120
125Phe Glu Val Val Met Glu Lys Asp Met Val Gly Ser Pro Ala His Asp
130 135 140Thr Asn His Arg Gly Pro Pro Thr Ser Trp Leu Ala Pro Gly
Arg Ala145 150 155 160Lys Thr Phe Arg Leu Lys Leu Pro Ala Leu Leu
Ala Leu Thr Ala Arg 165 170 175Glu Ser Ser Val Arg Ser Gly Gly Ala
Gly Gly Ala Gly Ala Pro Gly 180 185 190Ala Val Val Val Asp Val Asp
Leu Thr Pro Ala Ala Pro Ser Ser Glu 195 200 205Ser Leu Ala Leu Asp
Glu Val Thr Ala Met Asp Asn His Val Ala Gly 210 215 220Leu Gly Pro
Ala Glu Glu Arg Arg Ala Leu Val Gly Pro Gly Ser Pro225 230 235
240Pro Arg Ser Ala Pro Gly Gln Leu Pro Ser Pro Arg Ala His Ser Leu
245 250 255Asn Pro Asp Ala Ser Gly Ser Ser Cys Ser Leu Ala Arg Thr
Arg Ser 260 265 270Arg Glu Ser Cys Ala Ser Val Arg Arg Ala Ser Ser
Ala Asp Asp Ile 275 280 285Glu Ala Met Arg Ala Gly Val Leu Pro Pro
Pro Pro Arg His Ala Ser 290 295 300Thr Gly Ala Met His Pro Leu Arg
Ser Gly Leu Leu Asn Ser Thr Ser305 310 315 320Asp Ser Asp Leu Val
Arg Tyr Arg Thr Ile Ser Lys Ile Pro Gln Ile 325 330 335Thr Leu Asn
Phe Val Asp Leu Lys Gly Asp Pro Phe Leu Ala Ser Pro 340 345 350Thr
Ser Asp Arg Glu Ile Ile Ala Pro Lys Ile Lys Glu Arg Thr His 355 360
365Asn Val Thr Glu Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp Val
370 375 380Leu Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp
Thr Ile385 390 395 400Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp
Trp Leu Ile Leu Leu 405 410 415Leu Val Ile Tyr Thr Ala Val Phe Thr
Pro Tyr Ser Ala Ala Phe Leu 420 425 430Leu Lys Glu Thr Glu Glu Gly
Pro Pro Ala Thr Glu Cys Gly Tyr Ala 435 440 445Cys Gln Pro Leu Ala
Val Val Asp Leu Ile Val Asp Ile Met Phe Ile 450 455 460Val Asp Ile
Leu Ile Asn Phe Arg Thr Thr Tyr Val Asn Ala Asn Glu465 470 475
480Glu Val Val Ser His Pro Gly Arg Ile Ala Val His Tyr Phe Lys Gly
485 490 495Trp Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu
Leu Ile 500 505 510Phe Gly Ser Gly Ser Glu Glu Leu Ile Gly Leu Leu
Lys Thr Ala Arg 515 520 525Leu Leu Arg Leu Val Arg Val Ala Arg Lys
Leu Asp Arg Tyr Ser Glu 530 535 540Tyr Gly Ala Ala Val Leu Phe Leu
Leu Met Cys Thr Phe Ala Leu Ile545 550 555 560Ala His Trp Leu Ala
Cys Ile Trp Tyr Ala Ile Gly Asn Met Glu Gln 565 570 575Pro His Met
Asp Ser Arg Ile Gly Trp Leu His Asn Leu Gly Asp Gln 580 585 590Ile
Gly Lys Pro Tyr Asn Ser Ser Gly Leu Gly Gly Pro Ser Ile Lys 595 600
605Asp Lys Tyr Val Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser
610 615 620Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys
Ile Phe625 630 635 640Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met
Tyr Ala Ser Ile Phe 645 650 655Gly Asn Val Ser Ala Ile Ile Gln Arg
Leu Tyr Ser Gly Thr Ala Arg 660 665 670Tyr His Thr Gln Met Leu Arg
Val Arg Glu Phe Ile Arg Phe His Gln 675 680 685Ile Pro Asn Pro Leu
Arg Gln Arg Leu Glu Glu Tyr Phe Gln His Ala 690 695 700Trp Ser Tyr
Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys Gly Phe705 710 715
720Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu Asn Arg Ser Leu
