RU2838410C1 - Methods of using mass spectroscopy in multiplex evaluations of targets - Google Patents

Abstract

FIELD: measuring.

SUBSTANCE: group of inventions relates to methods for assessing affinity of ligands for a specific receptor target molecule. Disclosed is a multiplex method for quantifying binding of a test compound with a predetermined target molecule and with non-target molecules, involving the following steps: (a) obtaining a mixture of target molecules from a crude extract; (b) incubating said mixture of target molecules in a plurality of mixtures of ligands and test compounds; (c) removing unbound ligands from said plurality of target molecules; (d) isolating ligands associated with said target molecules in said mixture of target molecules; (e) determining the amount of said ligand bound by the target molecule using mass spectrometry and a calibration curve; (f) determining the affinity of the test compound for said target molecules; (g) measuring binding of said test compound with a predetermined target molecule and comparing said binding with binding of said test compound with non-target molecules; (h) calculation K_on and K_off said test compound for said target molecule. Also disclosed are multiplex methods for quantifying binding affinity and a multiplex method for determining K_on and/or K_off.

EFFECT: group of inventions provides a multiplex method for assessing binding affinity of test compounds.

26 cl, 6 dwg, 15 tbl, 8 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Patent Application Serial No. EP19306104, filed September 13, 2019, and Provisional Patent Application Serial No. EP19306110, filed September 16, 2019, the entire contents of which are hereby incorporated by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present methods relate to characterizing the binding of various compounds to target molecules using label-free technology such as mass spectrometry (MS). They further relate to assessing the affinity of ligands for a specific target receptor molecule.

LEVEL OF TECHNOLOGY

[0003] Below is provided background information for specific aspects of the present invention as they may relate to technical features referenced in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in more detail in the documents discussed below, and these materials may provide additional guidance to those skilled in the art to make or use specific aspects of the present invention as claimed. The contents of such documents are hereby incorporated by reference. The discussion below should not be construed as an admission of the applicability of this information to any claim in the present description or to the effect of the described prior art material.

SCIENTIFIC PATENTS AND PUBLICATIONS

[0004] Wanner et al ., WO 2002095403 (US 7,074,334), "Method for determining the binding behavior of ligands which specifically bind to target molecules", describes an invention relating to a method for determining the binding behavior of ligands that specifically bind to target molecules at least at one binding site, wherein the markers are present in native form and the concentrations of K4 and K5 or the amounts of M2 and M1 are determined by mass spectrometry.

[0005] Wanner et al ., U.S. Patent Publication 2006/0201886, "Method for determining the binding behavior of ligands which specifically bind to target molecules," describes a method for determining the binding behavior of ligands that specifically bind to target molecules at least at one binding site. The markers are present in native form, and concentrations are determined by mass spectrometry. The use of µ-opioid receptors as target molecules, morphine as a marker, and naloxone as a ligand at various concentrations is described.

[0006] Dollinger et al . US 5891742, "Affinity selection of ligands by mass spectroscopy", describes a method in which compounds are selected from a combinatorial library by contacting the library with a target (human urokinase plasminogen activator), separating unbound compounds from compound-target complexes, and analyzing the complexes or eluted compound by mass spectroscopy.

[0007] Neiens et al ., “Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters,” ChemMedChem 13(5) 453–463 (2018), describe label-free, mass spectrometry-based binding assays (MS-based binding assays) targeting monoamine transporters. Human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) are used in simultaneous binding experiments.

[0008] Grimm et al ., “Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays,” Anal Bioanal Chem. 2015 Jan; 407(2):471-85 describe a method for quantifying indatraline, a highly active non-selective inhibitor of three monoamine transporters (for dopamine, DAT; for norepinephrine, NET; for serotonin, SERT), by LC-MS/MS and its use in MS binding assays.

[0009] In de Jong et al ., "Development of a multiplex non-radioactive receptor assay: the benzodiazepine receptor, the serotonin transporter and the β-adrenergic receptor," Rapid Comm. Mass Spectrom. 21:567–572 (2007), a method was described in which a pool of receptors from rat cortical tissue, i.e., homogenized cortex, was combined with flunitrazepam (which binds to benzodiazepine binding sites [receptors]), MADAM (2-[2-[(dimethylamino)methyl]phenyl]sulfanyl-5-methylaniline; dihydrochloride, which binds to the serotonin transporter), and pindolol (a beta-blocker [adrenergic beta-antagonist]). Each ligand was incubated with a known displacing agent for it.

[0010] Bowes et al ., “Reducing safety-related drug attrition: the uses of in vitro pharmacological profiling”; Nat. Rev. Drug Discov. 2012 Dec;11(12):909–22 describe the rationale for in vitro pharmacological profiling used by four major pharmaceutical companies. Proposed targets include GPCRs, ion channels, enzymes, neurotransmitter transporters, and nuclear receptors.

ESSENCE OF THE INVENTION

[0011] The following summary is neither intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.

[0012] The present invention, in various embodiments, relates to a multiplexed method for quantifying binding of a test compound to a target molecule and binding to non-target target molecules, comprising the steps of: (a) providing a mixture comprising target molecules from at least one of (i) healthy or unhealthy human or non-human tissue, and (ii) a synthetic protein preparation; (b) incubating said target molecules in a plurality of mixtures comprising ligands and test compounds, wherein said target molecules are incubated with different ligands; (c) after incubation, removing unbound ligands from said plurality of mixtures; then (d) isolating ligands bound to target molecules in said mixture of target molecules, ligands, and test compounds; (e) determining the amount of ligand bound to the target molecule by measuring the ligands obtained in step (d) using mass spectrometry and a calibration curve; and (f) determining the affinity of the test compound for the target molecule in said mixture of target molecules using the data obtained in step (e); and (g) measuring the binding of said test compound to a predetermined target molecule and comparing said binding to the binding of said test compound to non-target molecules.

[0013] In various embodiments, the present invention relates to a multiplexed method for quantifying binding of a test compound to a predetermined target molecule, as well as binding to non-target target molecules, comprising the steps of: (a) providing a mixture comprising target molecules from at least one of (i) healthy or unhealthy human or non-human tissue, and (ii) a synthetic protein preparation; (b) incubating said target molecules in a plurality of mixtures comprising ligands and test compounds, wherein said target molecules are incubated with different ligands; (c) removing unbound ligands from said plurality of mixtures; then (d) isolating ligands bound to target molecules in said mixture of target molecules; (e) determining the amount of ligand bound to target molecules by measuring the ligands obtained in step (d) using mass spectrometry and a calibration curve; (f) determining the affinity of a test compound for target molecules in said mixture of target molecules using the data obtained in step (e); and (g) measuring the binding of said test compound to a predetermined target molecule and comparing said binding to the binding of said test compound to non-target molecules.

[0014] Multiplexing in the present methods may include multiple target molecules in the same mixture, where the target molecules do not exist in the same preparation in nature. In various embodiments, the present invention relates to a heterologous mixture of target molecules. In other specific embodiments, the present invention relates to a mixture of target molecules comprising at least one human target molecule or more than one human target molecule.

