U.S. patent application number 11/436689 was filed with the patent office on 2006-09-14 for method for determining the binding behavior of ligands which specifically bind to target molecules.
Invention is credited to Wolf Bertling, Georg Hofner, Klaus Wanner.
Application Number | 20060201886 11/436689 |
Document ID | / |
Family ID | 7685946 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060201886 |
Kind Code |
A1 |
Wanner; Klaus ; et
al. |
September 14, 2006 |
Method for determining the binding behavior of ligands which
specifically bind to target molecules
Abstract
The invention relates to a method for determining the binding
behavior of ligands which specifically bind to target molecules at
at least one binding site, whereby the markers are present in a
native form and the determination of the concentrations K4 and K5
or of the quantities M2 and M1 is carried out by means of mass
spectrometry.
Inventors: |
Wanner; Klaus; (Munchen,
DE) ; Hofner; Georg; (Holzkirchen, DE) ;
Bertling; Wolf; (Erlangen, DE) |
Correspondence
Address: |
Heribert Muensterer
1317 Village Green Drive
Southlake
TX
76092
US
|
Family ID: |
7685946 |
Appl. No.: |
11/436689 |
Filed: |
May 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10478025 |
Nov 15, 2003 |
7074334 |
|
|
PCT/EP02/05543 |
May 21, 2002 |
|
|
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11436689 |
May 19, 2006 |
|
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Current U.S.
Class: |
210/656 ;
204/450; 210/806; 424/400; 424/450; 514/1 |
Current CPC
Class: |
C40B 30/04 20130101;
G01N 33/537 20130101; G01N 33/6845 20130101; G01N 33/536
20130101 |
Class at
Publication: |
210/656 ;
514/001; 424/400; 424/450; 210/806; 204/450 |
International
Class: |
B01D 15/08 20060101
B01D015/08; A61K 9/00 20060101 A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2001 |
DE |
101 25 258.7 |
Claims
1. A species with a pharmaceutical activity, which pharmaceutical
activity has been detected by a method of determining the ability
of the species to bind specifically to one or more target molecules
at at least one binding site thereof, the method comprising: (A)
preparing a first mixed phase wherein the species is contacted,
alone or in combination with one or more other species, with (i)
one or more markers which bind specifically to the one or more
target molecules, and (ii) with the one or more target molecules,
wherein the species is (are) present in a concentration K1, the one
or more markers are present in a concentration K2 and the one or
more target molecules are present in a concentration K3; (B)
preparing a second mixed phase wherein the one or more markers are
contacted with the one or more target molecules; (C) incubating the
first and second mixed phases under identical conditions, which
conditions permit a binding of the species and the one or more
markers to the one or more target molecules, resulting in (a) a
quantity M1 of one or more markers GM1 that are bound to one or
more target molecules and a concentration K4 of one or more unbound
markers in the first mixed phase and (b) a quantity M2 of one or
more markers GM2 that are bound to one or more target molecules and
a concentration K5 of one or more unbound markers in the second
mixed phase; (D) separating the one or more markers GM1 from the
first mixed phase and separating the one or more markers GM2 from
the second mixed phase; (E1) determination of the concentrations K4
and K5, and calculating a concentration K6 from K5, wherein K6 is a
concentration of one or more unbound markers in the second mixed
phase under the assumption that the one or more markers have been
contacted therein in the concentration K2 with the one or more
target molecules in the concentration K3, and determining the
binding behavior of the species from the ratio of the
concentrations K6 and K4, or (E2) determination of the quantities
M1 and M2, and calculating a quantity M3 from M2, wherein M3 is a
quantity of one or more bound markers in the second mixed phase
under the assumption that the one or more markers have been
contacted therein in the concentration K2 with the one or more
target molecules in the concentration K3, and determining the
binding behavior of the species from the ratio of the quantities M3
and M1; wherein the one or more markers are present in a native
form, the determination of K4 and K5 or M1 and M2 is carried out by
mass spectrometry, and the species is a previously unknown
species.
2. The species of claim 1, wherein in (B) the one or more markers
are present in a concentration K2 and the one or more target
molecules are present in a concentration K3.
3. The species of claim 1, wherein the one or more markers GM1
comprise specifically bound markers SGM1 and the one or more
markers GM2 comprise specifically bound markers SGM2 and wherein
SGM1 and SGM2 are released after (D), and quantities thereof are
used in (E2) instead of M1 and M2.
4. The species of claim 1, wherein the method further comprises
repeating (A) to (E1) or (A) to (E2) wherein in at least one of (A)
and (B) an agent which competes with the one or more markers for a
specific binding to the one or more target molecules is added to at
least one of the first and second mixed phases in an amount which
substantially prevents or cancels a specific binding of the one or
more markers to the one or more target molecules.
5. The species claim 1, wherein the method further comprises
repeating (A) to (E1) or (A) to (E2) at least once with a different
ratio of K1 and K3.
6. The species of claim 1, wherein (D) comprises at least one of
dialysis, precipitation, adsorption, binding to immobilized
molecules which have an affinity to at least one of the one or more
target molecules, complexes of the one or more target molecules and
the one or more markers and complexes of the one or more target
molecules and the one or more species, centrifugation,
chromatography, and filtration.
7. The species of claim 1, wherein (D) comprises an
ultrafiltration.
8. The species of claim 1 , wherein (D) is carried out under
conditions under which at least one of (a) the concentrations of
the one or more unbound markers in the first and the second mixed
phases and (b) the amounts of the one or more bound markers GM1 and
GM2 remain constant.
9. The species of claim 8, wherein the mass spectrometry is carried
out in one of the MS, MS/MS and MS.sup.n modes.
10. The species of claim 9, wherein the mass spectrometry is
preceded by at least one of a capillary electrophoresis, a gas
chromatography and a liquid chromatography.
11. The species of claim 1, wherein the mass spectrometry is
preceded by a HPLC.
12. The species of claim 11, wherein the HPLC comprises reverse
phase chromatography.
13. The species of claim 1, wherein prior to the mass spectrometry
a sample preparation by means of at least one of extraction,
filtration, gas chromatography, capillary electrophoresis, liquid
chromatography, FPLC.RTM., reverse phase chromatography, size
exclusion chromatography and affinity chromatography is carried
out.
14. The species of claim 1, wherein the species has been generated
by a method of combinatorial chemistry.
15. The species of claim 1, wherein the one or more target
molecules comprise at least one target molecule that is embedded in
a membrane structure.
16. The species of claim 1, wherein at least one of the one or more
target molecules and the one or more markers is selected from
peptides, proteins, hormones, nucleic acids, sugars, polymers, low
molecular weight compounds of natural or synthetic origin and
structures on or inside cells, cell fragments, cell homogenates,
synaptosomes, liposomes, vesicles, tissue cuts, viruses, components
and fragments thereof, capsides, components and fragments thereof,
and mixtures of natural substances.
17. The species of claim 1, wherein at least one of the one or more
target molecules and the one or more markers is selected from
receptors, prions, enzymes, transport proteins, ion channels,
antibodies, synaptic plasma membrane vesicles and vesicles found
inside cells.