725 730 735Leu Gln His Cys Lys Pro Phe Arg Gly Ala Thr Lys Gly Cys
Leu Arg 740 745 750Ala Leu Ala Met Lys Phe Lys Thr Thr His Ala Pro
Pro Gly Asp Thr 755 760 765Leu Val His Ala Gly Asp Leu Leu Thr Ala
Leu Tyr Phe Ile Ser Arg 770 775 780Gly Ser Ile Glu Ile Leu Arg Gly
Asp Val Val Val Ala Ile Leu Gly785 790 795 800Lys Asn Asp Ile Phe
Gly Glu Pro Leu Asn Leu Tyr Ala Arg Pro Gly 805 810 815Lys Ser Asn
Gly Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His Lys 820 825 830Ile
His Arg Asp Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Glu Phe 835 840
845Ser Asp His Phe Trp Ser Ser Leu Glu Ile Thr Phe Asn Leu Arg Asp
850 855 860Thr Asn Met Ile Pro Gly Ser Pro Gly Ser Thr Glu Leu Glu
Gly Gly865 870 875 880Phe Ser Arg Gln Arg Lys Arg Lys Leu Ser Phe
Arg Arg Arg Thr Asp 885 890 895Lys Asp Thr Glu Gln Pro Gly Glu Val
Ser Ala Leu Gly Pro Gly Arg 900 905 910Ala Gly Ala Gly Pro Ser Ser
Arg Gly Arg Pro Gly Gly Pro Trp Gly 915 920 925Glu Ser Pro Ser Ser
Gly Pro Ser Ser Pro Glu Ser Ser Glu Asp Glu 930 935 940Gly Pro Gly
Arg Ser Ser Ser Pro Leu Arg Leu Val Pro Phe Ser Ser945 950 955
960Pro Arg Pro Pro Gly Glu Pro Pro Gly Gly Glu Pro Leu Met Glu Asp
965 970 975Cys Glu Lys Ser Ser Asp Thr Cys Asn Pro Leu Ser Gly Ala
Phe Ser 980 985 990Gly Val Ser Asn Ile Phe Ser Phe Trp Gly Asp Ser
Arg Gly Arg Gln 995 1000 1005Tyr Gln Glu Leu Pro Arg Cys Pro Ala
Pro Thr Pro Ser Leu Leu 1010 1015 1020Asn Ile Pro Leu Ser Ser Pro
Gly Arg Arg Pro Arg Gly Asp Val 1025 1030 1035Glu Ser Arg Leu Asp
Ala Leu Gln Arg Gln Leu Asn Arg Leu Glu 1040 1045 1050Thr Arg Leu
Ser Ala Asp Met Ala Thr Val Leu Gln Leu Leu Gln 1055 1060 1065Arg
Gln Met Thr Leu Val Pro Pro Ala Tyr Ser Ala Val Thr Thr 1070 1075
1080Pro Gly Pro Gly Pro Thr Ser Thr Ser Pro Leu Leu Pro Val Ser
1085 1090 1095Pro Leu Pro Thr Leu Thr Leu Asp Ser Leu Ser Gln Val
Ser Gln 1100 1105 1110Phe Met Ala Cys Glu Glu Leu Pro Pro Gly Ala
Pro Glu Leu Pro 1115 1120 1125Gln Glu Gly Pro Thr Arg Arg Leu Ser
Leu Pro Gly Gln Leu Gly 1130 1135 1140Ala Leu Thr Ser Gln Pro Leu
His Arg His Gly Ser Asp Pro Gly 1145 1150 1155Ser
210575DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 2cgattgacat tgattattga ctagttatta
atagtaatca attacggggt cattagttca 60tagcccatat atggagttcc gcgttacata
acttacggta aatggcccgc ctcgtgaccg 120cccaacgacc cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata 180gggactttcc
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
240catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg
taaatggccc 300gcctggcatt atgcccagta catgacctta cgggactttc
ctacttggca gtacatctac 360gtattagtca tcgctattac catggtgatg
cggttttggc agtacaccaa tgggcgtgga 420tagcggtttg actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg 480ttttggcacc
aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg
540caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg
tttagtgaac 600cgtcagatcg cctggagacg ccatccacgc tgttttgacc
tccatagaag acaccgggac 660cgatccagcc tccgcggccg ggaacggtgc