[0015] The present invention, in particular aspects, relates to methods as described above, wherein step (a) comprises obtaining a target molecule or molecules from a crude extract. The present invention, in particular aspects, relates to a method as described above, wherein said step of obtaining target molecules comprises obtaining human target molecules. The extract may be present ex vivo on membranes from cerebral cortex, cerebellum, ventricular and liver membrane preparations.

[0016] In various embodiments, the binding of the test compound to the predetermined target molecule can occur in any of the cerebral cortex, cerebellum, ventricular, and liver membrane preparations. For example, the test compound is of interest for binding to the A1 receptor. Stimulation of the A1 receptor has a myocardial depressant effect by reducing the conduction of electrical impulses. This makes adenosine a useful drug for the treatment and diagnosis of excessively rapid heart rate.

[0017] In various embodiments, binding of a test compound to other target molecules may be considered off-target binding.

[0018] The present invention, in particular embodiments, relates to methods as described above, wherein step (c) comprises removing unbound ligands from mixtures or a plurality of mixtures using a glass filter. The present invention, in particular embodiments, relates to a method as described above, wherein step (d) comprises eluting bound ligand from a glass filter using a solvent, then concentrating samples from the filter.

[0019] The present invention, in particular aspects, relates to methods as described above, wherein said mass spectroscopy comprises the use of liquid chromatography/electrospray ionization tandem mass spectrometry. The present invention, in particular aspects, relates to a method as described above, further comprising the step of determining K _on and K _off of a test compound for a target molecule.

[0020] The present invention, in particular aspects, relates to the methods as described above, wherein said target molecules are present in a mixture of receptor target molecules that do not exist in nature. The present invention, in particular aspects, relates to a method as described above, wherein said target molecules are selected from the group consisting of a Na ⁺ channel, an alpha-1-beta adrenergic receptor, an alpha-2-beta adrenergic receptor, A1 (adenosine receptor), M1 (muscarinic receptor), 5-HT _2A (serotonin receptor), Alpha 1 ns (adrenergic receptor), Alpha 2 ns (adrenergic), D1 (dopamine receptor), and 5H-trans (serotonin receptor).

[0021] In specific embodiments, the present invention relates to a method as described above, comprising the step of determining K _on and K _off of a test compound for a target molecule, wherein K _off is determined by a displacement method or a dilution method.

[0022] The present invention, in specific embodiments, relates to a method as described above, wherein the ligands used to assay the target molecules can be selected from the group consisting of CPX, pirenzepine, prazosin, RX821002, SCH233900, 8-OH-DPAT, EMD281014, paroxetine, D600, MK801, and naloxone.

[0023] The present invention, in various embodiments, relates to a multiplexed method for quantifying the binding of at least two different test compounds (test compound C1, C2, etc.) to at least two different receptor target molecules (target receptor RT1 for C1, RT2 for C2, etc.), based on competitive binding between the test compounds and known binders for RT1 and RT2 (known binders B1, B2, etc.), comprising: (a) providing a mixture comprising (i) test compounds C1 and C2; (ii) known binders B1, B2; and (iii) receptor target molecules RT1, RT2; (b) allowing complexes to form in said mixture between test compounds C1, C2, etc., RT1 and RT2, as well as B1 and B2; (c) separating compounds that do not form complexes with RT1, RT2, etc., from said complexes; (d) isolating binders B1, B2, etc. from the complexes obtained in step (c) and passing the isolated binders through a mass spectrometer to measure binding of test compounds C1 and C2 using mass spectroscopy; and (e) determining the relative affinities of C1 and C2 for RT1 and RT2, respectively.

[0024] For further clarification, the expression "etc." refers to series of members from series of materials that can be represented as C _n , B _n , and RT _n , where n is between 2 and 40 or between 1 and 40 or between 2 and 50. This shows, for example, that if n=10, there are 10 C, 10 B, and 10 RT.

[0025] For purposes of clarification, it is envisaged that the group of target receptor molecules (RT), test compounds (C), and known binders (B) comprises between two and approximately 20 members (or more) in a single multiplex reaction.

[0026] The present invention, in various embodiments, relates to a method as described above, wherein the RT1-RT _n target receptor molecules are present in a mixture not found in nature in the same mixture or in the same tissue.

[0027] In many embodiments, the present invention relates to a multiplexed method for quantifying the binding affinity of at least two different test compounds (test compound C1- _Cn ) for at least two different receptor target molecules (receptor RT1 for C1, _RTn for _Cn ), based on competitive binding between the test compounds and known binders for RT1 and RT2 (known binder B1- _Bn ), comprising: (a) providing a mixture comprising (i) test compounds C1- _Cn ; (ii) known binders B1- _Bn ; and (iii) receptor target molecules RT1- _RTn ; (b) allowing complexes to form in said mixture between test compounds C1- _Cn , RT1- _RTn , and B1- _Bn ; (c) separating compounds that do not form complexes with their target molecules from said complexes; (d) isolating known binders from the complexes obtained in step (c) and passing the isolated binders through a mass spectrometer to measure binding of test compounds using mass spectroscopy; and (e) determining the relative affinities of the compounds C1- _Cn for RT1- _RTn , respectively, where _Cn , _Bn , and _RTn represent a series of members where n is between 2 and 40.

[0028] The present invention, in various embodiments, relates to a method as described above, wherein step (a) comprises obtaining target receptor molecules from a crude extract. The present invention, in particular aspects, relates to a method as described above, wherein said step of obtaining RT1-RT _n target receptor molecules comprises obtaining human target receptor molecules. The present invention, in particular aspects, relates to a method as described above, wherein step (c) comprises separating using a glass filter and washing. The present invention, in particular aspects, relates to a method as described above, wherein step (d) comprises eluting the bound ligand from the filter using a solvent, then concentrating the samples from the filter.

[0029] The present invention, in various embodiments, relates to a method as described above, wherein said mass spectroscopy comprises the use of liquid chromatography/electrospray ionization tandem mass spectrometry. The present invention, in various other embodiments, relates to a method as described above, further comprising the step of determining K _on and K _off of a test compound for a target molecule.

[0030] The present invention further relates to a multiplexed method for quantitatively assessing the binding affinity of a test compound for a target molecule, comprising the steps of: (a) providing at least three target molecules as outlined in the scheme below (Table 1); (b) incubating said target molecules in a plurality of mixtures containing ligands and test molecules; (c) removing unbound ligands from the mixtures; (d) recovering ligands bound to the target molecules; (e) determining the amount of each ligand present on the target molecules by measuring the ligands obtained in step (d) by mass spectrometry using a calibration curve obtained using known concentrations of the ligand; and (f) calculating the affinity of the test compound for the target molecule from the data obtained in step (e). A method as described, wherein the same test compound is used with each target molecule. The method further comprises using target molecules with ligands as shown in Table 2.