18. The species of claim 1, wherein the species is selected from
peptides, proteins, hormones, nucleic acids, sugars, polymers, low
molecular weight compounds of natural or synthetic origin and
structures on or inside cells, cell fragments, cell homogenates,
synaptosomes, liposomes, vesicles, tissue cuts, viruses, components
and fragments thereof, capsides, components and fragments thereof,
and mixtures of natural substances.
19. The species of claim 1, wherein the species is selected from
receptors, prions, enzymes, transport proteins, ion channels,
antibodies, synaptic plasma membrane vesicles and vesicles found
inside cells.
20. A pharmaceutical composition which comprises the species of
claim 1 and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 10/478,025, the entire disclosure whereof is expressly
incorporated by reference herein, which is a U.S. National Stage of
International Application No. PCT/EP02/05543, filed May 21, 2002,
which claims priority under 35 U.S.C. .sctn. 119 of German Patent
Application No. 101 25 258.7, filed May 23, 2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a method for determining the
binding behavior of ligands which specifically bind to target
molecules at at least one binding site.
[0004] 2. Discussion of Background Information
[0005] DE 198 14 775 A1 discloses a method for determining the
binding constants of dissolved agents and substances on surfaces
constituted by amphiphilic molecules. Therein, layers of
amphiphilic molecules are bonded to a solid carrier so that a
solid-supported membrane is formed. A defined quantity of this
solid-supported membrane is contacted with a mobile phase. The
mobile phase contains a defined quantity of substances which are to
be examined with respect to lipid binding. After an incubation, the
solid-supported membranes are separated from the mobile phase. The
concentrations of the substances in the mobile phase or the
solid-supported membrane are determined. The method can be carried
out with solid-supported membranes and substances that are present
in different quantity ratios. From the determined concentrations,
the binding constant can be calculated.
[0006] From WO 99/50669 A1 there is known a method for screening a
compound library to determine the relative affinity of a plurality
of putative ligands to a target receptor in relation to an
indicator compound by means of frontal chromatography. Therein, the
target receptors are immobilized on the solid phase of a
chromatographic column. The compound library is applied onto the
chromatographic column and equilibrated therewith at least
partially. Subsequently, the indicator compound is applied and its
break through time, i.e., the time until it appears in the effluent
of the chromatographic column, is determined by means of mass
spectrometry. The break through time of the indicator compound is
affected by its affinity, the affinity of ligands present to the
target receptor, and the number of target receptors which are not
occupied by the ligands. From the change in the break through time
in comparison to the known break through time of the indicator
compound, it can be determined whether compounds of the compound
library have an affinity to the target receptors, and whether this
affinity is higher or lower than that of the indicator
compound.
[0007] A disadvantage of this method is that the target receptors
are invariably immobilized. They are not present in the native
state, whereby it is possible to only a limited extent to say
something about the binding behavior on native target receptors. A
further disadvantage of the method is that in kinetic studies, it
leads to incorrect results regarding the binding behavior between
ligands and the indicator compound. If a ligand and the indicator
compound had the same affinities to the target receptor, the
affinity of the ligand would be determined as too low if the ligand
shows slower binding kinetics relative to the indicator compound. A
further disadvantage is that the quantity of the indicator compound
which is bound or unbound, respectively, in the binding equilibrium
state cannot be determined.
[0008] WO 01/22078 A1 relates to a method for the identification of
an active chemical substance from a mixture of active and inactive
chemical substances, where a target substance is added to the
mixture. Complexes formed of the active substance and the target
substance are separated from the mixture. For identification, the
active chemical substance may be released from the complexes and
identified by, for example, mass spectrometry. Alternatively, the
active substance may also be identified by comparing a chromatogram
or a mass spectrum, respectively, of each of the mixture of active
and inactive substances and the mixture after separation of the
complex. A disadvantage thereof is that it must be possible to
determine each of the substances of the mixture.
[0009] WO 97/43301 A2 relates to a method for characterizing the
members of a combinatorial library which bind to a certain domain.
Therein, the domain is mixed with the members of the combinatorial
library to permit a binding of the members to the domain. The
formed complexes are separated from the unbound members. The bound
members are eluted from the complexes and analyzed by mass
spectrometry. Therein, it is possible to elute more weakly bound
members separately from more strongly bound members. A disadvantage
thereof is that each of the members of the mixture must be
determinable by mass spectrometry.
[0010] From WO 96/22530 A1 and U.S. Pat. No. 5,891,742 A there is
known a method for determining a compound which binds to a target
compound from a mixture of compounds. Therein, the target compound
is contacted with the mixture of compounds. Unbound compounds are
separated from formed complexes of the compounds with the target
molecules. The formed complexes are analyzed by mass spectrometry.
It is of disadvantage therein that all compounds and complexes,
respectively, of the binding assay must be determinable by mass
spectrometry.
[0011] From WO 00/00823 A1 there is known a method for identifying
a ligand which covalently binds to a target molecule. In this
method, a target molecule is contacted with several compounds of a
substance library. The compound which forms a covalent bond with a
chemically reactive group of the target molecule is identified by
mass spectrometry. For this purpose, the conjugate formed by the
covalent bond may be fragmented. The bound compound may as well be
released by cleaving a disulfide bond. For identification, the mass
spectroscopic characteristics of the compounds to be determined or
of the fragment to be determined, respectively, must be known in
this method.
[0012] From WO 99/45150 A1 there is known a method for determining
the relative binding affinity to a target substance of compounds of
a combinatorial mixture. Therein, a first complex of the target
substance and a standard compound binding thereto is contacted with
the combinatorial mixture of compounds, whereby second complexes
are formed from these compounds and the target substance. The
mixture is analyzed by mass spectrometry. From the quantity of
first and second complexes contained therein, the relative binding
affinity can be determined. Also in this assay, it must be possible
to analyze all compounds which potentially form complexes with the
target molecules, and the complexes formed therefrom,
respectively.
[0013] From WO 00/47999 A1 there is known a method for screening
complex biological samples for a ligand which binds to a target
protein. Therein, the target protein is mixed with the complex
biological sample and incubated under conditions which permit the
formation of complexes of ligands with the target protein. Formed
ligand-target protein complexes are separated and caused to
dissociate. Released ligands can be analyzed by mass spectrometry
following the separation of the target protein. The separation is
effected in each case by means of size exclusion processes.
Furthermore, WO 00/47999 A1 discloses the use of a reference
substance and of a known competitive ligand. The competitive ligand
serves to determine whether a ligand binds specifically or
non-specifically to the selected target protein. For this purpose,
the competitive ligand is mixed with the complex biological sample,
the process is carried out as described, and it is determined
whether the mass spectroscopic signal of the ligand is affected by
the presence of the competitive ligand. The method discloses a
comparison of the binding assays in the presence and absence of the
competitive ligand. However, all bound ligands are invariably
determined by mass spectrometry.
[0014] Moreover, it is generally known form the prior art to
indirectly determine the binding of unlabeled ligands to a target
molecule by means of ligands which are labeled with a marker
substance. Therein, the unlabeled ligands are incubated with the
target molecules together with a defined portion of labeled
ligands. The subsequent determination of the quantity of the marker
substance which is bound to the target molecules through the
labeled ligands allows the determination of the binding behavior of
the ligands which are bound to the target molecules.