attggaacgc ggattccccg tgccaagagt 720gacgtaagta ccgcctatag
actctatagg cacacccctt tggctcttat gcatgctata 780ctgtttttgg
cttggggcct atacaccccc gctccttatg ctataggtga tggtatagct
840tagcctatag gtgtgggtta ttgaccatta ttgaccactc ccctattggt
gacgatactt 900tccattacta atccataaca tggctctttg ccacaactat
ctctattggc tatatgccaa 960tactctgtcc ttcagagact gacacggact
ctgtattttt acaggatggg gtcccattta 1020ttatttacaa attcacatat
acaacaacgc cgtcccccgt gcccgcagtt tttattaaac 1080atagcgtggg
atctccacgc gaatctcggg tacgtgttcc ggacatgggc tcttctccgg
1140tagcggcgga gcttccacat ccgagccctg gtcccatgcc tccagcggct
catggtcgct 1200cggcagctcc ttgctcctaa cagtggaggc cagacttagg
cacagcacaa tgcccaccac 1260caccagtgtg ccgcacaagg ccgtggcggt
agggtatgtg tctgaaaatg agctcggaga 1320ttgggctcgc accgtgacgc
agatggaaga cttaaggcag cggcagaaga agatgcaggc 1380agctgagttg
ttgtattctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg
1440gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac
cagacataat 1500agctgacaga ctaacagact gttcctttcc atgggtcttt
tctgcagaag cttgaattgc 1560tcgaggctag cgttttaaac ccagcacagt
ggtctagatt cgaagaattc atgcctgtaa 1620gaagaggtca cgtggctccg
caaaacacat tcctggacac gattataaga aagtttgagg 1680ggcagtctag
aaagttcatc attgcaaatg ctcgcgtaga gaactgcgca gtgatctact
1740gtaatgatgg tttctgtgag ctctgtggat attcacgggc cgaagttatg
cagcgcccat 1800gcacatgtga cttcctccat ggtccccgga cccagcggag
agctgccgct cagattgccc 1860aggcgttgct gggagcagaa gaaaggaagg
tggagatcgc cttttaccgg aaagatggct 1920cctgcttcct ctgcttggtc
gacgtagtcc ctgtgaagaa cgaagatggt gctgtgataa 1980tgttcatcct
gaatttcgag gttgtgatgg agaaagatat ggtgggctca cccgctcacg
2040acactaacca tcgcgggcct cctacctctt ggctcgcccc agggcgggcc
aagactttta 2100gactcaaatt gcccgcactg ctcgctctta ccgcacggga
gtctagtgtc aggagcggtg 2160gagctggagg cgcgggcgcc cctggagctg
tcgtggtgga cgtggatctc acaccggcag 2220cgccgtcttc cgagagcctt
gccctggatg aggtgaccgc aatggacaac cacgtggctg 2280gactcgggcc
tgccgaggag agacgcgcct tggttggacc cgggagtcct cctcggtccg
2340ctcccggcca acttccttcc ccgcgggctc attccctcaa ccctgatgca
tctggaagct 2400cctgttccct ggcccggact cggtcacgag agagttgtgc
ttccgtacgg cgcgcttcta 2460gcgctgatga catagaagct atgagagccg
gggtgttgcc gcctcctcca cgccacgcct 2520ccacgggtgc tatgcaccct
ctgcggtccg gcctgctcaa tagcacgtcc gacagcgatc 2580tggtgaggta
tcgcactata tcaaagatcc cacaaattac attgaatttt gtcgatctca
2640aaggggatcc attcctcgct tctcccacta gcgataggga aattatcgcg
cccaagatca 2700aggagcggac tcacaacgtg accgagaagg taactcaagt
tctgagcttg ggagcggatg 2760tcctgcctga gtacaaactt caggccccga
ggattcatag atggacgatt ttgcattatt 2820ctccctttaa ggctgtttgg
gactggttga tattgctgct tgtgatttac actgccgtgt 2880ttacgccata
cagcgcagct ttcctgctga aggagactga agagggacct cctgcaactg
2940agtgtggcta cgcttgccag ccccttgccg tcgtggacct gatagttgat
ataatgttca 3000ttgtagatat tctcataaat tttcggacaa cctatgtaaa
tgctaacgaa gaggttgtct 3060cccaccccgg tagaatcgcc gtccattatt
ttaaaggatg gtttctcatt gatatggtgg 3120ccgcaattcc ttttgatctc
cttatctttg gatctggctc cgaggaactc atcggactgc 3180tgaaaacagc