Target molecule Adenosine A1 receptor Muscarinic acetylcholine receptor 5-HT _2A (serotonin) Alpha-1A-adrenergic receptor Alpha-2A adrenergic receptor Dopamine receptor D1 Transporter 5HT 5HT1a receptor 5HT2a receptor Ca channel Cave PCP receptor Opioid receptor

Table 1

Target molecule Ligand Adenosine A1 receptor CPX Muscarinic acetylcholine receptor Pirenzepine 5-HT _2A (serotonin) EMD281014 Alpha-1A-adrenergic receptor Prazosin Alpha-2A adrenergic receptor RX82102 Dopamine receptor D1 SCH23390 Transporter 5HT paroxetine 5HT1a receptor 8-OH-DPAT 5HT2a receptor EMD281014 Ca channel Cave D600 PCP receptor MK801 Opioid receptor naloxone

Table 2

[0031] The present invention, in specific aspects, relates to a method as described above using the following combinations of target receptor molecules and ligands (Table 3):

Target receptor molecule Ligand Adenosine A1 receptor CPX Muscarinic acetylcholine receptor M1 pirenzepine 5HT1a 8-OH-DPAT/5 5-HT _2A (serotonin) EMD281014 Alpha-1A-adrenergic receptor Prazosin Alpha-2A adrenergic receptor RX82102 Dopamine receptor D1 SCH23390 Transporter 5HT paroxetine Ca++ channel ("Cave") D600 Opioid mu receptor Naloxone Sigma receptor/PCP receptor MK801

Table 3

[0032] The above target receptor molecules may be assayed with other ligands not listed in the above Table 3, or other target receptor molecules not listed in the above Table 3 may be assayed with the ligands shown above.

[0033] In various embodiments, the present methods comprise a multiplex method for determining the K _on and K _off values of a test compound for a target molecule, comprising the steps of: (a) providing a mixture of target molecules from at least one of (i) healthy or unhealthy human or non-human tissue, and (ii) a synthetic protein preparation; (b) incubating said target molecules in a plurality of mixtures comprising ligands and test compounds, wherein said target molecules are bound to different ligands and incubated with the different target molecules; (c) after incubation, removing unbound ligands from the mixtures; (d) isolating bound ligands bound to the target molecules; (e) determining the amount of ligand bound to the target molecules by measuring the ligands obtained in step (d) at specific time points in the reaction using mass spectrometry and a calibration curve; and (f) calculating K _on or K _off of the test compound for the target molecule using the data obtained in step (e).

[0034] In various embodiments, the present methods include a method wherein K _on and K _off are determined in mixtures of various membranes ex vivo consisting of at least two rat cortex, cerebellum, ventricular, and liver membrane preparations. In particular aspects, the present methods include a method wherein the membrane mixtures comprise at least two of A1, A2A (h), A3 (h), M1, M2 (h), Alpha1ns, Alpha2ns, D1, D2S (h), 5HT1a, 5HT2a, 5H-trans, Cave, PCP, opioid ns, AT2 (h), B2 (h), CB1 (h), CCK1 (CCKA), H4 (h), and CysLT1 (LTD4) (h) receptors. In particular aspects, the present methods include a membrane mixture comprising all of these receptors. In other specific embodiments, the present methods include a method wherein K _off is determined by a displacement method. In specific aspects, the present methods include a method wherein K _off is determined by a dilution method. In various embodiments, (h) is human.

[0035] In various embodiments, the target molecules are receptors.

[0036] As described below, the same test compound can be used with the above-mentioned different target receptor molecules and different ligands, obtaining information on target and off-target binding by the test compound.

[0037] Other features will become apparent from the accompanying figures and from the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

[0038] Examples of embodiments are illustrated by way of example and not limitation in the tables and accompanying figures, like references designating like elements, and in them:

[0039] Fig. 1A and 1B show an exemplary workflow for MS binding assays used to characterize the binding of various ligands (target molecules or receptor target molecules) to a test compound.

[0040] Fig. 2A-2C show the correlation between radioligand binding and the present MS binding mode in rat cortex sodium channels. Fig. 2A is a graph showing the results of a radioligand binding assay for sodium channels. Fig. 2B is a graph showing the results of an MS binding assay for veratridine. Fig. 2C is a graph showing the results of an MS binding assay for batrachotoxin.

[0041] Fig. 3A is a graph showing the effect of WB4101 concentration in the presence of prazosin and RX821002. Fig. 3B is a graph showing the effect of yohimbine concentration in the presence of RX821002 and prazosin.

[0042] Fig. 4A-K are a series of graphs showing the results of a simultaneous binding assay using rat cortex target molecules. Fig. 4A is a graph showing the effect of NECA concentration in the presence of CPX at 5 nM on rat cortex. Fig. 4B is a graph showing the effect of atropine concentration in the presence of pirenzepine at 1 nM on rat cortex. Fig. 4C is a graph showing the effect of serotonin concentration in the presence of 8-OH-DPAT at 1 nM on rat cortex. Fig. 4D is a graph showing the effect of WB4101 concentration in the presence of prazosin at 1 nM on rat cortex. Fig. 4E is a graph showing the effect of yohimbine concentration in the presence of RX821002 at 1 nM on rat cortex. Fig. 4F is a graph showing the effect of butaclamol concentration in the presence of SCH23390 at 1 nM on rat cortex. Fig. 4G is a graph showing the effect of zimelidine concentration in the presence of paroxetine at 1 nM on rat cortex. Fig. 4H is a graph showing the effect of serotonin concentration in the presence of EMD281014 at 1 nM on rat cortex. Fig. 4I is a graph showing the effect of D888 concentration in the presence of D600 at 5 nM on rat cortex. Fig. 4J is a graph showing the effect of DAMGO concentration in the presence of naloxone at 1 nM on rat cortex. Fig. 4K is a graph showing the effect of SKF10047 concentration in the presence of MK801 at 5 nM on rat cortex.

[0043] Fig. 5 is a schematic workflow for using MS to determine the binding kinetics of a test compound to its cognate receptor molecule.

[0044] Fig. 6A is a graph showing the results of an MS analysis for determining the kinetic curve of binding of CGP54626 to GABAB1b/2. Fig. 6B is a graph showing the results of an MS analysis for determining the kinetic curve of dissociation of GABAB1b/2 from CGP542626 at a concentration of 1 nM by the displacement method by adding 10 μM CPG52432. Fig. 6C is a graph showing the results of an MS analysis for determining the kinetic curve of dissociation of GABAB1b/2 from CGP54626 at a concentration of 5 nM by the dilution method.

