U.S. patent application number 10/483617 was filed with the patent office on 2004-08-19 for cardiovascular safety assay.
Invention is credited to Heylen, Godelieve Irma Christine, Janssen, Cornelus Gerardus Maria, Jurzak, Mirek, Van Assouw, Henricus Petrus Martinus Maria.
Application Number | 20040161805 10/483617 |
Document ID | / |
Family ID | 8180635 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040161805 |
Kind Code |
A1 |
Heylen, Godelieve Irma Christine ;
et al. |
August 19, 2004 |
Cardiovascular safety assay
Abstract
The present invention provides assays and kits for the screening
of test compounds for their capability to induce cardiotoxicity in
a subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict cardiotoxicity of compounds during the
development of new therapeutics and other agents.
Inventors: |
Heylen, Godelieve Irma
Christine; (Westmalle, BE) ; Janssen, Cornelus
Gerardus Maria; (Vosselaar, BE) ; Jurzak, Mirek;
(Frankfurt, DE) ; Van Assouw, Henricus Petrus Martinus
Maria; (JE Oirschot, NL) |
Correspondence
Address: |
PHILIP S. JOHNSON
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
8180635 |
Appl. No.: |
10/483617 |
Filed: |
January 13, 2004 |
PCT Filed: |
July 2, 2002 |
PCT NO: |
PCT/EP02/07364 |
Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
G01N 2800/32 20130101;
G01N 33/6893 20130101; G01N 33/60 20130101; G01N 33/5014 20130101;
G01N 2500/02 20130101; G01N 33/6887 20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 033/53; G01N
033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 13, 2001 |
EP |
01202689.4 |
Claims
37. (new) An assay for screening test compounds, comprising: a)
incubating a source containing HERG or a fragment thereof with: i)
radiolabeled astemizole of formula (III) 4ii) the test compound;
and b) measuring the effect of the test compound on the amount of
reference compound bound to HERG.
38. (new) An assay for screening test compounds for their ability
to induce cardiotoxicity in a subject, comprising: a) incubating a
source containing HERG or a fragment thereof with: i) radiolabeled
astemizole of formula (III) 5ii) the test compound; and b)
measuring the effect of the test compound on the amount of
reference compound bound to HERG.
39. (new) An assay for screening test compounds for their
capability to induce cardiac arrhythmia in a subject, comprising:
a) incubating a source containing HERG or a fragment thereof with:
i) radiolabeled astemizole of formula (III) 6ii) the test compound;
and b) measuring the effect of the test compound on the amount of
reference compound bound to HERG.
40. (new) An assay according to claim 37, wherein the source
containing HERG is selected from the group consisting of: i) an
isolated and purified nucleic acid which encodes HERG having an
amino acid sequence that is at least 80% identical to that of SEQ
ID NO:2 or a fragment thereof; ii) an isolated and purified nucleic
acid which encodes HERG comprising the amino acid sequence of SEQ
ID NO:2 or a fragment thereof; iii) cells expressing on the surface
thereof the HERG polypeptide channel or a fragment thereof; or iv)
membrane preparations of cells expressing on the surface thereof
the HERG polypeptide channel or a fragment thereof.
41. (new) An assay according to claim 37, wherein the source
containing HERG are membrane preparations of cells expressing on
the surface thereof the HERG polypeptide channel consisting of SEQ
ID NO:2.
42. (new) Radiolabeled astemizole of formula (III) 7
43. (new) A process for preparing radiolabeled astemizole as in
claim 42 comprising: a) demethylating astemizole of formula (I)
using a suitable reagent such as 48% aqueous hydrobromic acid: 8b)
reacting the intermediate of formula (II) with
[.sup.3H]-methyliodide (CT.sub.3I) optionally in a reaction inert
solvent and in the presence of a base to obtain said radiolabeled
astemizole: 9
44. (new) A high-throughput assay for screening test compounds, the
assay comprising: a) contacting membrane preparations of cells
expressing on the surface thereof HERG polypeptide channels having
an amino acid sequence that is at least 80% identical to that of
SEQ ID NO:2 of fragments thereof, with radiolabeled astemizole as
claimed in claim 42, for a time sufficient to allow binding of the
reference compound with the HERG polypeptide channel; b) contacting
membrane preparations of cells expressing on the surface thereof
HERG polypeptide channels having an amino acid sequence that is at
least 80% identical to that of SEQ ID NO:2 or fragments thereof,
with radiolabeled astemizole of step a) together with the test
compound for a time sufficient to allow binding of the reference
compound and of the test compound with the HERG polypeptide
channel; c) measuring the amount of radiolabeled astemizole bound
to the HERG channel in step a); d) measuring the amount of
radiolabeled astemizole bound to the HERG channel in step b); and
e) compare the amount of radiolabeled astemizole bound to the HERG
channel measured in step a) with the amount of radiolabeled
astemizole bound to the HERG polypeptide channel measured in step
b).
45. (new) A high-throughput proximity detection assay for screening
test compounds the assay comprising: i) HERG labeled with a first
label capable of participating in a proximity detection assay; ii)
Radiolabeled astemizole as claimed in claim 6 capable of
participating in a proximity detection assay; iii) contacting HERG
of step i) and radiolabeled astemizole of step ii) together with a
test compound for a time sufficient to allow binding of the
reference compound and of the test compound to HERG; and iv) detect
an interaction between HERG of step i) and radiolabeled astemizole
of step ii) by means of proximity of the first label with the
second label when HERG and the reference compound interact.
46. (new) A kit comprising: a) a source containing HERG; b)
radiolabeled astemizole as claimed in claim 42.
47. (new) A kit according to claim 46 wherein the source containing
HERG is being selected from: i) an isolated and purified nucleic
acid which encodes HERG having an amino acid sequence that is at
least 80% identical to that to SEQ ID NO:2 or a fragment thereof;
ii) an isolated and purified nucleic acid which encodes HERG
comprising the amino acid sequence of SEQ ID NO:2 or a fragment
thereof; iii) cells expressing on the surface thereof the HERG
polypeptide channel or a fragment thereof; or iv) membrane
preparations of cells expressing on the surface thereof the HERG
polypeptide channel or a fragment thereof.