[0015] The methods known in the prior art make it necessary to
directly or indirectly quantify either the bound or the unbound
ligands. With direct quantification, this may involve a substantial
expenditure, depending on the ligand. For quantification, the
employed detection system must be calibrated for each of the
ligands to be quantified. This is necessary also for ligands of
which it is not known whether they show an affinity to the target
molecules. The indirect quantification by means of labeled ligands
involves substantial expenditure as well. Initially it is necessary
to label the ligands with a marker substance so that the labeled
ligands still have sufficient affinity to the target molecules.
Frequently, various labeling methods with various marker substances
must be tested for this purpose. Often, only radioactive marker
substances prove to be suitable. Handling these marker substances
and the ligands labeled therewith is dangerous, and because of the
required safety precautions, it involves substantial expenditure.
Furthermore, not every ligand is suitable for labeling by a certain
desired method. This may necessitate a search for suitable
ligands.
[0016] Object of the present invention is the elimination of the
disadvantages of the prior art. In particular, there is to be
provided a method which makes it possible to determine, with little
expenditure, the specific binding behavior of any ligands to any
desired target molecules, unaffected to a large extent by the
kinetics of the binding of the ligands to the target molecules.
SUMMARY OF THE INVENTION
[0017] In accordance with the invention, there is provided a method
for determining the binding behavior of ligands which specifically
bind to target molecules at at least one binding site, which method
comprises the following steps: [0018] (a) preparation of a first
mixed phase wherein the ligands in a concentration K1 are contacted
with markers, in a concentration K2, which specifically bind to the
target molecules, and with the target molecules in a concentration
K3, and of a second mixed phase wherein the markers are contacted
with the target molecules, wherein after the contacting, the first
and second mixed phases are incubated under identical conditions
which permit a binding of the ligands and the markers to the target
molecules, [0019] (b) separation of the bound markers GM1 contained
in the first mixed phase from the first mixed phase, and of the
bound markers GM2 contained in the second mixed phase from the
second mixed phase, and [0020] (c1) determination of a
concentration K4 of the unbound markers in the first mixed phase,
and of a concentration K5 of the unbound markers in the second
mixed phase, wherein a concentration K6 is determined from the
concentration K5, which concentration K6 corresponds to the
concentration of the unbound markers in the second mixed phase
under the assumption that the markers in the concentration K2 have
been contacted therein with the target molecules in the
concentration K3, and determination of the binding behavior of the
ligands from the ratio between the concentrations K6 and K4, or
[0021] (c2) determination of an quantity M1 of the bound markers
GM1 and of a quantity M2 of the bound markers GM2, wherein a
quantity M3 is determined form the quantity M2, which quantity M3
corresponds to the quantity of the bound markers in the second
mixed phase under the assumption that the markers in the
concentration K2 have been contacted therein with the target
molecules in the concentration K3, and determination of the binding
behavior of the ligands from the ratio of the quantities M3 and M1,
wherein the markers are present in a native form, and the
determination of the concentrations K4 and K5 or of the quantities
M2 and M1 is effected by means of mass spectrometry.
[0022] The binding behavior is to be construed to mean any behavior
which characterizes the specific binding of the ligands to the
target molecules at at least one binding site. It may be a relative
binding behavior. In this case, the binding behavior indicates
whether the ligands bind better or worse than the markers to the
target molecules, and optionally, to which extent they bind
thereto. It may as well be an absolute binding behavior. In this
case, the affinity of the ligands to the target molecules, or the
quantity of ligands which are bound to the target molecules are
determined. The determination of the binding behavior may as well
consist of a mere approximation of the binding behavior.
[0023] The term mixed phase comprises both a pure solution and a
suspension. The mixed phase may contain membrane structures such
as, e.g., vesicles. The target molecules may be present in
solution, suspension, immobilized or embedded in membrane
structures, in particular, vesicles. Further, instead of the target
molecules, the ligands may be immobilized. The target molecules or
the ligands may be present immobilized on, e.g., the wall of a
vessel or on particles. Further, in the case of target molecules
which are embedded in vesicles, the vesicles may be immobilized,
e.g., by binding to a solid phase. The conditions according to lit.
a are selected so that a binding of the ligands and the markers to
the target molecules can take place, in particular, as regards
temperature and duration of the incubation. The incubation may be
carried out until a binding equilibrium is at least almost
reached.
[0024] Markers are understood to be known, quantifiable agents
which at the employed concentrations K2 and K3 and under the
conditions according to lit. a, bind specifically to the target
molecules. The binding of the markers to the target molecules is
inhibited by the binding of the ligands. This may occur due to,
e.g., an allosteric inhibition of the binding, or because the
markers and the ligands compete for the binding to the binding site
of the ligands on the target molecules. The concentration K6 or the
quantity M3, respectively, may be determined by calculation or
graphically from the binding properties of the markers and the
concentration K5 or the quantity M2, respectively. The
concentration K6 is that concentration of unbound marker, and the
quantity M3 is that quantity of bound marker, which would be
present in the second mixed phase after the incubation if the
markers at the concentration K2 had been contacted therein with the
target molecules at the concentration K3. From the ratio between
the concentrations K6 and K4 or the quantities M3 and M1,
respectively, the quantity of the markers whose binding to the
target molecules is inhibited, and therefrom the binding behavior
of the ligands, may be determined. The ratio in the sense of the
invention may be both a relation and a difference. Both the markers
and the ligands and the target molecules may be of a uniform type,
or may differ from each other.
[0025] The separation according to lit. b may take place, for
example, by centrifugation of formed complexes. The complexes may
contain target molecules and ligands, target molecules and markers,
or target molecules, ligands and markers. It is to be understood
that instead of a concentration, a quantity, and instead of a
quantity, a concentration may be determined.
[0026] "Native form" means that the markers do not have any
modifications which are necessary for the detection, quantification
or identification. Such modifications may, for example, consist in
a labeling with a marker substance.
[0027] The advantage of the method is that the influence of kinetic
differences in the binding behavior between the ligands and the
markers on the result can to a large extent be eliminated in that
incubation is conducted until a binding equilibrium is at least
almost reached. A further advantage of the method according to the
present invention is that the target molecules may be present in a
native, in particular, not immobilized form. Thereby the method
permits to say something about the native binding behavior.
[0028] A further advantage of the method is that in the case of
ligands which are difficult to quantify directly, only the markers
need to be quantified. Thus the method is not subject to
limitations which rest in the nature of the ligands and cause
problems in the quantification. With respect to the markers, there
is the advantage that a development of markers from agents which
bind specifically to the target molecules is eliminated, because
there is no need for the markers to be labeled. Thus a search for
agents which are suitable for labeling by a particular labeling
method and bind specifically to the target molecules is not
necessary. By the same token, there is no need for a search for
agents which bind specifically to the target molecules and which
still have a sufficient affinity after the labeling. Moreover, the
expenditure which is necessary due to the handling of the utilized
marker substances is eliminated, which expenditure may be quite
high, in particular, with radioactive marker substances. A health
hazard associated with the employment of radioactive marker
substances is not present, either.