DETAILED DESCRIPTION

REVIEW

[0045] The present invention relates to a method for measuring the binding activity of a test compound to a target receptor molecule using a mixture of biologically significant target molecules. The present invention further relates to methods for measuring the binding activity of test compounds to various receptor (target) molecules using a heterologous mixture of biologically significant target molecules. The target molecules in this assay can be used to assess off-target binding. In one aspect of the method, a competitive binding assay is used using a target molecule or tissue known to bind to the ligand. As is known from the principles of radioimmunoassays (RIA), dilution curves are constructed using different concentrations of a known ligand (or "marker") and its binding to the target molecule. Unlike RIA, the markers in the present method do not need to be labeled or otherwise chemically modified. The binding of the test compound to the ligand and tissue (target molecules) is then measured at a known concentration; the MS signal is then compared with the MS signals obtained in the dilution curve. The potency of the test compound in binding to the target molecule is then known, and the IC ₅₀ or EC ₅₀ can be determined.

[0046] In the present methods, the binding characteristics of test compounds to different target molecules can be determined in a multiplexed manner. The present methods also relate to in vitro methods for testing drug candidates.

[0047] The present methods can use commercially available high performance liquid chromatography (HPLC) and MS equipment. The MS format can be electrospray from a well, or matrix assisted laser desorption/ionization (MALDI) format, or use another ionization method.

[0048] The present methods can be automated using laboratory robotic equipment. All separations and reactions in the method are contained in the same sample well until the isolated molecules are introduced into the HPLC. A sample plate with any desired number of wells can be used.

[0049] A variety of target molecules can be obtained for use in the present methods. Crude or purified extracts can be used, for example, by the methods described in US 4,446,122, "Purified human prostate antigen"; US 6,548,019, "Device and methods for single step collection and assaying of biological fluids"; Magomedova et al ., "Quantification of Oxysterol Nuclear Receptor Ligands by LC/MS/MS"; Methods Mol. Biol. 2019;1951:1-14; and Wang, "Purification and autophosphorylation of insulin receptors from rat skeletal muscle", Biochim Biophys Acta. 1986 Aug 29;888(1):107-15, the contents of each of which are hereby incorporated by reference.

[0050] The use of a glass filter to obtain a sample for MS analysis can be carried out as described, for example, in Merck Millipore, “Perfection in preparation for better mass spectra,” Merck Millipore, product description 2012, retrieved from (colon slash slash www. merckmillipore.com/INTERSHOP/web/WFS/Merck-JP-Site/ja_JP/-/JPY/ShowDocument-Pronet id=201306.10657.

DEFINITIONS

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. In general, the nomenclature used in connection with cellular and molecular biology and chemistry, and in the methods thereof, is well known and conventional in the art. Specific experimental methods, not specifically defined, are generally carried out according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. For purposes of clarity, the following terms are defined below.

[0052] When a range of values is presented, it is understood that each intermediate value, up to a tenth of a unit of the lower limit, unless the context clearly requires otherwise, between the upper and lower limits of that range, and any other recited or intermediate value in that recited range, is included in the methods, cells, compositions and kits. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included in the methods, cells, compositions and kits limited by any specifically excluded limit in the recited range. When a recited range includes one or both limits, ranges excluding either or both of those included limits are also included in the methods, cells, compositions and kits.

[0053] Specific ranges are provided herein using numerical values preceded by the term "about." The term "about" is used herein to provide a literal justification for the exact amount it precedes, as well as for an amount that is about or approximately the amount the term precedes. In determining whether an amount is about or approximately a specifically recited amount, an unlisted amount of about or approximately may be an amount that, in the context in which it is presented, provides a substantial equivalent to the specifically recited amount.

[0054] The contents of all publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference herein and are herein incorporated by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. Citation of any publication is for its contents prior to the filing date and should not be construed as an admission that the present methods, cells, compositions, and kits are not provided to backdate such publication, since the publication date provided may differ from the actual publication date, which may require independent confirmation.

[0055] It should be noted that, as used in the present specification and the appended claims, the singular forms include plural reference unless the context clearly requires otherwise. It should be further noted that the claims may be drawn to exclude any optional element. Thus, this statement is intended to serve as a precursor to the use of such exclusive terminology as "solely," "only," etc., in connection with the recitation of claimed elements, or the use of a "negative" limitation.

[0056] The term " affinity " is used in the conventional sense to refer to binding affinity. Binding affinity is the strength of the binding interaction between a single biomolecule (e.g., a protein) and its ligand/binding partner (e.g., a drug or inhibitor). Binding affinity is typically measured and reported by the equilibrium dissociation constant (Kd), which is used to evaluate and rank the strengths of bimolecular interactions. Accordingly, binding kinetics describes how quickly a compound binds to its target and how quickly it dissociates from it. It thus measures two things - the rate of binding and the rate of dissociation. See, US 5,324,633A, "Method and apparatus for measuring binding affinity".

[0057] The term " ligand " or " binding substance " is used herein to refer to a material that is known to bind to a given receptor or other target molecule. This term can be further understood by reference to Siimans et al ., US 5,814,498, "Methods of enumerating receptor molecules for specific binding partners on formed bodies and in solution", the contents of which are hereby incorporated by reference as providing the concept of competitive binding.

[0058] " Mixture of targets " or target molecules means a mixture of structurally distinct targets or other receptor target molecules. As a non-limiting example, the mixture may include glutamate receptors, dopamine D1 receptors, and nicotinic acetylcholine receptors. These receptors may be present in a single tissue type, such as the cerebral cortex of an animal, or may not be present in a single tissue type. The mixture of targets may also include, for example, glutamate receptors (from the cerebral cortex) and VEGF receptors (from endothelial cells). See "heterologous mixture of receptor target molecules" below.

[0059] " Heterologous mixture of target receptor molecules" refers to a mixture of different target molecules that are not found naturally in the same tissue or, if present in the same tissue, have different biological functions. As a non-limiting example, the mixture may comprise more than one tissue selected from the group consisting of modified cells expressing G protein-coupled receptors (GPCRs), cerebral cortex from animal sources (having 15 different target molecules, as described, for example, in Zilles et al., “Multiple Transmitter Receptors in Regions and Layers of the Human Cerebral Cortex,” Front Neuroanat. 11:78 (2017)), cerebellum, heart, muscle (including cardiac ion channels), biological enzymes (e.g., COX2, COX1, MAO, PDE4, Ache, LCK), nuclear receptors (e.g., AR and NR3C1), and nucleic acid molecules.

[0060] Target molecules may have desirable binding and binding that is not desirable, known as off-target binding. As discussed above, off-target binding is generally avoided for safety reasons. See Bowes et al . and Eurofins Safety Panels, htt-ps-:slash-slash www(dot).eurofinsdiscoveryservices.com/cms/cms-content/services/safety-and-efficacy/safety- pharmacology/safety-panels/, for a selection of in vitro Safety panels.

[0061] The term " MS " means mass spectrometry. In the present method, a variety of mass spectrometry methods can be used, such as AMS (accelerator mass spectrometry), gas chromatography-MS, liquid chromatography-MS, ICP-MS (inductively coupled plasma mass spectrometry), IRMS (isotope ratio mass spectrometry), ion mobility spectrometry-MS, MALDI-TOF, SELDI-TOF, tandem MS, TIMS (thermal ionization mass spectrometry), and SSMS (spark emitter mass spectrometry).