48. (new) A kit according to claim 46 wherein the source containing
HERG is an isolated and purified HERG polypeptide channel or a
fragment thereof, bound to a solid support.
49. (new) A kit according to claim 48 wherein the solid support is
a fluorescer comprising solid support.
50. (new) A kit according to claim 46 wherein the source containing
HERG consists of membrane preparations of cells expressing on the
surface thereof HERG polypeptide channels encoded by the nucleic
acid sequence consisting of SEQ ID NO:2.
51. (new) A kit according to claim 50 wherein said cells are HEK293
cells.
52. (new) A kit according to claim 51 optionally comprising means
to remove the excess of unbound labeled reference compound from the
incubation mixture.
53. (new) A kit according to claim 51 wherein the separating means
consist of GF/B filtration.
54. (new) Use of an isolated and purified polynucleotide which
encodes HERG comprising the nucleic acid sequence of SEQ ID NO:2 or
a fragment thereof in an assay according to claim 37.
55. (new) Use of an isolated an purified polynucleotide which
encodes HERG comprising the nucleic acid sequence of SEQ ID NO:1 or
a fragment thereof in an assay according to claim 37.
56. (new) Use of cells expressing on the surface thereof the HERG
polypeptide channel comprising the amino acid sequence of SEQ ID
NO:2 or a fragments thereof in an assay according to claim 37.
57. (new) Use of membrane preparations of cells expressing on the
surface thereof the HERG polypeptide channel comprising the amino
acid sequence of SEQ ID NO:2 or a fragment thereof in an assay
according to claim 37.
58. (new) Use of membrane preparations of cells expressing on the
surface thereof HERG polypeptide channels encoded by the amino acid
sequence consisting of SEQ ID NO:2 in an assay according to claim
37.
59. (new) Use of radiolabeled astemizole as claimed in claim 6 in
an assay according to claim 37.
Description
[0001] The present invention relates to the field of cardiovascular
safety assays and provides assays and kits for the screening of
test compounds for their capability to induce cardiotoxicity in a
subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict potential cardiotoxicity of compounds during
the development of new therapeutics and other agents. The present
invention finds particularly advantageous use in high throughput
screening of chemical compound libraries.
BACKGROUND OF THE INVENTION
[0002] Evidence has accrued that several drugs may prolong cardiac
repolarisation (hence, "measured as" the QT interval of the surface
electrocardiogram) to such a degree that potentially
life-threatening ventricular arrhythmias e.g. torsades de pointes
(TdP) may occur, especially in case of overdosage or
pharmacokinetic interaction.
[0003] The number of drugs reported to induce QT interval
prolongation with or without TdP continues to increase (W.
Haverkamp et al. (2000) Cardiovascular Research 47, 219-233). As
many as 50 clinically available or still investigational
non-cardiovascular drugs and cardiovascular non-anti-arrhythmic
drugs have been implicated. A number of drugs, both old and new,
have either been withdrawn from the market or have had their sale
restricted. Of concern is the interval, usually measured in years,
from the marketing of these drugs to initial recognition of their
association with QT interval prolongation and/or TdP. It would
therefore be beneficial to investigate any new chemical entity for
this potential side effect before its first use in man at an early
stage of the development of new therapeutics and/or other
agents.
[0004] A key component in the present development of new
therapeutic agents consists of High Throughput Screening (HTS) of
chemical compound libraries. Pharmaceutical companies have
established large collections of structurally distinct compounds,
which act as the starting point for drug target lead identification
programs. A typical corporate compound collection now comprises
between 100,000 and 1,000,000 discrete chemical entities. While A
few years ago a throughput of a few thousand compounds a day and
per assay was considered to be sufficient, pharmaceutical companies
nowadays aim at ultra high throughput screening techniques with
several hundreds of thousands of compounds tested per week. In a
typical HTS related screen format, assays are performed in
multi-well microplates, such as 96, 384 or 1536 well plates. The
use of these plates facilitates automation such as the use of
automated reagent dispensers and robotic pipetting instrumentation.
Further, to reduce the cycle time, the costs and the resources for
biochemicals such as recombinant proteins, HTS related screens are
preferably performed at room temperature with a single measurement
for each of the compounds tested in the assay.
[0005] A decisive criterion in the lead evaluation process will be
an early recognition of their potential association with QT
prolongation and/or TdP. However, there are currently no reliable,
fast, easy screening methods available to assess cardiotoxicity,
which can cope with the number of compounds identified in the
currently deployed HTS techniques. It is an object of this
invention to solve this problem in the art by providing assays and
kits which are based on the finding that the interaction of
astemizole with the HERG potassium channel can be exploited to
predict cardiotoxicity of compounds during the development of new
therapeutics and other agents.
[0006] The currently available in vitro models comprise
heterologous expression systems, disaggregated cells, isolated
tissues and the isolated intact (Lagendorf-perfused) heart. In all
models the effect of potassium current blockade is assessed by
measurement of either ionic currents using two-electrode voltage
clamp recordings (Dascal N. (1987) Crit.Rev.Biochem 22, 341-356) or
patch-clamp recordings (Zhou Z. et al., (1998) Biophysical Journal
74, 230-241), of membrane potentials using microelectrodes or
confocal microscopy (Dall'Asta V. et al. (1997) Exp.Cell Research
231, 260-268). None of the aforementioned methods can be used in an
HTS screen in view of the complexity of the experimental
procedures, the slow cycling times, the nature of the source
materials (i.e. isolated tissues and disaggregated cells thereof)
and the reliability of the test results.