[0029] The use of mass spectrometry is advantageous in that markers
which are present in a native form can be detected, quantified and,
when different markers are used at the same time, identified.
[0030] In the method according to the present invention only the
affinity of the markers, and not the affinity of the ligands to be
studied, to the target molecules is of relevance. If the markers
have a sufficient affinity to the target molecules, the binding
behavior of ligands which have very low affinities to the target
molecules may be determined by selecting a correspondingly high
concentration K1. The utilized markers may be selected so that they
can still be detected in low concentrations as well. A further
advantage of the method is that an unspecific binding of the
ligands to be studied has almost no impact on the result of the
measurement. Substantially only the non-specific binding of the
markers must be taken into account.
[0031] In an advantageous embodiment, in step lit. a the markers in
the second mixed phase are contacted in a concentration K2 with the
target molecules in a concentration K3. Thereby, the determination
of the concentration K6 in step lit. c1 or of the quantity M3 in
step lit. c2 is simplified, because under these conditions the
concentration K6 is the same as the concentration K5, and the
quantity M3 is the same as the quantity M2.
[0032] In a further advantageous embodiment, after step lit. b the
specifically bound markers SGM1 among the bound markers GM1 and the
specifically bound markers SGM2 among the bound markers GM2 are
released after the step lit. b. In step lit. c2, the released
specifically bound markers SGM1 take the place of the bound markers
GM1, and the released specifically bound markers SGM2 take the
place of the bound markers GM2. The release may be effected, for
example, by adding an excess of molecules which specifically
inhibit the binding of the markers. Such molecules may, for
example, be competitors which displace the specifically bound
markers from the binding site. Another possibility consists in
removing by a washing step non-specifically bound markers from the
target molecules so that only specifically bound markers remain
bonded. The specifically bound markers may then be released by
means of non-specific methods. By releasing the specifically bound
markers the proportion of non-specifically bound markers, can be
taken into account.
[0033] Further, for taking into account a non-specific binding of
the markers to the target molecules the steps lit. a to c1 or c2
may be carried out an additional time, wherein in step lit. a a
material which competes with the markers for the specific binding
to the target molecules is added to the first and/or the second
mixed phase in such an amount that a specific binding of the
markers to the target molecules is largely prevented or terminated.
The concentration K4 determined thereby in steps lit. c1 or c2 is
identified as K4', K5 as K5' and K6 as K6', and the quantity M1 as
M1', M2 as M2' and M3 as M3'. The values determined without the
agent are still identified as K4, K5, K6, M1, M2, and M3. The
difference between K4' and K4, K5' and K5, and K6' and K6 in each
case is the concentration of the specifically bound markers. M1',
M2' and M3' each are the quantities of the non-specifically bound
markers. The difference between M1 and M1', M2 and M2' and M3 and
M3' is the quantity of the specifically bound markers.
[0034] In a preferred embodiment of the invention, for the
determination of the affinity the steps lit. a to c1 or c2 are
carried out at least twice with a different ratio of the
concentrations K1 to K3. Preferably, in this case the incubation
according to step lit. a is carried out until the binding
equilibrium is at least almost reached. From the quantities of the
markers whose binding has been inhibited at the different ratio of
the concentrations, the affinity can be determined. This can be
done by means of a non-linear regression analysis of the
competition or displacement graph. The competition or displacement
graph represents the inhibition of the binding of the markers by
ligands as a function of the concentration of unbound ligands.
[0035] Preferably, the separation according lit. b is effected by
dialysis, precipitation, adsorption, binding to immobilized
molecules which have an affinity to the target molecules or to
formed complexes of the target molecules and the markers and/or the
ligands, centrifugation, chromatography, or filtration, in
particular, ultrafiltration. The separation according to lit b. is
preferably carried out under conditions under which the
concentration of the unbound markers in the first and in the second
mixed phases and/or the quantity of the bound markers GM1 and GM2
remains constant. Any method wherein the separation of bound
ligands is carried out under conditions under which the proportions
of the bound and the unbound markers and ligands remain constant is
suitable for this purpose. As such a method, the separation of
formed complexes of target molecules and markers and/or ligands
under binding conditions, e.g., by means of centrifugation or
filtration, may be contemplated. The advantage of the separation
under the mentioned conditions is that a cancellation of a binding
equilibrium which is caused by the separation, for example in a
washing step, is avoided. A dissociation of the complexes which are
formed from the target molecules and the markers and/or the ligands
which is caused by the cancellation of the binding equilibrium does
not take place. An error in the quantification of the bound ligands
resulting therefrom is avoided. A further advantage of the
separation under the mentioned conditions is that the first and the
second mixed phases do not have to be separated from the bound
markers GM1 and GM2 quantitatively. It is sufficient to separate a
defined portion of each of the first and the second mixed phases.
From this portion and the concentrations K4 and K5 determined in
step lit. c1 the amount of marker whose binding has been inhibited
by the ligands may be determined.
[0036] Advantageously, the mass spectrometry is carried out in the
MS, MS/MS or MS.sup.n mode. In the MS mode, ions formed by the
analyte in the ion source of a mass spectrometer, in the MS/MS mode
selectively daughter ions of the ions formed by the analyte, and in
the MS.sup.n mode selectively further daughter ions of the ions
formed by the analyte are quantified. Mass spectrometry may be
preceded by a capillary electrophoresis or a gas or liquid
chromatography, preferably HPLC, in particular, by using reverse
phase chromatography. A separation of molecules which can be
distinguished in the mass spectrometry only with difficulty can be
achieved thereby. The molecules can then separately be analyzed in
the mass spectrometry.
[0037] Preferably, prior to the mass spectrometry a sample
preparation by means of extraction, preferably solid phase or
liquid-liquid extraction, filtration, in particular,
ultrafiltration, gas chromatography, capillary electrophoresis,
liquid chromatography, preferably HPLC, FPLC.RTM., reverse phase
chromatography, size exclusion chromatography and/or affinity
chromatography is carried out. Sample preparation means the work-up
of a sample from which the concentrations K4 and K5 or the
quantities M2 and M1 are to be determined. This may be necessary,
for example, if certain components must be removed from the sample.
These may be components which are harmful to a mass spectrometer,
for example, because they would plug or attack the feed lines, or
which reduce the sensitivity of a mass spectrometric measurement,
e.g., because they reduce the signal of the marker to be analyzed.
Furthermore, these may be components which would interfere with a
method which precedes the mass spectrometry.
[0038] The first and the second mixed phases according to lit. a
may preferably contain more than 10, in particular, more than 100
various target molecules and/or markers. In a preferred embodiment
the first and the second mixed phase according to lit. a contain a
common marker which is specific to various target molecules. If
various markers are used, each marker specifically binds to at
least one binding site of a predetermined target molecule.
Preferably, the various markers differ from each other at least as
regards the molar mass, the formation of ions or daughter ions in
the mass spectrometry, or the chromatographic or electrophoretic
behavior. Advantageously, the first mixed phase according to lit. a
preferably contains more than 10, in particular, more than 100
different ligands. The ligands may have been generated by methods
of combinatorial chemistry. Such methods are known, for example,
from Felder, E. R., Chimia 48 (1994), 521-541, Gallop et al., J.