[0062] The term " multiplex " refers to an assay in which multiple different assays are performed in a single procedure using different target molecules having different ligands. The method may also include the presence of different test compounds. Binding of a test compound to different target molecules that do not exist together in nature can be performed simultaneously in a multiplex assay. In addition, in a multiplex assay, multiple results can be obtained from a single mixture of target receptors and a binding profile can be obtained with different target molecules that can clarify target binding and thus safety.

[0063] The term " liquid chromatography/electrospray ionization tandem mass spectrometry " can also be understood with reference to, for example, Bandu et al ., "Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS/MS) Study for the Identification and Characterization of In Vivo Metabolites of Cisplatin in Rat Kidney Cancer Tissues: Online Hydrogen/Deuterium (H/D) Exchange Study", PLosOne 2015 Aug 5:10(8).

[0064] The term " target receptor molecule " or " target molecule " or "receptor molecule" refers to a biological compound for which binding of a test compound is to be measured. The target receptor molecule may be present in a target tissue obtained from a cell of an animal (human or non-human). It may be produced by recombinant DNA or otherwise synthesized to contain one or more target molecules of interest. It may be membrane-bound or exist in a liquid mixture such as an enzyme. Potential tissues for target receptors as used herein may be cerebral cortex, brain astrocytes, neuronal tissues (including neuronal stem cells), cardiac tissue, liver tissue, blood tissue, kidney tissue, eye tissue, intestinal tissue, etc. The target tissue may be normal or diseased. It may be from an animal source or a human source. The term " heterologous mixture of target molecules" refers to tissues or cell lines from different sources, as illustrated above. Tissues may be different tissues if they are derived from the same tissue, but the tissues have different structures, due to disease, developmental state, etc.

[0065] The term " synthetic protein preparation " means a protein preparation that has been synthesized rather than obtained from a native cell or tissue. A synthetic protein preparation may be synthesized by recombinant DNA methods, peptide synthesis, or the like.

[0066] The term " test compound " means a material being tested for its binding affinity for target molecules. It may interact with and compete with a known ligand (marker) if it binds to a target molecule that is also bound to the marker. The test compound may be a potential drug, as well as metabolites of such a drug. It may be a small molecule or a protein, or a polynucleotide. It may also be a molecule being tested for its potential for in vivo diagnostic use.

GENERALIZED METHOD AND APPARATUS

[0067] The present methods can be adapted to a wide variety of test compounds and a wide variety of targets for which the binding characteristics of the test compounds are to be examined. Of particular interest is the examination of test compounds that are drug candidates for use in vivo in humans. The binding of the test compounds to various target molecules expressed in various tissues is examined in the present methods. The binding is either desirable for a therapeutic effect or undesirable to avoid off-target effects, for drug safety purposes. Thus, the present methods find application, for example, in identifying potential human therapeutics and their potential undesirable binding to various human tissues expressing potential targets for binding of the test compound.

EXAMPLES

EXAMPLE 1: Process flow

[0068] Referring now to Figs. 1A and 1B, it is shown that the present methods include a series of incubation, separation, and washing steps resulting in direct or indirect quantification of test compounds that dissociate from a target molecule due to competition with a known binding ligand. See the inset in Fig. 1A illustrating a single test well 107. The ligands are labeled 1, 2, and 3 to represent different ligands 105 binding to different target receptor molecules 104 in a single well.

[0069] Figs. 1A-1B show the incubation of a heterologous mixture of target receptor molecules with ligands (known binders) and various test compounds. Wells, vials, or other containers contain target molecules. As shown in 101, step (a), a given well in a multi-well plate can contain mixtures of target molecules 104, ligands (known binders) 105, and various test compounds 106. As shown in inset 107, target molecules 104 can bind to different ligands 105, designated 1, 2, and 3. The discrimination and identification of ligands is accomplished by MS. The different wells contain different amounts of molecules, so that the results from the wells in Figs. 1A and 1B can be used to construct concentration curves as shown in Figs. Then, as shown in Fig. 1A, step (b), unbound ligands are separated from the complexes in the wells. Then, as shown in (c), ligands bound to the target molecules are separated from the mixture and recovered from the well for use in Fig. 1B, step (d). Recovery of bound ligands in step (c) can be facilitated by using acetonitrile 103 and a glass filter allowing only unbound ligands to pass through. Various organic solvents can be used in this step, as well as in other isolation steps, to prepare ligands for use in step (d).

[0070] After the previously bound ligand molecules are isolated, the amount of ligand recovered from each well is quantified by liquid chromatography and electrospray MS (mass spectroscopy) (step (d) in Fig. 1B). The LC/ESI-MS/MS method is used so that liquid chromatography can reduce the amount of irrelevant mass spectroscopic peaks when the mass spectrometer is used to identify and quantify different ligands.

[0071] Fig. 1B shows a device for HPLC 108, solvents for obtaining a mobile phase 109, a module for obtaining mixtures of components 110 and a column for HPLC 111 having an output to an ion source 112 and a mass spectrometer 113. Illustrative analysis of the chromatogram and mass spectrum reveals an absolute quantitative assessment of the eluted target molecules 114.

[0072] In another embodiment of the present methods, a fixed amount of test compound can be measured at different concentrations of ligands (known binders). That is, an excess of test compound is used, if available, and different amounts of ligands are used. The ligand competitively causes dissociation of the test compound-target molecule complex to determine the binding behavior of the test compound to the target molecule.

[0073] In addition, Fig. 1A and 1B show the preparation of target receptor molecules placed in test wells, vials, or other containers. They may be a crude extract of tissue containing target receptor molecules. The tissue may be blood, serum, cerebrospinal fluid, a brain fragment (cerebral cortex, cerebellum, brain stem, etc.), gland extracts (adrenal, pituitary, thymus, pancreas, ovary, thyroid, testes, hypothalamus, etc.), or organ tissue such as heart, skeletal muscle, kidney, lung, etc. The tissue may be human or non-human tissue, or animal tissue. It may be normal or diseased. Receptor target molecules do not have to be purified and are selected based on the intended use of the test compound, the availability of known ligands, and the objective of the study. The objective of the study may be to obtain a safety profile, where a wide variety of potential target molecules can be tested using the test compound to assess for unwanted binding.

[0074] In addition, the target receptor molecules can be produced without using endogenous tissue, but instead, produced by rDNA or protein synthesis. Known cloned receptors that can be used in the present methods include histamine H3 receptors, opioid receptors, G protein-coupled receptors, vanilloid receptors, glutamate receptors, etc.

[0075] The multiplex methods herein are performed in a plurality of reaction regions (wells), shown as F, G, and H, for an 8-row, 96-well plate (as shown in 101). A 384-well plate or other multi-well formats can be used. In this example, target receptor molecules were prepared with ligand and test compounds and multiplexed at 2 hours, 37°C in a 96-well plate. As shown in inset 107 below panel (a), a well contains a row of receptors bound to ligands 105 and a row of receptors 104 bound to test compound 106 in place of ligand 105.