[0007] The present inventors surprisingly found that a binding
assay using labeled astemizole as a specific ligand for the HERG
channel can be used to predict the potential association of
compounds with QT prolongation and/or TdP. This binding assay
solves the aforementioned problems and can be deployed in an HTS
related screen format.
[0008] A similar assay has been described by Chadwick C. et al.
(Chadwick C. et. al., (1993) Circulation Research 72, 707-714)
wherein [.sup.3H]-dofetilide has been identified as a specific
radioligand for the cardiac delayed rectifier K.sup.+-channel. This
article further demonstrates a good correlation between dofetilide
displacement and potassium channel blocking activity of a number of
antiarrhytmic compounds. This binding assay facilitates the
characterization of drug-channel interactions at the molecular
level.
[0009] In this assay labeled dofetilide has been prepared from the
dibromo precursor by .sup.3H-exchange yielding the incorporation of
two .sup.3H-labels per molecule. There is a direct correlation
between the number of .sup.3H-labels per molecule and the
sensitivity of the binding assay. The present invention provides an
improved binding assay over the above, as the use of a
desmethylastemizole precursor in a reaction with
[3.sup.3H]-methyliodide resulted in the incorporation of three
.sup.3H-labels per molecule astemizole. The specific activity of
the thus obtained radioligand is 1.5-2 times higher than the
specific activity of [.sup.3H]-dofetilide.
[0010] Further, the dofetilide assay could not be used in an HTS
related screen format since the ventricular myocytes isolated from
adult male guinea pigs had to be used within 6 hours of isolation.
In addition only 36% of the isolated cells were viable and could be
used in the binding assay. In order to be used in an HTS related
screen, the starting material should be readily available and in
sufficient amounts. The present invention solves this problem as
membrane preparations of HEK293 cells, stably expressing the HERG
potassium channel, are used. Said cells can be maintained in
culture in sufficient amount avoiding the need and supply of animal
models and as cell membranes are used in the binding assay, the
latter can be stored in binding assay ready aliquots at -80.degree.
C. for later use. A further drawback of the dofetilide binding
assay described by Chadwick et al. has to do with the incubation
protocol. As viable myocytes are used, the incubation has to be
performed at the physiological temperature of 34.degree. C. The
latter undoubtedly increases the costs, possible cycle time and
complexity of the assay if to be performed ill an HTS related
screen format. The present invention solves this problem as it was
surprisingly demonstrated that the membrane preparations could be
incubated at room temperature. Especially in light of a study by
Zhoe Z. et al. Zhou Z. et al., (1998) Biophysical Journal 74,
230-241) which concluded that the kinetic properties measured for
HERG are markedly dependent on temperatura and that differences
observed in several reports, are diminished when studies are
performed at physiological temperatures, i.e. 35.degree. C.
[0011] This and other aspects of the invention will be described
herein below.
SUMMARY OF THE INVENTION
[0012] The present invention provides an assay for screening test
compounds for their capability to induce cardiotoxicity in a
subject, the method comprising incubating a source containing HERG
or a fragment thereof with a reference compound and the test
compound, for a time sufficient to allow binding of the reference
compound and of the test compound with the HERG polypeptide channel
and measuring the effect of the test compound on the amount of
reference compounds bound to HERG.
[0013] In a preferred embodiment of this invention, the assay
consists of incubating membrane preparations of cells, preferably
HEK293 cells, expressing on the surface thereof the HERG
polypeptide channel comprising the amino acid sequence of SEQ ID
NO:2 or a fragment thereof; with a labeled reference compound.
Wherein said labeled reference compound is a drug capable to induce
cardiac arrhythmia in a subject, preferably said labeled reference
compound is [.sup.3H]-astemizole. Incubating the above together
with the test compound and measure the effect of the test compound
on the amount of reference compound bound to the HERG polypeptide
channel. In a further embodiment the means of measurement consist
of separating means to remove the excess of unbound labeled
reference compound from the incubation mixture and of means for
detection of the labeled reference compound wherein the latter
preferably consists of radiolabeled measurement using scintillation
counting.
[0014] The invention further provides a high-throughput assay for
screening compounds for their capability to induce cardiotoxicity
in a subject, the assay comprising;
[0015] a) contacting membrane preparations of cells expressing on
the surface thereof HERG polypeptide channels having an amino acid
sequence that is at least 80% identical to that of SEQ ID NO:2 or
fragments thereof, with a labeled reference compound for a time
sufficient to allow binding of the reference compound with the HERG
polypeptide channel;
[0016] b) contacting membrane preparations of cells expressing on
the surface thereof HERG polypeptide channels having an amino acid
sequence that is at least 80% identical to that of SEQ ID NO:2 or
fragments thereof, with the labeled reference compound of step a)
together with the test compound for a time sufficient to allow
binding of the reference compound and of the test compound with the
HERG polypeptide channel;
[0017] c) measuring the amount of labeled reference compound bound
to the HERG channel in step a);
[0018] d) measuring the amount of labeled reference compound bound
to the HERG channel in step b); and
[0019] e) compare the amount of labeled reference compound bound to
the HERG channel measured in step a) with the amount of labeled
reference compound bound to the HERG polypeptide channel measured
in step b).
[0020] In a preferred embodiment of the high-throughput screening
assay, the membrane preparations are derived from cells, preferably
HEK293 cells, expressing on the surface thereof HERG polypeptide
channels encoded by the amino acid sequence consisting of SEQ ID
NO:2. In a further embodiment of the high-throughput screening
assay the labeled reference compound is astemizole, preferably
[.sup.3H]-astemizole.
[0021] The present invention also encompasses kits for screening
compounds for their capability to induce cardiotoxicity in a
subject as well as the use of reagents, including polynucleotides,
polypeptides and suitable reference compounds in the assays of the
present invention.
BRIEF DESCRIPTION OF THE DRAWING
[0022] FIG. 1A shows the saturation binding of [.sup.3H]-astemizole
to cell membrane preparations of HEK293 cells stably transfected
with the HERG channel cDNA. TB represents Total Binding measured,
NSB represents Non Specific Binding measured and SB represents
Specif Binding measured.