Med. Chem. 37 (1994), 1233-1251, Houghten, R. A., Trends Genet. 9
(1993), 235-239, Houghten et al., Nature 354 (1991), 84-86, Lam et
al., Nature 354 (1991), 82-84, Carell et al., Chem. Biol. 3 (1995),
171-183, Madden et al., Perspectives in Drug Discovery and Design 2
(1994), 269-285, Cwirla et al., Biochemistry 87 (1990), 6378-6382,
Brenner, S. and Lerner, R. A., Proc. Natl. Acad. Sci. USA 89
(1992), 5381-5383, Gordon et al., J. Medicinal Chemistry 37 (1994),
1385-1401 and Lebi et al., Biopolymers 37 (1995), 177-198.
[0039] By using several different ligands, target molecules and/or
markers, the efficiency of the method can be increased
significantly. When the method is carried out with different
markers, only each of the markers has to be quantified in order to
determine if ligands which have an affinity to the target molecules
were present in the first mixed phase. Thus the method is suitable
for high throughput screening. To find out which of the ligands
have an affinity to the target molecules, a deconvolution strategy
may be pursued. Here, the method is carried out anew with a reduced
number or a different combination of ligands to be studied. By
comparing the results it can then be determined which of the
ligands has bonded to the target molecules in which amount. Also,
the different markers may have different graduated affinities to
the target molecules. By determining or approximating the amount of
those markers whose binding to the target molecules has been
inhibited, the affinity of the ligands to the target molecules may
be determined or at least approximated.
[0040] In one embodiment, following step lit. c1 or c2 the bound
ligands are identified by mass spectrometry and/or the amount of
bound ligands is determined by mass spectrometry. For this, the
bound ligands may be specifically released prior to mass
spectrometry, for example, by the addition of specific competitors.
Such a method is very efficient in a high throughput screening in
which many substances which predominantly do not bind to the target
molecules are studied at the same time with respect to their
binding behavior. The identification of the bound ligands or the
determination of the quantity of the bound ligands, respectively is
necessary only if it results from step lit. c1 or c2 that bound
ligands are present at all.
[0041] For the determination of the concentrations K4 and K5 or of
the quantities M1 and M2 the markers may be used as external
standard. Also, it is possible to add to the first and/or the
second mixed phase after step lit. b as internal standard at least
one marker which has a marking substance. Such an internal standard
facilitates the determination of the concentrations K4 and K5
because it is affected to the same extent as the marker itself.
Thus, losses in the sample preparation or a possibly necessary
concentration of the sample may be taken into account as readily as
interfering factors which affect the mass spectrometric
determination.
[0042] The target molecules may be present embedded in membrane
structures. This may be advantageous, for example, with target
molecules which are naturally present embedded in membrane
structures, and whose binding properties are altered when they are
not embedded in membrane structures. In an advantageous embodiment,
the target molecules are not bound to a solid support. Thereby a
change in the binding behavior of the target molecules by the
binding to a solid support and and/or an influence on the kinetics
of the binding are avoided.
[0043] It is possible to regenerate the target molecules after the
steps lit. b or c2 have been carried out. This permits a reuse
thereof. This makes sense, in particular, with valuable target
molecules, e.g., target molecules on cells on which several studies
are to be conducted. The target molecules, markers or ligands may
be selected from the following group: peptides, proteins, in
particular, receptors, prions, enzymes, transport proteins, ion
channels or antibodies, hormones, nucleic acids, sugars, polymers,
low molecular weight compounds of natural or synthetic origin and
structures on or inside cells, cell fragments, cell homogenates,
synaptosomes, liposomes, vesicles, in particular, synaptic plasma
membrane vesicles and vesicles which are found in cells, tissue
cuts, viruses, their components or fragments of these components,
capsides, their components or fragments of these components and
mixtures of natural substances.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] In the following, the invention is explained in greater
detail by the drawing and by means of working examples. There are
shown in
[0045] FIG. 1 The result of a method carried out according to the
present invention, wherein there were employed .mu.-opioid
receptors as target molecules, morphine as a marker and naloxone as
a ligand in different concentrations, and
[0046] FIG. 2 The result of a control experiment wherein there were
employed .mu.-opioid receptors as target molecules, [.sup.3H]DAMGO
as a marker and naloxone as a ligand.
DETAILED DESCRIPTION OF THE INVENTION
[0047] In all working examples, human .mu.-opioid receptors served
as target molecules in the binding assays. The target molecules
were provided by means of a membrane preparation of transfected
human .mu.-opioid receptors which express CHO-K1 cells of the
company Biotrend, Cologne.
WORKING EXAMPLE 1
Screening of Substance Libraries by Determination of an Unbound
Marker
1.1. Binding Assays
[0048] An assay B0 contained the .mu.-opioid receptors in a
concentration of 5.5 nmol/L, 5 mmol/L of MgCl.sub.2 and 50 mmol/L
of Tris HCl, pH 7.4, in a total volume of 250 .mu.L. The assay B0
was incubated in a 1.5 ml PP reaction vessel at 25.degree. C. for
150 minutes. Subsequently, the membrane particles were centrifuged
off at 50,000 g at 4.degree. C. for 20 minutes. 200 .mu.L were
taken from the resultant supernatant U0. The following binding
assays were treated like B0: [0049] A binding assay B1 which was
composed like binding assay B0 and additionally contained 10 nmol/L
of morphine as a marker. [0050] A binding assay B2 which was
composed like binding assay B1 and additionally contained
(.+-.)-methadone in a concentration of 50 .mu.mol/L. [0051] A
binding assay B3 which was composed like binding assay B1 and
additionally contained the substances of a library 1, consisting of
4-chloroaniline, 2,3-dichloroaniline, methyl benzoate,
acetoanilide, amitryptiline, (+)-bicuculine, phenol, naloxone,
benzoic acid and tramadol, each in a concentration of 1 .mu.mol/L.
[0052] A binding assay B4 which was composed like binding assay B1
and additionally contained the substances of a library 2, each in
concentration of 1 .mu.mol/L. The library 2 [0053] consists, except
for naloxone, of the substances of the library 1.
[0054] From B1-B4 supernatants U1-U4 were recovered.
1.2. Quantification of the Marker
[0055] For quantification, a mass spectrometric determination was
carried out under the following conditions on an Applied Biosystems
API 2000 with TurbolonSpray source in the MRM mode to which a
Hewlett Packard HP1100 LC system was coupled: [0056] Column:
Superspher 60 RP select B4 .mu.m 125.times.2 mm [0057] Eluent: 0.1%
HCOOH in water/acetonitrile (80/20) [0058] Flow: 0.4 ml/minute
(isocratic) [0059] Sample volume: 20 .mu.L (injected by means of
Autosampler)
[0060] Morphine was directly measured in the supernatants U1-U4 at
the mass transitions 286.1/115.1 and 286.1/128.1. It was quantified
in comparison to morphine which was dissolved in U0 in
concentrations of 2 nmol/L to 10 nmol/L as external standard. With
each sample two measurements were carried out.