[0076] Following incubation in step (a), the target receptor molecule complexes bound to the target molecules are separated from unbound ligands and free target molecules by filtration. Vacuum filtration is applied to the entire plate simultaneously (Fig. 1A, step (b). Alternatively, wells containing a plunger or syringe can be used in step (b) to separate the bound complexes from the unbound molecules. Alternatively, the target receptor molecules can be labeled to separate from the wells. In this embodiment, unbound molecules can be easily removed.

[0077] After separation of the bound ligand from the free ligands, the complexes can be washed using a low ionic strength buffer and finally eluted using an organic buffer or a high ionic strength buffer, effectively isolating the ligand-bound receptors for processing in step (c). The receptors can also be labeled using magnetic beads and processed as described above. Accordingly, as shown in Fig. 1A step (b) 102, the isolated ligand-receptor complex is further processed to separate the ligand from the bound receptor target molecules, such as by elution with acetonitrile (Fig. 1A, step (c), 103). In step (d), (Fig. 1B), the isolated ligand molecules from step (c) are analyzed directly using LC-MS with electrospray ionization in positive ion mode-tandem mass spectrometry.

[0078] Referring now to Fig. 1B, step (d), the ligand mixture is purified by liquid chromatography and analyzed by mass spectroscopy. The image used is from Wikipedia, “Liquid chromatography-mass spectrometry,” https(colon slash slash en.wikipedia(dot)org/wiki/Liquid_chromatography-mass_spectrometry, accessed 6-28-2019. As described herein, mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of charged particles (ions). Although there are many different types of mass spectrometers, they all use electric or magnetic fields to manipulate the movement of ions formed from the analyte of interest and determine their m/z ratio. The main components of a mass spectrometer are an ion source, a mass analyzer, a detector, and data and vacuum systems. The ion source is where the components of the sample introduced into the MS system are ionized using electron beams, photon beams (UV light), laser beams, or corona discharge. In In electrospray ionization, the ion source moves ions that exist in a liquid solution into the gas phase. The ion source converts and fragments the neutral sample molecules into gas phase ions, which it sends to a mass analyzer. While the mass analyzer applies electric and magnetic fields to sort the ions by their mass, the detector measures and amplifies the ion current to calculate the abundance of each mass-resolved ion. The data system records, processes, stores, and displays the data in a computer to produce a mass spectrum that can be easily seen by the human eye. In the example, electrospray ionization MS is used.

[0079] A calibration curve with known concentrations is used to quantify the amount of test compound that has, through competition, caused the ligand to dissociate and bound to the test receptor molecule. Various other mass spectroscopy methods, as detailed above, may be used, provided that they do not generate excessive extraneous data.

[0080] It should be noted that the known binder, i.e. the marker, is unlabeled (as is the test compound). This is a key advantage of the present MS method over the RIA (radioimmunoassay) method. RIA is also based on competition between a known binder and the test compound, but requires that the marker be radiolabeled to achieve the desired sensitivity. In an alternative embodiment, a label such as deuterium can be added to increase sensitivity.

[0081] Further details of liquid chromatography/electrospray ionization tandem mass spectrometry can be found in Becker, US 6835927, “Mass spectrometric quantification of chemical mixture components,” the contents of which are hereby incorporated by reference.

[0082] Thus, Fig. 1A and 1B show a series of incubation and wash steps for the described assay, for direct or indirect quantification of test compounds, wherein in step (a), a given well 101 in a multi-well plate contains a mixture of a target receptor molecule 104, a ligand (known binder) 105, and a test compound 106. In addition, as shown in inset 107, the target molecule can bind to different ligands, designated 1, 2, and 3. The mixtures are allowed to incubate for 2 hours at 37°C in a multi-well plate. Following incubation in step (a), vacuum filtration is applied 102 to the multi-well plate to separate the bound receptor target molecule from unbound ligands and free target molecules, as shown in step (b). Step (c) shows that the separated ligand-receptor complex is further washed with a low ionic strength buffer, such as by elution with acetonitrile 103, so as to separate the ligand from the bound receptor target molecule before proceeding to step (d) of the described binding assay. In step (d), (Fig. 1B), the isolated ligand molecules from step (c) are analyzed directly using LC-MS with electrospray ionization-positive ion tandem mass spectrometry (ESIM).

EXAMPLE 2: Comparability between the present MS method and the RIA method

[0083] With reference now to Fig. 2A, the analysis of radioligand binding to the sodium (Na) channel and its comparison with the present MS method is shown in Figs. 2B and 2C. Fig. 2B shows the specific binding of veratridine in the presence of batrachotoxin at 50 nM. This experiment was performed using sodium channels as target receptor molecules. It can be considered that the ligand (a known binder) is batrachotoxin, which binds to and irreversibly opens sodium channels of nerve cells and prevents their closure. The test compound is the neurotoxin veratridine, which acts by binding to and preventing the inactivation of voltage-gated sodium ion channels in cell membranes of the heart, nerves and skeletal muscle.

[0084] SNR (signal to noise ratio) was determined as follows (Table 4):

SNR 6 8 Batrachotoxin (Kd=91 nM) 143 nM (EC50) Veratridine 5.6 μM (IC50) 12.2 μM (IC50)

Table 4

[0085] Figure 2C shows an indication of the specificity of binding. Curve 201 in Figure 2C shows the total signal, curve 202 shows the signal associated with non-specific binding in the presence of veratridine, and curve 203 shows the specific signal. In this case, batrachotoxin binding was determined in membranes that had been pre-incubated with a competitor (veratridine) known to bind to the same site. In this way, it can be determined whether a specific ligand binds non-specifically to the filter, plastic, or other sites.

Materials and methods:

Obtaining rat cortex membrane

[0086] Rat cortices from male Wistar rats were collected and transferred to 50 mM Tris-HCl (pH 7.4) and homogenized with polyton. The homogenate was centrifuged at 50,000 g for 15 min at 4°C. The resulting pellet was washed in lysis buffer containing 50 mM Tris-HCl (pH 7.4) containing 1 μg/ml leupeptin and 1 μM pepstatin and centrifuged at 50,000 g for 15 min at 4°C. The pellet was finally resuspended in a smaller volume of lysis buffer and the final protein concentration was determined according to the Bradford method using bovine serum albumin as a standard.

Filtration and elution of samples

[0087] The incubation was stopped by filtration after transferring the binding/reaction mixture (200 μl aliquots per well) to 96-well glass filter plates and then rapidly filtered under vacuum, the membrane fraction bound to the filters was washed several times with wash buffer (50 mM Tris-HCl and 150 mM NaCl) in a vacuum manifold. The membrane filters were pretreated for 1 hour with 50 mM Tris-HCl and 0.3% polyethyleneimine (PEI) solution.