[0023] FIG. 1B shows the Scatchard plot for the saturation binding
experiments. From the fitted line a K.sub.D of 3.07.+-.2.26 nM
(n=11) could be determined with a B.sub.max (Maximal Binding) of
3260.+-.900 fmol/mg protein (n=11).
[0024] FIG. 2 shows the binding affinities of 42 reference
compounds compared to the electrophysiological patch clamp data. A
Superman rank correlation coefficient of 0.87 could be
obtained.
DETAILED DESCRIPTION
[0025] The present invention relates to the field of cardiovascular
safety assays and provides assays and kits for the screening of
test compounds for their capability to induce cardiotoxicity in a
subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict cardiotoxicity of compounds during the
development of new therapeutics and other agents. The present
invention finds particularly advantageous use in high throughput
screening of chemical compound libraries.
[0026] In one embodiment of the present invention, the assay for
screening test compounds comprises: a) incubating a source
containing HERG or a fragment thereof with i) a reference compound,
ii) the test compound; and b) measuring the effect of the test
compound on the amount of reference compound bound to HERG.
[0027] In a specific embodiment of the present invention the assays
are used to assess the capability of the test compounds to induce
cardiac arrhythmia in a subject.
[0028] As used herein the term "test compound" refers to a
chemically defined molecule whose cardiac arrhythmia inducing
capability is assessed in an assay according to the invention. Test
compounds include, but are not limited to, drugs, ligands (natural
or synthetic), polypeptides, peptides, peptide mimics,
polysaccharides, saccharides, glycoproteins, nucleic acids,
polynucleotides and small organic molecules. In one embodiment test
compounds comprise an existing library of compounds. In another
embodiment, test compounds comprise a novel library of
compounds.
[0029] As used herein the term "reference compound" refers to a
drug capable to induce cardiotoxicity in a subject. Reference
compounds include, but are not limited to, astemizole, terfenadine,
erythromycin, sparfloxain, probucol, terodiline and sertindole.
[0030] As used herein the term "HERG" refers to the Human
Ether-a-go-go-Related Gene channel. It is a delayed rectifier
potassium channel that plays a role in the control of action
potential repolarization in many cell types. HERG was originally
cloned from human hippocampus and it is strongly expressed in the
heart. The HERG polypeptides according to the invention include
isolated and purified proteins having an amino acid sequence that
is at least 80% identical to that of SEQ ID NO:2 or a fragment
thereof. In a further embodiment the HERG polypeptide channel
according to the invention has an amino acid sequence comprising
the amino acid sequence of SEQ ID NO:2. In a preferred embodiment
the HERG polypeptide according to the invention consists of SEQ ID
NO:2.
[0031] Variants of the defined sequence and fragments also form
part of the invention. Variants include those that vary from the
parent sequence by conservative amino acid changes, wherein
"conservative amino acid changes" refers to the replacement of one
or more amino acid residue(s) in the parent sequence without
affecting the biological activity of the parent molecule based on
the art recognized substitutability of certain amino acids (See
e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol.5,
Supp. 3, pgs 345-352, 1987). Further variants are variants in which
several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted or
added in any combination.
[0032] Methods for comparing the identity and similarity of two or
more sequences are well known in the art. Thus for instance,
programs available in the Winconsin Sequence Analysis Package,
version 9.1 (Devreux J. et al, Nucleic Acid Res., 12, 387-395,
1984), for example the programs BESTFIT and GAP, may be used to
determine the % identity between two polynucleotides and the %
identity and the % similarity between two polypeptide sequences.
BESTFIT uses the "local homology" algorithm of Smith and Waterman
(J. Mol. Biol., 147, 195-197, 1981) and finds the best single
region of similarity between two sequences. BESTFIT is more suited
to compare two polynucleotide or two polypeptide sequences that are
dissimilar in length, the program assuming that the shorter
sequence represents a portion of the longer. In comparison, GAP
aligns two sequences, finding a "maximum similarity", according to
the algorithm of Neddleman and Wunsch (J.Mol.Biol., 48, 443-453,
1970). GAP is more suited to compare sequences that are
approximately the same length and an alignment is expected over the
entire length. Preferably, the parameters "Gap-Weight" and "Length
Weight" used in each program are 50 and 3 for polynucleotide
sequences, and 12 and 4 for polypeptide sequences, respectively.
Preferably, % identities and similarities are determined when the
two sequences being compared are optimally aligned. Other programs,
for determining identity and/or similarity between sequences are
also known in the art, for instance the BLAST family of programs
(Altschul S F et al, Nucleic Acids Res., 25:3389-3402, 1997).
[0033] Those skilled in the art will recognize that the
polypeptides according to the invention, i.e. the HERG polypeptide
channel, could be obtained by a plurality of recombinant DNA
techniques including, for example, hybridization, polymerase chain
reaction (PCR) amplification, or de novo DNA synthesis (See e.g.,
T. Maniatis et al. Molecular Cloning: A Laboratory Manual, 2d Ed.
Chap.14 (1989)). Thus, in a further embodiment the present
invention provides the use of the isolated and purified
polynucleotides encoding the HERG polypeptide or a fragment
thereof, in an assay or kit according to the invention. In another
embodiment the present invention provides the use of the isolated
and purified polynucleotide encoding the HERG polypeptide channel
or a fragment thereof comprising the polynucleotide sequence of SEQ
ID NO:1. In a preferred embodiment the present invention provides
the use of the isolated and purified polynucleotide encoding the
HERG polypeptide channel consisting of the polynucleotide sequence
of SEQ ID NO:1.