1.3. Analysis
[0061] In U1-U4 the following concentrations of unbound morphine
were determined as average of two measurements: [0062] U1: 2.25
nmol/L [0063] U2: 7.66 nmol/L [0064] U3: 7.92 nmol/L [0065] U4:
2.03 nmol/L
[0066] From each difference of the employed concentrations of
morphine and the concentrations of unbound morphine determined for
U1-U4, the following concentrations of bound morphine were
determined for B1-B4: [0067] B1: 7.75 nmol/L [0068] B2: 2.34 nmol/L
[0069] B3: 2.08 nmol/L [0070] B4: 7.97 nmol/L
[0071] In B1, the binding of morphine was determined without
additional competitor as total binding. By contrast, in B2 the
binding of morphine was determined in the presence of 50 .mu.mol/L
of methadone under conditions which could be expected to
approximate a total occupation of the target molecules by
methadone. Morphine has merely bound non-specifically in this case.
Since in B3 the concentration of bound morphine is only of the
order of magnitude of the non-specific binding, there is at least
one substance in library 1 which has a significant affinity to the
target molecules at a concentration of 1 .mu.mol/L. Since in B4 the
concentration of bound morphine is of the order of magnitude of the
total binding, there is no substance in library 2 which at a
concentration of 1 .mu.mol/L, has a significant affinity to the
target molecules. Since the libraries 1 and 2 differ only in that
naloxone is contained in library 1, but not in library 2, the
affinity exhibiting substance can only be naloxone.
WORKING EXAMPLE 2
Determination of the Affinity of a Ligand by Measurement of the
Unbound Marker
2.1. Binding Assays
[0072] A first binding assay B1 contained the .mu.-opioid receptors
in a concentration of 5.5 nmol/L, 10 nmol/L of morphine as a
marker, 5 mmol/L of MgCl.sub.2 and 50 mmol/L of Tris HCl, pH 7.4,
in a total volume of 250 .mu.L. The binding assay B1 was incubated
in a 1.5 ml PP reaction vessel at 25.degree. C. for 150 minutes.
Subsequently, the membrane particles were centrifuged off at 50,000
g at 4.degree. C. for 20 minutes. 200 .mu.L were taken from the
resultant supernatant U1. The following binding assays were treated
like B1: [0073] A second binding assay B2 which was composed like
binding assay B1 and additionally [0074] contained (.+-.)-methadone
in a concentration of 50 .mu.mol/L. [0075] Five further binding
assays B3-B7 which were composed like B1 and additionally contained
naloxone in the following concentrations: B3: 1 nmol/L of naloxone,
[0076] B4: 10 nmol/L of naloxone, B5: 30 nmol/L of naloxone, B6:
100 nmol/L of naloxone, [0077] B7: 1 .mu.mol/L of naloxone.
[0078] In a second test series which was carried out in an
identical manner, the binding assays B1-B7 were substituted by the
identical binding assays B1*-B7*. From B1-B7 the supernatants
U1-U7, and from B1*-B7* the supernatants U1*-U7* were
recovered.
2.2. Quantification of the Marker
[0079] The mass spectrometric determination was carried out as
described under 1.2. Additionally, naloxone was quantified at the
mass transition 328.1/310.1. With each sample two measurements were
carried out.
2.3 Analysis
[0080] In U1-U7 and U1*-U7* the following concentrations of unbound
morphine and naloxone were determined as the average of two
measurements: TABLE-US-00001 Morphine Naloxone U1: 2.25 nmol/L --
U2: 7.66 nmol/L -- U3: 2.78 nmol/L 0.35 nmol/L U4: 4.15 nmol/L 4.54
nmol/L U5: 4.75 nmol/L 15.2 nmol/L U6: 7.00 nmol/L 77.2 nmol/L U7:
8.23 nmol/L 858 nmol/L U1*: 2.24 nmol/L -- U2*: 6.29 nmol/L -- U3*:
2.18 nmol/L 0.14 nmol/L U4*: 3.16 nmol/L 2.96 nmol/L U5*: 4.43
nmol/L 13.0 nmol/L U6*: 5.50 nmol/L 61.7 nmol/L U7*: 5.86 nmol/L
668 nmol/L
[0081] From each difference of the employed concentrations of
morphine and the concentrations of unbound morphine determined for
U1-U7 and U1*-U7*, the following concentrations of bound morphine
were determined for B1-B7 and B1*-B7*: TABLE-US-00002 B1: 7.75
nmol/L B1*: 7.76 nmol/L B2: 2.34 nmol/L B2*: 3.71 nmol/L B3: 7.22
nmol/L B3*: 7.82 nmol/L B4: 5.85 nmol/L B4*: 6.84 nmol/L B5: 5.15
nmol/L B5*: 5.57 nmol/L B6: 3.00 nmol/L B6*: 4.50 nmol/L B7: 1.77
nmol/L B7*: 4.14 nmol/L
[0082] In B1 and B1*, the binding of morphine was determined
without additional competitor as total binding. By contrast, in B2
and in B2* the binding of morphine was determined in the presence
of 50 .mu.mol/L of methadone under conditions which could be
expected to approximate a total occupation of the target molecules
by methadone. Morphine has merely bound non-specifically in this
case. The specific binding results from the difference of the
concentrations determined for B1 and B2 and B1* and B2*,
respectively. In FIG. 1 the concentration of the bound morphine is
represented as a function of the concentration of the unbound,
i.e., free naloxone.
[0083] By means of a non-linear regression analysis carried out
with the program Prism 2.01 Graph Pad, San Diego, USA, the
concentration of free naloxone at which 50% of the morphine are
specifically bound was determined. This value is called IC.sub.50
value. For the binding assays B1-B7, an IC.sub.50 value of 11.9
nmol/L, and for the binding assays B1*-B7* an IC.sub.50 value of
11.6 nmol/L was determined.
[0084] The K.sub.i value as a measure of the affinity of naloxone
to the target molecules may be determined as follows according to
Hulme, E. C. and Birdsall, N. J. M., Strategy and tactics in
receptor-binding studies in Receptor-Ligand-Interactions, ed.
Hulme, E. C., Oxford University Presss, Oxford (1992), 63-176, in
binding experiments in which the concentration of the free marker
is different to a non-negligible extent from the starting
concentration of the marker: K i = IC 50 2 .times. ( L * - L 0 * )
L 0 * + 1 + L * K d ##EQU1##
[0085] The meanings are: [0086] L*: Concentration of the marker at
the IC.sub.50 value. [0087] L.sub.0*: Concentration of the free
marker in the absence of the competitor and [0088] K.sub.d:
Affinity of the marker to the target molecules.
[0089] From Raynor, K., Kong, H., Mestek, A., Bye, L. S., Tian, M.,
Liu, J., Yu, L., and Reisine, T., Characterisation of the cloned Mu
opioid Receptor, J. Pharmacol. Exp. Ther. 272 (1995), 423-428, it
is known that the K.sub.d value for morphine is 2.0 nmol/L. From
this, there results for B1-B7 at [0090] IC.sub.50=11.9 nmol/L
[0091] L*=4.8 nmol/L [0092] L.sub.0*=2.25 nmol/L a K.sub.i for
naloxone of 1.4 nmol/L with respect to the human .mu.-opioid
receptors.