[0088] The filters were dried for one hour at 50°C and cooled to room temperature before elution of batrachotoxin using acetonitrile (containing 100 pM antipyrine as an internal standard) via a vacuum manifold. Relative quantification of ligand in each sample was performed by UHPLC-MS-MS using the area ratio of ligand to internal standard.

Development of UHPLC-MS/MS method

[0089] UHPLC-QQQ analysis was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA).

[0090] Chromatographic separation was performed on a C18 column (Poroshell 120 EC-C18, Agilent). The injection volume was 20 μl (full loop injection). The mobile phase consisted of two solutions, solvent A (0.1% formic acid and 6 mM ammonium acetate in water) and solvent B (0.1% formic acid and 6 mM ammonium acetate in acetonitrile), the column was thermostatted in an oven at 35°C, and the flow rate was 650 μl/min.

[0091] Chromatographic gradient used for a C18 column; initial composition B was 0% for 0.3 min and increased to 80% from 0.3 to 0.9 min, then 100% was reached from 1 min to 1.3 min, followed by re-equilibration to the initial state for 0.3 min.

[0092] For MS analysis, data were acquired using electrospray ionization (ESI) in positive ion mode with the ion spray voltage set to 5500 V. Source desolvation was performed using the following set parameters: temperature (TEM) at 600 °C, gas ion source 1 (GS1) at 40 psi (276 kPa), gas ion source 2 (GS2) at 50 psi (345 kPa), and curtain gas (CUR) at 50 psi (345 kPa). Specific MRM method parameters allowing quantification and monitoring of the ligand (batrachotoxin) are described in Table 5. Raw data were processed in Sciex Analyst and the individual AUC (area under the curve) for each analyte in each sample was determined using MultiQuant software.

Q1, mass (Yes) Q3, mass (Yes) Time (ms) ID DP
(volt) EP
(volt) CE
(volt) CXP
(volt) 539.2 400.2 150 Batrachotoxin 140 10 23 12

DP: declustering potential, EP: input potential, CE: collision energy and CXP: collision cell output voltage.

Table 5: MRM Method

Linking via MS experiments

Determination of the optimal ligand concentration

[0093] Cortical membrane preparations containing the sodium channel (Na ⁺ ) site 2 receptor and batrachotoxin were incubated in triplicate in assay buffer (50 mM Hepes/Tris-HCl, 0.8 mM MgSO4, 5 mM KCl, 7.5 mg/L scorpion venom, 2 mM MgCl2, 10 μg/mL trypsin, 1 g/L glucose, 130 mM choline, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 0.1% BSA) in polypropylene 96-well deep-well plates at 37°C. First, 12 concentrations (ranging from 10 pM to 300 nM) of batrachotoxin were co-incubated for 60 min at 37°C, with 1 concentration (200 μg/well) of rat cortical membrane preparation.

[0094] Non-specific binding was determined by co-incubation with 10 μM verapamil.

[0095] The incubation was stopped by filtration after the entire volume of the binding reaction mixture had been transferred to a filter plate. The remaining amount of batrachotoxin was determined by UHPLC-MS/MS.

For saturation analyses:

[0096] Aliquots of membrane containing 200 μg of rat cortical membrane preparation were incubated in triplicate in the presence of 50 nM batrachotoxin in a total volume of 200 μl of assay buffer. Incubation was stopped by filtration after incubation for 60 min at 37°C.

[0097] Non-specific binding was determined by co-incubation with 10 μM verapamil.

[0098] The incubation was stopped by filtration after the entire volume of the binding reaction mixture had been transferred to a filter plate. The remaining amount of batrachotoxin was determined by UHPLC-MS/MS.

Competitive mass binding assays:

[0099] Ligand displacement assays were performed using eight concentrations of the competitive ligand, veratridine (ranging from 0.1 nM to 100 μM), in triplicate. Incubation was stopped by filtration after incubation for 60 min at 37°C. The remaining amount of batrachotoxin was determined by UHPLC-MS/MS.

EXAMPLE 3: Multiplexing using 2 simultaneous targets

[00100] Fig. 3A is a graph showing a simultaneous binding experiment using alpha 1 and alpha 2 beta adrenoceptors. The target molecules are contained in rat cortex, which contains both alpha 1 and alpha 2 beta adrenoceptors. The test compound is WB4101 and the ligands are prazosin and RX821002. Fig. 3B shows a simultaneous binding assay to α1 and α2 beta adrenoceptors using yohimbine as the test compound and the same target molecules and ligands as in Fig. 3A.

[00101] Detailed reference is now made to Fig. 3A. A rat cortex preparation was used to measure the effect of compounds on two different target molecules, in this case, the α1B-adrenergic receptor and the α2B-adrenergic receptor. The two receptors are structurally and functionally distinct. The human α-1A adrenergic receptor (ADRA1A) has a canonical length of 466 amino acids and a mass of 51,487 Da. The human α-2A adrenergic receptor (ADRA2A) has a canonical length of 450 amino acids and a mass of 48,957 Da.

[00102] WB4101 is a known α1B-adrenergic receptor antagonist. Prazosin is a drug known to bind to the alpha-1 (α1) adrenergic receptor, which is a G protein-coupled receptor (GPCR). These receptors are found on vascular smooth muscle. RX821002 is a potent, selective α2-adrenergic receptor antagonist.

[00103] In this example, a target molecule containing both α1 and α2-beta-adrenergic receptors was incubated with WB4101 (test compound) in the presence of prazosin (ligand, or "marker", for α1) and RX821002 (ligand, or "marker", for α2), as shown in Fig. 3A. In Fig. 3B, yohimbine (test compound) was tested in the presence of prazosin (marker for α1) and RX821002 (marker for α2). Signals are designated ns for non-specific.

[00104] As shown in Fig. 3A, both prazosin and RX821002 were shown to bind specifically to the α1 and α1 adrenergic receptor (respectively). Fig. 3B shows a simultaneous determination of α1 and α1 binding using yohimbine as a test compound and the same targets and ligands as in Fig. 3A.

Obtaining rat cortex membrane

[00105] Rat cortices from male Wistar rats were collected and transferred to 50 mM Tris-HCl (pH 7.4) and homogenized with polyton. The homogenate was centrifuged at 50,000 g for 15 min at 4°C. The resulting pellet was washed in lysis buffer containing 50 mM Tris-HCl (pH 7.4) containing 1 μg/ml leupeptin and 1 μM pepstatin and centrifuged at 50,000 g for 15 min at 4°C. The pellet was finally resuspended in a smaller volume of lysis buffer and the final protein concentration was determined according to the Bradford method using bovine serum albumin as a standard.

Filtration and elution of samples

[00106] The incubation was stopped by filtration after transferring the binding/reaction mixture (200 μl aliquots per well) to 96-well glass filter plates and then rapidly filtered under vacuum, the membrane fraction bound to the filters was washed several times with wash buffer (50 mM Tris-HCl and 150 mM NaCl) in a vacuum manifold. The membrane filters were pretreated for 1 hour with 50 mM Tris/HCl and 0.3% polyethyleneimine (PEI) solution.