tagactcctg cggctcgtgc gggtggcacg gaagctggac cgatactctg
3240aatatggtgc agcggtcctc ttcttgttga tgtgtacatt cgcgctcatc
gcccattggc 3300tcgcgtgtat ttggtatgcc attggtaaca tggagcaacc
ccacatggac agtagaatcg 3360gttggctcca caatctgggc gaccagattg
gcaagccgta caattcctct ggcctcggag 3420ggccatctat caaggacaag
tacgtgaccg ctctgtattt taccttttcc tctctgacta 3480gcgtcgggtt
cggcaatgtg tctcccaata cgaactccga gaagatattc agcatttgcg
3540tgatgttgat cggatccctc atgtatgcgt caatcttcgg caacgtgtct
gcaatcattc 3600agcgcctgta ttcagggacc gctcggtatc atacacaaat
gctgagggtt agggagttca 3660taagattcca ccaaataccc aacccactcc
ggcagcgact tgaggaatat ttccagcatg 3720cctggtccta caccaatgga
atagatatga atgccgtcct caaggggttt cctgagtgct 3780tgcaggcgga
tatttgcctc catctcaacc gatccctgtt gcagcattgt aagccattta
3840ggggtgctac taaaggctgt ctccgcgcgt tggccatgaa gttcaagacc
acccacgctc 3900cgcctggaga cactctggta cacgcaggtg atctcctcac
cgccctgtac ttcatctcca 3960ggggttccat tgaaattctc agaggggacg
ttgttgtggc tattcttggt aagaatgaca 4020tcttcgggga accactgaac
ctgtatgccc ggcccgggaa aagcaacggg gacgtaagag 4080ccctgacgta
ttgtgacctg cataagatcc atagggacga cctgctcgag gtgctggata
4140tgtacccgga gttctccgat cacttctgga gctctctgga aattacattc
aacttgagag 4200ataccaacat gatccccggg agtccaggct caaccgagct
ggaaggcggc ttctctcggc 4260agaggaagcg aaaactttca ttccgccggc
gaaccgacaa ggatactgaa caaccaggag 4320aagtgtccgc cctcggcccc
ggaagagctg gagcaggtcc aagttctaga ggtcgaccag 4380gcggcccctg
gggcgaatct ccatctagtg gcccatcttc cccagagtct tcagaggacg
4440agggacccgg gcgatcttct tctccattga ggctggtgcc gtttagctca
ccccggccac 4500ctggcgagcc tcctggaggc gaaccgctta tggaggattg
tgagaaatca tcagatacat 4560gcaatccttt gtctggcgct tttagtggcg
tgtccaatat cttttccttc tggggtgatt 4620ctcggggacg acagtatcaa
gaactcccca gatgcccagc cccaacgccc agtctgttga 4680acattcctct
gagttcccca ggcaggcgcc cacggggcga cgtcgagtct cgactggacg
4740ctctccagag acaactgaat agactggaaa ctcgcctgtc agcagacatg
gcaacagtgc 4800tgcagctgct ccagagacag atgaccctgg tccctcctgc
ctactccgcc gtgacgacac 4860ctggaccagg ccccacaagc acatctcctc
tgctgccagt gagtccactg ccaaccctga 4920cactcgactc cttgagtcaa
gtgagccagt ttatggcatg tgaagagctc cctcccgggg 4980cacccgaact
ccctcaagag ggacctacac ggcggctcag tcttcctggc cagctcgggg
5040ccttgacctc ccaacctttg catcgacacg gctcagaccc cggctcctga
gaattccgcc 5100ccccccctaa cgttactggc cgaagccgct tggaataagg
ccggtgtgcg tttgtctata 5160tgttattttc caccatattg ccgtcttttg
gcaatgtgag ggcccggaaa cctggccctg 5220tcttcttgac gagcattcct
aggggtcttt cccctctcgc caaaggaatg caaggtctgt 5280tgaatgtcgt
gaaggaagca gttcctctgg aagcttctga agacaaacaa cgtctgtagc
5340gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
gccaaaagcc 5400acgtgtataa gatacacctg caaaggcggc acaaccccag
tgccacgttg tgagttggat 5460agttgtggaa agagtcaaat ggctctccta
agcgtattca acaaggggct gaaggatgcc 5520cagaaggtac cccatcgtat
gggatctgat ctggggcctc ggtgcacatg ctttacatgt 5580gtttagtcga
ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
5640tgaaaaacac gatgataata tggccacaac catggaacaa gagacggggg