[0093] For B1*-B7*, there results at [0094] IC.sub.50=11.6 nmol/L
[0095] L*=4.2 nmol/L [0096] L.sub.0*=2.24 nmol/L a K.sub.i for
naloxone of 1.7 nmol/L with respect to the human .mu.-opioid
receptors. 2.4. Control Experiment Competition
Naloxone/[.sup.3H]DAMGO on Human .mu.-Opioid Receptors
[0097] [.sup.3H]DAMGO is the .sup.3H labeled .mu.-opioid receptor
agonist Tyr-D-Ala-Gly-N-Methyl-Phe-Gly(o1)-encephaline. 22 .mu.g of
protein of a membrane preparation of transfected CHO K1 cells of
the company Biotrend, Cologne, which preparation contained 30 fmol
of human .mu.-opioid receptors, were incubated as described in
Wanner, K. T., Praschak, I., Hofner, G. and Beer, H., Asymmetric
Synthesis and Enantiospecificity of Binding of
2-(1,2,3,4-Tetrahydro-1-isoquinolinyl)-ethanol derivatives to .mu.-
and .kappa.-receptors, Arch. Pharm. 329 (1996), 11-12, with 4.5
nmol/L of [.sup.3H]DAMGO and 0 nmol/L, 1 nmol/L, 10 nmol/L, 30
nmol/L, 100 nmol/L and 1 .mu.mol/L of naloxone for 60 minutes at
25.degree. C. in a total volume of 250 .mu.L. The non-specific
binding was determined in the presence of 50 .mu.mol/L of
(.+-.)-methadone. Two test series were carried out in triplicate
each. In FIG. 2 the concentration of the formed [.sup.3H]DAMGO is
represented as a function of the concentration of the naloxone
employed. By means of a non-linear regression analysis carried out
with the program Prism 2.01 Graph Pad, San Diego, USA, the
IC.sub.50 value, i.e., the concentration of naloxone at which 50%
of the [.sup.3H]DAMGO are specifically bound was determined. From
Raynor, K., Kong, H., Mestek, A., Bye, L. S., Tian, M., Liu, J.,
Yu, L., and Reisine, T., Characterisation of the cloned Mu opioid
Receptor, J. Pharmacol. Exp. Ther. 272 (1995), 423-428, it is known
that the K.sub.d value for DAMGO is 1.4 nmol/L. According to Cheng,
Y. C. and Prusoff, W. H., Relationship between the inhibition
constant Ki and the concentration of inhibitor which causes 50 cent
inhibition IC50 of an enzymatic reaction, Biochem. Pharmacol. 22
(1973), 3099-3108, the following K.sub.i values of naloxone with
respect to human .mu.-opioid receptors were calculated from the
K.sub.d-value for DAMGO and the IC.sub.50 values of 18 nmol/L and
17 nmol/L from the first and second test series, respectively:
[0098] 1st Test series K.sub.i=4.2 nmol/L [0099] 2nd Test series
K.sub.i=4.1 nmol/L
WORKING EXAMPLE 3
Screening of Substance Libraries By Measuring the Bound Marker
After Its Controlled Release
[0099] 3.1. Binding Assays
[0100] The binding assays B1-B4 described in Working Example 1 were
used and treated as described under 1.1. Pellets P1-P4 obtained
from B1-B4 after centrifugation were treated as follows: P1-P4 were
each washed three times with 200 .mu.L of 5 mmol/L of MgCl.sub.2
and 50 mmol/L of Tris HCl, pH 7.4. Subsequently, P1-P4 were each
resuspended in 200 .mu.L of 5 mmol/L of MgCl.sub.2 and 50 mmol/L of
Tris HCl, pH 7.4, and incubated for 15 minutes at room temperature
in a shaking water bath and centrifuged at 50,000.times.g for 20
minutes at 4.degree. C. The resultant supernatants were discarded
and the obtained pellets P1'-P4' were each resuspended with 200
.mu.L of 50 .mu.mol/L of (.+-.)-methadone in 5 mmol/L of MgCl.sub.2
and 50 mmol/L of Tris HCl, pH 7.4 and once again incubated as
before for 15 minutes. The assays were subsequently centrifuged as
before, whereby the supernatants F1-F4 were recovered.
3.2. Quantification of the Marker
[0101] The morphine contained in F1-F4 was quantified as described
under 1.2.
3.3. Analysis
[0102] In F1-F4 the following concentrations of unbound, i.e.,
released morphine were measured as average of two measurements:
[0103] F1: 4.84 nmol/L [0104] F4: 4.80 nmol/L [0105] F2/F3:
morphine not detectable
[0106] In B1, the binding of morphine was determined without
additional competitor as total binding. By contrast, in B2 the
binding of morphine was determined in the presence of 50 .mu.mol/L
of methadone under conditions which could be expected to
approximate a total occupation of the target molecules by
methadone. Morphine has merely bound non-specifically in this case.
From P2' apparently no morphine could be released, since morphine
is not detectable in F2. Accordingly, it can be assumed that the
morphine which was detected in F1 and released from P1' was bound
specifically. Since about as much morphine was released from P4' in
F4 as from P1' into F1, the library 2 apparently does not contain a
ligand with pronounced affinity to the target molecules. Apparently
no morphine could be released from P3', because morphine was not
detectable in F3. Accordingly, the library 1 contains at least one
substance which has a significant affinity to the target molecules
at a concentration of 1 .mu.mol/L. Since the libraries 1 and 2
differ only in that naloxone is contained in library 1, but not in
library 2, the substance with affinity can only be naloxone.
WORKING EXAMPLE 4
Screening of Substance Libraries By Measuring the Unbound Marker
and Subsequent Identification of Ligands Having Affinity Following
Their Controlled Release
4.1. Binding Assays
[0107] With the exception of each containing the substances of the
library 1 and of the library 2 in a concentration of 10 nmol/L
instead of 1 .mu.mol/L, the binding assays B1#-B4# were the same as
the binding assays B1-B4 in Working Example 1. The binding assays
were treated as described under 1.1. By centrifugation,
supernatants U1#-U4# and pellets P1#-P4# were obtained from the
binding assays B1#-B4#. P4# was discarded. P1#-P3# were each washed
three times with 200 .mu.L of 5 mmol/L of MgCl.sub.2 and 50 mmol/L
of Tris HCl, pH 7.4. Subsequently, P1#-P3# were each resuspended in
200 .mu.L of 5 mmol/L of MgCl.sub.2 and 50 mmol/L of Tris HCl, pH
7.4, incubated for 15 minutes at room temperature in a shaking
water bath, and centrifuged at 50,000.times.g for 20 minutes at
4.degree. C. The resultant supernatants were discarded and the
obtained pellets P1#'-P3#' were each resuspended with 200 .mu.L of
50 .mu.mol/L of (.+-.)-methadone in 5 mmol/L of MgCl.sub.2 and 50
mmol/L of Tris HCl, pH 7.4, and once again incubated as before for
15 minutes. The assays were subsequently centrifuged as before,
whereby the supernatants F1#-F3# were recovered.