[0034] The term "fragments thereof" describes a piece, or
sub-region of protein or polynucleotide molecule whose sequence is
disclosed herein, such that said fragment comprises 5 or more amino
acids that are contiguous in the parent protein, or such that said
fragment comprises 15 or more nucleic acids that are contiguous in
the parent polynucleotide. The term "fragments thereof" is intended
to include "functional fragments" wherein the isolated fragment,
piece or sub-region comprises a funtionally distinct region such as
an active site, a binding site or a phosphorylation site of the
receptor protein. Functional fragments may be produced by cloning
technology, or as the natural products of alternative splicing
techniques.
[0035] As used herein, "isolated" refers to the fact that the
polynucleotides, proteins and polypeptides, or respective fragments
thereof in question, have been removed from its in vivo environment
so that it can be manipulated by the skilled artisan, such as but
not limited to sequencing, restriction digestion, site-directed
mutagenesis, and subcloning into expression vectors for a nucleic
acid fragment as well as obtaining the protein or protein fragments
in quantities that afford the opportunity to generate polyclonal
antibodies, monoclonal antibodies, amino acid sequencing, and
peptide digestion. Therefore, the nucleic acids as described herein
can be present in whole cells or in cell lysates or in a partially,
substantially or wholly purified form.
[0036] A polypeptide is considered "purified" when it is purified
away from environmental contaminants. Thus a polypeptide isolated
from cells is considered to be substantially purified when purified
from cellular components by standard methods while a chemically
synthesized polypeptide sequence is considered to be substantially
purified when purified from its chemical precursors. A
"substantially pure" protein or nucleic acid will typically
comprise at least 85% of a sample with greater percentages being
preferred. One method for determining the purity of a protein or
nucleic acid molecule, is by electrophoresing a preparation in a
matrix such as polyacrylamide or agarose. Purity is evidenced by
the appearance of a single band after staining. Other methods for
assessing purity include chromatography and analytical
centrifugation.
[0037] The term "time sufficient to allow binding" as used herein
refers to the time needed to generate a detectable amount of
labeled reference compound bound to the HERG polypeptide channel.
The time needed to generate this detectable amount will vary
depending on the assay system. One of skill in the art will know
the amount of time sufficient to generate a detectable amount of
labeled reference compound bound to the HERG polypeptide channel
based upon the assay system.
[0038] Assays
[0039] Assays of the present invention can be designed in many
formats generally known in the art of screening compounds for
binding polypeptide channels.
[0040] The assays of the present invention advantageously exploit
the fact that the interaction of astemizole with the HERG potassium
channel can be exploited to predict cardiotoxicity of compounds
during the development of new therapeutics and other agents.
[0041] Therefore, the present invention provides an assay for
screening test compounds, the assay comprising a) incubating a
source containing HERG or a fragment thereof with; i) a reference
compound, ii) the test compound; and b) measuring the effect of the
test compound on the amount of reference compound bound to
HERG.
[0042] In a first embodiment of this invention the source
containing HERG is an isolated and purified protein which encodes
HERG having an amino acid sequence that is at least 80% identical
to that of SEQ ID NO:2 or a fragment thereof.
[0043] In a second embodiment of this invention the source
containing HERG is an isolated and purified protein which encodes
HERG comprising the amino acid sequence of SEQ ID NO: 2 or a
fragment thereof.
[0044] In a further embodiment of this invention the source
containing HERG are cells expressing on the surface thereof the
HERG polypeptide channel or a fragment thereof.
[0045] In another embodiment of this invention the source
containing HERG are membrane preparations of cells expressing on
the surface therof the HERG polypeptide channel or a fragment
thereof.
[0046] In an alternative embodiment of this invention, the
reference compound is a compound capable to induce cardiotoxicity
in a subject, preferably selected from the group consisting of
astemizole, terfenadine, erythromycin, sparfloxain, probucol,
terodiline and sertindole. In a preferred embodiment the reference
compound is astemizole. It is a further object of this invention to
provide assays wherein the reference compound is labeled,
preferably radiolabeled.
[0047] In a preferred embodiment, the assay for screening test
compounds for their capability to induce cardiotoxicity in a
subject consists of a) incubating membrane preparations of cells
expressing on the surface thereof HERG polypeptide channels encoded
by the amino acid sequence consisting of SEQ ID NO:2 with i)
[.sup.3H]-astemizole, ii) the compound to be tested; and measuring
the effect of the test compound on the amount of reference compound
bound to HERG. The label of the reference compound is used to
measure this effect wherein said label can be measured using
amongst others scintillation counting.
[0048] A specific embodiment of the assays according to the
invention, consists of an high-throughput assay for screening test
compounds, the assay comprising: a) contacting membrane
preparations of cells expressing on the surface thereof HERG
polypeptide channels having an amino acid sequence that is at least
80% identical to that of SEQ ID NO:2 or fragments thereof, with a
labeled reference compound for a time sufficient to allow binding
of the reference compound with the HERG polypeptide channel; b)
contacting membrane preparations of cells expressing on the surface
thereof HERG polypeptide channels having an amino acid sequence
that is at least 80% identical to that of SEQ ID NO:2 or fragments
thereof, with the labeled reference compound of step a) together
with the test compound for a time sufficient to allow binding of
the reference compound and of the test compound with the HERG
polypeptide channel; c) measuring the amount of labeled reference
compound bound to the HERG channel in step a); d) measuring the
amount of labeled reference compound bound to the HERG channel in
step b); and e) compare the amount of labeled reference compound
bound to the HERG channel measured in step a) with the amount of
labeled reference compound bound to the HERG polypeptide channel
measured in step b).
[0049] In a further embodiment the membrane preparations of the
high-throughput screening assay consist of membrane preparations of
cells expressing on the surface thereof the HERG polypeptide
channel comprising the amino acid sequence of SEQ ID NO:2 or
fragments thereof.
[0050] In a preferred embodiment of this invention the membrane
preparations of the high-throughput screening assay consist of
membrane preparations of cells, preferably HEK 293 cells,
expressing on the surface thereof HERG polypeptide channels
consisting of the amino acid sequence of SEQ ID NO:2.