atccaccggt 5700cgccaccatg gccttaccag tgaccgcctt gctcctgccg
ctggccttgc tgctccacgc 5760cgccaggccg agccagttcc gggtgtcgcc
gctggatcgg acctggaacc tgggcgagac 5820agtggagctg aagtgccagg
tgctgctgtc caacccgacg tcgggctgct cgtggctctt 5880ccagccgcgc
ggcgccgccg ccagtcccac cttcctccta tacctctccc aaaacaagcc
5940caaggcggcc gaggggctgg acacccagcg gttctcgggc aagaggttgg
gggacacctt 6000cgtcctcacc ctgagcgact tccgccgaga gaacgagggc
tactatttct gctcggccct 6060gagcaactcc atcatgtact tcagccactt
cgtgccggtc ttcctgccag cgaagcccac 6120cacgacgcca gcgccgcgac
caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc 6180cctgcgccca
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga
6240cttcgcctgt gatatctaca tctgggcgcc cttggccggg acttgtgggg
tccttctcct 6300gtcactggtt atcacccttt actgcaacca caggaaccga
agacgtgttt gcaaatgtcc 6360ccggcctgtg gtcaaatcgg gagacaagcc
cagcctttcg gcgagatacg tctaaccctg 6420tgcggccgca ggtaagccag
cccaggcctc gccctccagc tcaaggcggg acaggtgccc 6480tagagtagcc
tgcatccagg gacaggcccc agccgggtgc tgacacgtcc acctccatct
6540cttcctcagg tctgcccggg tggcatccct gtgacccctc cccagtgcct
ctcctggccc 6600tggaagttgc cactccagtg cccaccagcc ttgtcctaat
aaaattaagt tgcatcattt 6660tgtctgacta ggtgtccttc tataatatta
tggggtggag gggggtggta tggagcaagg 6720ggcccaagtt aacttgttta
ttgcagctta taatggttac aaataaagca atagcatcac 6780aaatttcaca
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
6840caatgtatct tatcatgtct ggatccgctt caggcaccgg gcttgcgggt
catgcaccag 6900gtcgcgcggt ccttcgggca ctcgacgtcg gcggtgacgg
tgaagccgag ccgctcgtag 6960aaggggaggt tgcggggcgc ggaggtctcc
aggaaggcgg gcaccccggc gcgctcggcc 7020gcctccactc cggggagcac
gacggcgctg cccagaccct tgccctggtg gtcgggcgag 7080acgccgacgg
tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct
7140tccatctgtt gctgcgcggc cagccgggaa ccgctcaact cggccatgcg
cgggccgatc 7200tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg
tccagaccgc caccgcggcg 7260ccgtcgtccg cgacccacac cttgccgatg
tcgagcccga cgcgcgtgag gaagagttct 7320tgcagctcgg tgacccgctc
gatgtggcgg tccgggtcga cggtgtggcg cgtggcgggg 7380tagtcggcga
acgcggcggc gagggtgcgt acggcccggg ggacgtcgtc gcgggtggcg
7440aggcgcaccg tgggcttgta ctcggtcatg gtggcctgca gagtcgctcg
gtgttcgagg 7500ccacacgcgt caccttaata tgcgaagtgg acctgggacc
gcgccgcccc gactgcatct 7560gcgtgttaat tcgccaatga caagacgctg
ggcggggttt gtgtcatcat agaactaaag 7620acatgcaaat atatttcttc
cggggacacc gccagcaaac gcgagcaacg ggccacgggg 7680atgaagcagc
tagactcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
7740ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
agaatcaggg 7800gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
aggccaggaa ccgtaaaaag 7860gccgcgttgc tggcgttttt ccataggctc
cgcccccctg
acgagcatca caaaaatcga 7920cgctcaagtc agaggtggcg aaacccgaca
ggactataaa gataccaggc gtttccccct 7980ggaagctccc tcgtgcgctc
tcctgttccg accctgccgc ttaccggata cctgtccgcc 8040tttctccctt
cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
8100gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