4.2 Quantification of the Ligands
[0108] Unbound or free morphine, respectively, was quantified in
U1#-U4# and in F1#-F3# as described under 1.2. In F3#, also the
individual substances of the library 1 were additionally determined
at the respective specific mass transitions. However, except for
naloxone, none of the substances could be detected in F3#. The
released naloxone was quantified at the mass transition
328.1/310.1. Two measurements were carried out with each
sample.
4.3. Analysis
[0109] In U1#-U4# the following concentrations of unbound morphine
were determined as mean values: [0110] U1#: 2.39 nmol/L [0111] U2#:
7.80 nmol/L [0112] U3#: 5.02 nmol/L [0113] U4#: 2.76 nmol/L
[0114] Since B4# from the library 2 apparently did not contain a
ligand with significant affinity to the target molecules, P4# was
discarded.
[0115] In F1#-F3# the following average concentrations of released
morphine and naloxone were determined: TABLE-US-00003 Naloxone
Morphine F1: 4.63 nmol/L F2: morphine not detectable F3: 2.30
nmol/L 2.93 nmol/L
[0116] In F3#, naloxone and morphine could be detected in
approximately the same concentrations. Apparently, among the
substances of library 1 only naloxone has high affinity to the
target molecule, while the remaining substances have a distinctly
lower affinity. Since the identified ligand naloxone and the marker
morphine were contained in binding assay B3# in the same
concentrations, it can be inferred that the released identified
ligand naloxone has an affinity to target molecules which is in the
range of the affinity of the marker morphine.
WORKING EXAMPLE 5
Screening of Test Sustances with Regard to Their Affinity to
Different Target Molecules in Native Materials by Simultaneously
Determining the Unbound Portions of Two Different Markers
5.1. Conception
[0117] SCH23390 and spiroperidol possess high affinity to D.sub.1-
and D.sub.2-type receptors in the central nervous system, and are
used as markers for the respective target molecules. As native
material which contains both D.sub.1 and D.sub.2 receptors in
sufficient amount, a membrane fraction from the striatum of the
porcine brain is employed. Test substances are simultaneously
examined in a binding assay with respect to their ability to
inhibit the specific binding of SCH23390 to D.sub.1 receptors or
the specific binding of spiroperidol to D.sub.2 receptors,
respectively.
5.2. Recovery of the Receptor Material
[0118] The striata of 20 porcine brains (raw mass 20 g) were
homogenized with a Potter in the ten-fold volume of 0.32 mol/L of
sucrose, and were centrifuged at 1,000 g for 10 min at 4.degree. C.
The supernatant was centrifuged at 20,000 g for 10 min at 4.degree.
C. The pellets were resuspended in 50 mmol/L of Tris HCl, pH 7.4
(buffer), and centrifuged at 30,000 g for 10 min at 4.degree. C.
The last step was repeated. The resultant membrane fraction was
frozen in liquid N.sub.2.
5.3. Binding Assays
[0119] In a first binding assay (B1), an aliquot of the membrane
fraction which contained 1.7 mg of protein was incubated with 20
nmol/L of each of SCH23390 and spiroperidol in 50 mmol/L of Tris
HCl, pH 7.4 in a total volume of 250 .mu.L for 40 min at 25.degree.
C. in a 1.5 ml PP reaction vessel. Subsequently, the membrane
particles were centrifuged off at 50,000.times.g for 20 min at
4.degree. C. 200 .mu.L of the supernatant (U1) were taken off.
[0120] A second binding assay (B2) which additionally contained
butaclamol at a concentration of 100 .mu.mol/L was carried out
analogously to B1 and afforded U2. A third binding assay (B3) which
additionally contained haloperidol as a test substance at a
concentration of 1 .mu.mol/L was carried out analogously to B1 and
afforded U3. A fourth binding assay (B4) which additionally
contained chlorpromazine as a test substance at a concentration of
1 .mu.mol/L was carried out analogously to B1 and afforded U4. A
fifth binding assay (B5) which additionally contained methyl
benzoate as a test substance at a concentration of 1 .mu.mol/L was
carried out analogously to B1 and afforded U5. All binding assays
B1 to B5 were carried out as double determinations.
[0121] A further supernatant U0 which contained neither SCH23390
nor spiroperidol during the incubation was recovered in analogous
manner.
5.4. Quantification of the Marker
[0122] The mass spectroscopic determination of the marker was
carried out on an Applied Biosystems API 2000 with TurbolonSpray
source in the MRM mode, to which a Hewlett Packard HP1100 LC system
was coupled. The markers SCH23390 and spiroperidol were measured
directly side by side in the supernatants U1-U5 by means of the MRM
method which was based on the mass transitions of 288.1/91.1 for
SCH23390 and 395.9/122.9 for spiroperidol, respectively, and were
quantified in comparison to external standards (1 nmol/L to 5
nmol/L of SCH23390 and spiroperidol in U0). All samples were
measured twice. [0123] Column: Superspher 60 RP select B4 .mu.m
125.times.2 mm with 10.times.2 mm pre-column [0124] Eluent: 0.1%
HCOOH in water/acetonitrile (65/35) [0125] Flow: 0.35 ml/minute
(isocratic) [0126] Sample volume: 20 .mu.L (injected by means of
Autosampler) 5.5. Analysis
[0127] In U1-U5 the following concentrations of unbound markers
were determined (average values of doubles and double LCMS
determination): TABLE-US-00004 SCH23390 Spiroperidol U1: 2.88
nmol/L 1.93 nmol/L U2: 4.15 nmol/L 2.75 nmol/L U3: 2.77 nmol/L 2.78
nmol/L U4: 2.71 nmol/L 2.29 nmol/L U5: 2.71 nmol/L 1.89 nmol/L
[0128] Each of the differences of the markers in U1 and U2
describes the specific binding for SCH23390 and spiroperidol (for
SCH23390 1.27 nmol/L; for spiroperidol 0.82 nmol/L).
[0129] Among the studied test substances (haloperidol,
chlorpromazine and methyl benzoate), none is capable, at the
employed concentration of 1 .mu.mol/L, to inhibit the specific
binding of the marker SCH23390 to D.sub.1-type receptors.
Apparently, none of the studied test substances exhibits (in the
concentration employed) affinity to D.sub.1-type receptors.
[0130] In the employed concentration of 1 .mu.mol/L, methyl
benzoate is not capable of inhibiting the specific binding of
spiroperidol to D.sub.2-type receptors. Apparently, methyl benzoate
exhibits (in the concentration employed) no affinity to
D.sub.2-type receptors.
[0131] Haloperidol completely inhibits the specific binding of the
marker spiroperidol to D.sub.2-type receptors, while chlorpromazine
inhibits the specific binding of the marker spiroperidol to
D.sub.2-type receptors only at about 50%. Apparently, haloperidol
has a higher affinity to D.sub.2-type receptors than
chlorpromazine.
* * * * *