[0051] In a further preferred embodiment, the labeled reference
compound in the high-throughput screening assay consists of
[.sup.3H]-labeled astemizole. Said label can be measured using
amongst others scintillation counting.
[0052] In another specific embodiment the present invention
provides a high-throughput proximity detection assay for screening
test compounds the assay comprising:
[0053] i) HERG labeled with a first label capable of participating
in a proximity detection assay;
[0054] ii) a reference compound labeled with a second label capable
of participating in a proximity detection assay;
[0055] iii) contacting HERG of step i) and a reference compound of
step ii) together with a test compound for a time sufficient to
allow binding of the reference compound and of the test compound to
HERG; and
[0056] iv) detect an interaction between HERG of step i) and a
reference compound of step ii) by means of proximity of the first
label with the second label when HERG and the reference compound
interact.
[0057] The proximity of the first label to the second label,
brought about by the interaction of HERG and the reference compound
results in the production of a detectable signal. This may be
achieved by e.g. a scintillation proximity assay (SPA) system, in
which one of the labels is a radiolabel suitable for use in SPA and
the other label is a fluorescer comprised in a solid phase. The
detectable signal is light energy emitted when the labeled HERG
protein interacts with the labeled reference compound, bringing the
radiolabel sufficiently close to the fluorescer. Scintillation
proximity assay technology is described in U.S. Pat. No.
4,568,649.
[0058] Alternatively, the detectable signal may be a change in an
existing signal output, eg. fluorescence. Fluorescence resonance
energy transfer (FRET) is a method which works on this principle
and is described by Tsien R. et al. (Tsien R. et al. (1993) Trends
Cell Biol. 3: 242-245). It employs two different fluorescent
molecules, a donor and an acceptor, such that when these are in
sufficient proximity to one another the fluorescence of the donor
molecule is absorbed by the acceptor molecule and light of another
wavelength is emitted. Thus, when there is an interaction between
two molecules such as HERG and a reference compound, each of which
is labeled with one of these fluorescent molecules, a detectable
signal is produced.
[0059] By such proximity assays as are described above, the
screening assay according to the invention may be performed in a
single step, i.e. without the need of a separation step to remove
the excess of labeled reference compound from the incubation
mixture using separation means such as filtration.
[0060] In a preferred embodiment of the high-throughput proximity
detection assay, HERG is labeled with the fluorescer comprised in a
solid phase, such as coated scintillation proximity assay beads and
the reference compound is labeled with the radiolabel preferably
the reference compound is radiolabeled astemizole of formula (III).
1
[0061] It will be readily appreciated by the skilled artisan that
the binding of astemizole with HERG may also be used in a method
for the structure-based or rational design of compound which
affects the aforementioned binding, by:
[0062] a) probing the structure of the binding site of the HERG
polypeptide channel with astemizole;
[0063] b) identifying contacting atoms in the binding site of the
HERG polypeptide channel that interact with astemizole during
binding;
[0064] c) design test compounds that interact with the atoms
identified in (b) to affect the HERG--astemizole interaction;
and
[0065] d) contact said designed test compound with a source
containing HERG or a fragment thereof, to measure the capability of
said compound to affect the amount of labeled astemizole bound to
HERG.
[0066] It will be further appreciated that this will normally be an
iterative process.
[0067] Kits
[0068] The present invention also provides kits that can be used in
the above assays. In one embodiment the kit comprises a) a source
containing HERG; b) a reference compound.
[0069] In a first embodiment the kit comprises a source containing
HERG selected from: i) an isolated and purified protein which
encodes HERG having an amino acid sequence that is at least 80%
identical to that of SEQ ID NO:2 or a fragment thereof; ii) an
isolated and purified protein which encodes HERG comprising the
amino acid sequence of SEQ ID NO:2 or a fragment thereof, iii)
cells expressing on the surface thereof the HERG polypeptide
channel or a fragment thereof; or iv) membrane preparations of
cells expressing on the surface thereof the HERG polypeptide
channel or a fragment thereof.
[0070] In a further embodiment the kit comprises a reference
compound is selected from the group consisting of astemizole,
terfenadine, erythromycin, sparfloxain, probucol, terodiline and
sertindole. In a preferred embodiment the reference compound is
astemizole. It is a further object of this invention to provide
kits wherein the reference compound is labeled, preferably
radiolabeled.
[0071] In a specific embodiment the isolated and purified HERG
polypeptide channel is bound to a solid support, preferably to a
fluorescer comprising solid support such as coated scintillation
proximity beads.
[0072] Thus, in a specific embodiment the kit comprises a) an
isolated and purified HERG polypeptide channel or a fragment
thereof, bound to a solid support; and b) a labeled reference
compound. Preferably this specific embodiment consists of a kit
comprising a) an isolated and purified HERG polypeptide channel
consisting of the amino acid sequence of SEQ ID NO:2, bound to
fluorescer comprising solid support; and b) a radiolabeled
reference compound, preferably [.sup.3H ]-labeled astemizole.
[0073] In another specific embodiment the kit comprises a) membrane
preparations of cells, preferably HEK293 cells, expressing on the
surface thereof the HERG polypeptide channel consisting of the
amino acid sequence of SEQ ID NO:2; b) [.sup.3H]-labeled
astemizole; and c) means for measurement of the amount of labeled
reference compound bound to HERG.
[0074] The means of measurement consist of separating means to
remove the excess of unbound labeled reference compound from the
incubation mixture and of means for detection of the labeled
reference compound. The person skilled in the art will know the
separating means available for removing the excess of unbound
labeled reference compound from the incubation mixture. Said
separating means include, but are not limited to, magnetic beads,
centrifugation techniques and filtration techniques. The means for
detecting the labeled reference compound will be dependend on the
labeled used. Said labels may be fluorescent or radiolabels. The
skilled man will know the detection means available depending on
the label used.