gcccgaccgc 8160tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
taagacacga cttatcgcca 8220ctggcagcag ccactggtaa caggattagc
agagcgaggt atgtaggcgg tgctacagag 8280ttcttgaagt ggtggcctaa
ctacggctac actagaagga cagtatttgg tatctgcgct 8340ctgctgaagc
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
8400accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga 8460tctcaagaag atcctttgat cttttctacg gggtctgacg
ctcagtggaa cgaaaactca 8520cgttaaggga ttttggtcat gagattatca
aaaaggatct tcacctagat ccttttaaat 8580taaaaatgaa gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac 8640caatgcttaa
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
8700gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
tggccccagt 8760gctgcaatga taccgcgaga cccacgctca ccggctccag
atttatcagc aataaaccag 8820ccagccggaa gggccgagcg cagaagtggt
cctgcaactt tatccgcctc catccagtct 8880attaattgtt gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 8940gttgccattg
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
9000tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
aaaagcggtt 9060agctccttcg gtcctccgat cgttgtcaga agtaagttgg
ccgcagtgtt atcactcatg 9120gttatggcag cactgcataa ttctcttact
gtcatgccat ccgtaagatg cttttctgtg 9180actggtgagt actcaaccaa
gtcattctga gaatagtgta tgcggcgacc gagttgctct 9240tgcccggcgt
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
9300attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
gagatccagt 9360tcgatgtaac ccactcgtgc acccaactga tcttcagcat
cttttacttt caccagcgtt 9420tctgggtgag caaaaacagg aaggcaaaat
gccgcaaaaa agggaataag ggcgacacgg 9480aaatgttgaa tactcatact
cttctttttt caatattatt gaagcattta tcagggttat 9540tgtctcatga
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
9600cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
catgacatta 9660acctataaaa ataggcgtat cacgaggccc ctttcgtctc
gcgcgtttcg gtgatgacgg 9720tgaaaacctc tgacacatgc agctcccgga
gacggtcaca gcttgtctgt aagcggatgc 9780cgggagcaga caagcccgtc
agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 9840taactatgcg
gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
9900gcacagatgc gtaaggagaa aataccgcat caggaaattg taaacgttaa
tattttgtta 9960aaattcgcgt taaatttttg ttaaatcagc tcatttttta
accaataggc cgaaatcggc 10020aaaatccctt ataaatcaaa agaatagacc
gagatagggt tgagtgttgt tccagtttgg 10080aacaagagtc cactattaaa
gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 10140cagggcgatg
gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
10200cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
acggggaaag 10260ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
gagcgggcgc tagggcgctg 10320gcaagtgtag cggtcacgct gcgcgtaacc
accacacccg ccgcgcttaa tgcgccgcta 10380cagggcgcgt cgcgccattc
gccattcagg ctacgcaact gttgggaagg gcgatcggtg 10440cgggcctctt
cgctattacg ccagctggcg aaggggggat gtgctgcaag gcgattaagt
10500tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
tgaattgtcg 10560aggtctcgac ggtat 10575
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