[0075] In a specific embodiment the separating means consists of
GF/B filtration (Whatman Inc, Clifton, N.J.). In another specific
embodiment the detection means consists of scintillation counting
in a Topcount (Packard, Meriden, Conn.).
[0076] In a further embodiment the kits of the invention further
comprise instructions and/or multiple well plates for performing
the assay.
[0077] This invention will be better understood by reference to the
Experimental Details that follow, but those skilled in the art will
readily appreciate that these are only illustrative of the
invention as described more fully in the claims that follow
thereafter.
[0078] Additionally, throughout this application, various
publications are cited. The disclosure of these publications is
hereby incorporated by reference into this application to describe
more fully the state of the art to which this invention
pertains.
EXAMPLE 1
DNA Constructs and Stable Transfection of HEK293 Cells
[0079] HERG cDNA (Genbank Accession number: U04270 (SEQ ID NO:1))
was subcloned into bamHI/EcoRI sites of the pcDNA3 vector
(Invitrogen). This vector contains a CMV promotor and a SV40
promotor, which drive the expression of the inserted cDNA (HERG)
and neomycin resistance gene, respectively. The HEK293 cells were
transfected with this construct by a calcium phosphate precipitate
method (Gibco) or a lipofectamine method (Gibco). After selection
in 800 .mu.g/ml geneticin (G418; Gibco) for 15-20 days, single
colonies were picked with cloning cylinders and tested for HERG
current. The stably transfected cells were cultured in minimum
essential medium (MEM) supplemented with 10% fetal bovine serum and
400 .mu.g/ml geneticin.
[0080] For electrophysiological study, the cells were harvested
from the culture dish by trypsinization, washed twice with standard
MEM medium and seeded on small petri-dishes coated with
poly-L-lysine. Experiments were performed on the cells 1-2 days
after plating.
EXAMPLE 2
Membrane Preparations of HEK293 Cells Stably Transfected with the
HERG Potassium Channel
[0081] HEK293 cells stably transfected with the HERG channel cDNA,
were grown in DMEM culture medium enriched with 10% fetal calf
serum and antibiotics. Collected cells were homogenized in Tris-HCl
50 mM pH 7.4 using an Ultraturrax homogenizer and the homogenate
was centrifuged for 10 min at 23,500.times.g in a Sorvall
centrifuge. The cell membranes were washed once by
re-homogenization and re-centrifugation. The membranes were
supended in Tris-HCl 50 mM pH 7.4, aliquoted and stored at
-80.degree. C. 2
[0082] A solution of 4.6 g of astemizole (I) (10 mmol) in a 48%
aqueous hydrobromic acid solution (80 ml) was stirred and refluxed
for 2 hours. The reaction mixture was allowed to cool to room
temperature and the formed precipitate was filtered and washed with
water. The solids were dissolved in a mixture of
N,N-dimethylformamide (20 ml) and water (20 ml) and the mixture was
made alkaline by introducing slowly and with stirring a
concentrated aqueous ammoniumhydroxide solution. Then water (100
ml) was added and the mixture was stirred for 1 h. The precipitate
was filtered off and dried to the air for 18 h to yield
desmethylastemizole (II).
[0083] From this amount a fraction was taken and thoroughly
purified in portions via preparative HPLC on a Hypersyl ODS (5
.mu.m) bonded phase stainless steel column (7.1 mm ID.times.300 mm)
to yield astemizole free desmethylastemizole. Detection took place
at 282 nm and elution was performed isocratically with acetonitrile
water-diisopropylamine (56:44:0.2,v/v) at a flow rate of 4.0
ml/min. 3
[0084] A fraction of the HPLC purified desmethylastemizole (II)
(26.7mg, 60 .mu.mol) was dissolved in N,N-dimethylformamide (1.0
ml). To this solution 1N aqueous sodium hydroxide solution (60
.mu.l, 60 .mu.mol) was added. The mixture was stirred for 25
minutes at room temperature and added dropwise to a precooled
solution (-78.degree. C.) of [3.sup.3H]-methyliodide (370 MBq) in
toluene. The reaction mixture was vortexed and then left without
cooling for 3 hours. The toluene was evaporated from the reaction
mixture on a waterbath of 40.degree. C. at aspirator pressure and
the residue was purified in portions via preparative HPLC as
described above. The product containing fractions were combined and
depleted to 70 ml with methanol to give [.sup.3H]-astemizole (III)
with a total radioactivity of 198 MBq and a specific activity of
3.14 TBq/mmol (85 Ci/mmol).
EXAMPLE 4
Radioligand Binding Assay
[0085] Membranes were thawed and re-homogenized in incubation
buffer (Hepes 10 nM pH 7.4, 40 mM KCl, 20 mM KH.sub.2PO.sub.4, 5 mM
MgCl.sub.2, 0.5 mM KHCO.sub.3, 10 mM glucose, 50 mM glutamate, 20
mM aspartate, 14 mM heptanoic acid, 1 mM EGTA, 0.1% BSA) and 20-100
.mu.g protein was incubated with [.sup.3H]-astemizole for 60 min at
25.degree. C. with or without competitor followed by rapid
filtration over GF/B filter using a Filtermate196 harvester
(Packard, Meriden, Conn.). Filters were rinsed extensively with
ice-cold rinse-buffer (Tris-HCl 25 mM pH 7.4, 130 mM NaCl, 5.5 mM
KCl, 5 mM glucose, 0.8 mM MgCl.sub.2, 50 .mu.M CaCl.sub.2, 0.1%
BSA). Filter bound radioactivity was determined by scintillation
counting in a Topcount (Packard, Meriden, Conn.) and results were
expressed as counts per minute (cpm).
[0086] Initially, various parameters including buffer, radioligand
and compound to determined non-specific binding, were investigated
in order to select the optimal conditions.
[0087] In a saturation binding experiment, increasing
concentrations of [.sup.3H]-astemizole were incubated with
membranes, resuspended in buffer. Non-specific binding was measured
in the presence of 10 .mu.M R66204 (FIG. 1).
[0088] The effect of BSA and/or cyclodextrine present in the
incubation buffer, and of various ways of compound addition prior
to the experiment, was investigated by comparing the binding
affinities of 22 reference compounds to the electrophysiology data.
Compounds were dissolved in DMSO and further diluted in the same
solvent using a MultiprobeII pipetting station (Packard, Meriden,
Conn.). The final DMSO concentration in all experiments was 1%.
From this analysis it appears that compounds can be added directly
from the DMSO stock solution. Attempts to increase the solubility
of the compounds by addition of BSA and/or cyclodextrin did not
improve the correlation significantly.
EXAMPLE 5
Whole-cell Voltage Clamp Technique (Patch Clamp)
[0089] Solutions: The bath solution contained (in mM) 150 NaCl, 4
KCl, 5 glucose, 10 HEPES, 1.8 CaCl.sub.2 and 1 MgCl.sub.2 (pH 7.4
with NaOH). The pipette solution contained (in mM) 120 KCl, 5 EGTA,
10 HEPES, 4 MgATP, 0.5 CaCl.sub.2 and 2 MgCl.sub.2 (pH 7.2 with
KOH). Compounds were dissolved in DMSO to obtain a stock solution
of 10.sup.-2 M or 10.sup.-1 M. Control (=bath solution+DMSO) and
test solutions (=bath solution+DMSO+compound to be tested)
contained 0.3%, 0.1% or 0.03% DMSO. Test and control solutions were
applied to the cell under study using an Y-tube system, allowing to
rapidly change solutions (less than 0.5 s) in the vicinity of the
cell under study.
[0090] Electrophysiological measurements: A Petri dish containing
attached HEK293 cells expressing HERG was fixed on the stage of a
Patch Clamp Tower. An inverted microscope was used to observe the
cells. The Petri dish was constantly perfused with the bath
solution at room temperature.
[0091] Patch pipettes were pulled from borosilicate glass
capillaries using a horizontal Flaming/Brown micropipette puller
without further fire-polishing. The microelectrodes used had an
input resistance between 1.5 and 3 M.OMEGA. when filled with the
pipette solution.
[0092] The membrane current of the cells was measured at distinct
membrane potentials with the patch clamp technique by means of an
EPC-9 patch clamp amplifier. Data were acquired and analysed using
the programs Pulse and Pulsefit (HEKA), DataAccess (Bruxton) and
Igor (Wavemetrics). The current signals were low-pass filtered and
subsequently digitised. The liquid junction potential was
electronically corrected, before establishing the seal. After
disruption of the membrane, the cell capacitance and the series
resistance were compensated using the circuit of the EPC-9 patch
clamp amplifier.
[0093] The holding potential was -80 mV. The HERG current
(K.sup.+-selective outward current) was determined as the maximal
tail current at -40 mV after a 2 second depolarization to +60 mV.
Pulse cycling rate was 15 s. Before each test pulse a short pulse
(0.5 s) from the holding potential to -60 mV was given to determine
leak current. After establishing whole-cell configuration a 5
minute equilibration period allowed for internal perfusion of the
cell with the pipette solution. Thereafter test pulses were given
for 5 minutes to quantify the HERG current under control
conditions. While continuing the pulse protocol, perfusion was
switched from control solution to drug-containing solution. The
effect of the drug was measured after 5 minutes of drug
application. One to three concentrations of the drug were tested
per cell (applied cumulatively).
[0094] Parameter analysis of the measurements: The HERG current was
determined as the maximal tail current at -40 mV after a 2 second
depolarization to +60 mV, starting from a holding potential of -80
mV.
[0095] During the initial 5 minutes measured in the presence of the
control solution, the amplitude of the HERG-mediated membrane
K.sup.+ current gradually decreased with time (run-down). In order
to quantify accurately the extent of block by the compounds, this
continuous run-down of the K.sup.+ current has to be taken into
account. Therefore the time course of the K.sup.+ current (measured
at -40 mV) was fitted exponentially to the initial period of 5
minutes in control solution and extrapolated for the remainder of
the experiment. These extrapolations give the estimated amplitude
of the current if no drug would have been given. To determine the
extent of block by the compounds, the ratio of the measured current
was calculated by dividing each measured current amplitude by the
value of the fitted current at the same point in time.
EXAMPLE 6
Pharmacological Evaluation of the Binding Assay
[0096] For the pharmacological evaluation of the binding assay, 322
compounds were tested at 8 concentrations, for their ability to
inhibit [.sup.3H]-astemizole binding to the HERG channel and
pIC.sub.50-values were calculated by non-linear regression
analysis. If pIC.sub.50 values were available, the rank order
(Spearman) of the potencies for binding and patch clamp was
compared.
[0097] If in the patch clamp assay, compounds only have been tested
at <4 concentrations, a score was assigned to both binding- and
patch clamp data according to the following criteria:
[0098] score 1: pIC50<6 or % blockade <50% at 10.sup.-6 M or
higher
[0099] score 2: pIC50 between 6-8 or % blockade <50% between
10.sup.-6 and 10.sup.-8 M
[0100] score 3: pIC50>8 or % blockade >50% at 10.sup.-8 M or
lower
[0101] The rank order of potencies of 42 reference compounds to
displace the [.sup.3H]-astemizole binding from the HERG channel,
correlates well with the electrophysiological data for the
functional blockade of the rapid activating delayed rectifier
K.sup.+ current (r.sub.sp=0.87) (FIG. 2).
[0102] For 94% of the compounds tested, the binding data correlate
with the patch clamp data. In 2% of the cases the binding assay
scored higher than the patch clamp assay, for the remaining 4% it
is the other way around, i.e. the patch clamp assay scores higher
than the binding assay.
[0103] In view of this good correlation between binding data and
electrophysiological data it may be concluded that the radioligand
binding assay can be used as a primary screening tool for the
prediction of potential cardiovascular side-effects.
Sequence CWU 0
0
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