[00107] The filters were dried for one hour at 50°C and cooled to room temperature before elution of the ligands using acetonitrile (containing 100 pM antipyrine as an internal standard) via a vacuum manifold. Relative quantification of the ligand in each sample was performed by UHPLC-MS-MS using the area ratio of the ligand to the internal standard.

Development of UHPLC-MS/MS method

[00108] UHPLC-QQQ analysis was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA).

[00109] Chromatographic separation was performed on a C18 column (Poroshell 120 EC-C18, Agilent). The injection volume was 20 μl (full loop injection). The mobile phase consisted of two solutions, solvent A (0.1% formic acid and 6 mM ammonium acetate in water) and solvent B (0.1% formic acid and 6 mM ammonium acetate in acetonitrile), the column was thermostatted in an oven at 35°C, and the flow rate was 650 μl/min.

[00110] Chromatographic gradient used for a C18 column; initial composition B was 0% for 0.3 min and increased to 80% from 0.3 to 0.9 min, then 100% was reached from 1 min to 1.3 min, followed by re-equilibration to the initial state for 0.3 min.

[00111] For MS analysis, data were acquired using electrospray ionization (ESI) in positive ion mode, with the ion spray voltage set to 5500 V. Source desolvation was performed using the following set parameters: temperature (TEM) at 600 °C, gas ion source 1 (GS1) at 40 psi (276 kPa), gas ion source 2 (GS2) at 50 psi (345 kPa), and curtain gas (CUR) at 50 psi (345 kPa). Specific MRM assay parameters allowing quantification and monitoring of prazosin (the ligand, or “marker,” for α1) and RX821002 (the ligand, or “marker,” for α1) are described in Table 6. Raw data were processed in Sciex Analyst and individual AUC (area under the curve) for each analyte in each sample was determined using MultiQuant software.

Q1, mass (Yes) Q3, mass (Yes) Time (ms) ID DP
(volt) EP
(volt) CE
(volt) CXP
(volt) 384,300 231,162 100 Prazosin 140 10 56 9 235,100 203,000 100 RX821002 40 10 21 23

DP: declustering potential, EP: input potential, CE: collision energy and CXP: collision cell output voltage.

Table 6

Linking via MS experiments

Determination of the optimal ligand concentration

[00112] Rat cortical membrane preparations containing both alpha 1 non-selective (α1 NS) and alpha 2 non-selective (α2 NS) receptors were co-incubated with prazosin (a specific α1 NS ligand) and RX821002 (a specific α2 NS ligand) simultaneously. The assay was performed in triplicate in assay buffer (50 mM Tris-HCl, 5 mM EDTA/Tris, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 0.1% BSA) in polypropylene 96-well deep-well plates at 22°C. First, 12 concentrations (ranging from 0.1 nM to 300 nM) of prazosin and RX821002 were co-incubated for 60 min at 22°C, with 3 concentrations (200 μg/well) of rat cortical membrane preparations.

[00113] Non-specific binding was determined by co-incubation with 10 μM WB 4101 and yohimbine.

[00114] The incubation was stopped by filtration after the entire volume of the binding reaction mixture had been transferred to a filter plate. The remaining amount of both prazosin and RX821002 was determined by UHPLC-MS/MS.

Competitive mass binding assays :

[00115] Ligand displacement assays were performed using 12 concentrations of the competitive ligands, WB4101 (an α1 HC inhibitor) and yohimbine (an α2 HC inhibitor) (ranging from 0.1 nM to 100 μM), and 0.3 nM prazosin and 1 nM RX821002. They were coincubated with 200 μg/well rat cortical membrane in assay buffer, in triplicate. Incubation was stopped by filtration after incubation for 60 min at 22°C. The remaining amount of both prazosin and RX821002 was determined by UHPLC-MS/MS as an alpha-2 adrenergic receptor antagonist.

EXAMPLE 4: Multiplexing of different target molecules

[00116] Figs. 4A-K are a series of graphs showing the results of a simultaneous binding assay using rat cortex target molecules. Ligands and test compounds are shown in each figure. The target molecules are the following receptor molecules: A1 (adenosine receptor) (Fig. 4A); M1 (muscarinic receptor) (Fig. 4B); 5-HT 2A (serotonin receptor) (Fig. 4C); Alpha 1 ns (adrenergic receptor) (Fig. 4D); Alpha 2 ns (adrenergic receptor) (Fig. 4E); D1 (dopamine receptor) (Fig. 4F); 5H-trans (serotonin receptor) (Fig. 4G); 5-HT2A receptor (Fig. 4H); Ca++ channel (Fig. 4I); mu opioid receptor (Fig. 4J); PCP (sigma opioid receptor) (Fig. 4K).

[00117] As shown in Fig. 4A-K, 11 different target molecules were examined simultaneously. Various tissues can be used. For example, the first receptor target molecule, the adenosine A1 receptor, is also found in smooth muscle in the vascular system.

[00118] The experiment in Fig. 4A-K can be summarized as follows (Table 7):

Figure Target molecule Marker ligand Tested connection 4A Adenosine A1 receptor CPX NECA 4B Muscarinic acetylcholine receptor pirenzepine Atropine 4C 5-HT2A (serotonin) 8-OH-DPAT Serotonin 4D Alpha-1A-adrenergic receptor Prazosin WB4101 4E Alpha-2A adrenergic receptor RX82102 Yohimbine 4F Dopamine receptor D1 SCH23390 Butaclamol 4G 5HT transporter paroxetine Zimelidine 4H 5-HT (serotonin) EMD281014 Serotonin 4I Ca++ channel D600 D888 4J Opioid receptor naloxone DAMGO 4K PCP (sigma-type opioid receptor) MK801 SKF10047

Table 7

Materials and methods:

Obtaining rat cortex membrane

[00119] Rat cortexes from male Wistar rats were collected and transferred to 50 mM Tris-HCl (pH 7.4) and homogenized with polyton. The homogenate was centrifuged at 50,000 g for 15 min at 4°C. The resulting pellet was washed in lysis buffer containing 50 mM Tris-HCl (pH 7.4) containing 1 μg/ml leupeptin and 1 μM pepstatin and centrifuged at 50,000 g for 15 min at 4°C. The pellet was finally resuspended in a smaller volume of lysis buffer and the final protein concentration was determined according to the Bradford method using bovine serum albumin as a standard.

Filtration and elution of samples

[00120] The incubation was stopped by filtration after transferring the binding sample (200 μl aliquots per well) to 96-well glass filter plates and then rapidly filtered under vacuum, the membrane fraction bound to the filters was washed several times with wash buffer (50 mM Tris-HCl and 150 mM NaCl) in a vacuum manifold. The membrane filters were pretreated for 1 hour with 50 mM Tris/HCl and 0.3% polyethyleneimine (PEI) solution.

[00121] The filters were dried for one hour at 50°C and cooled to room temperature before elution of the specific ligands using acetonitrile (containing 100 pM antipyrine as an internal standard) via a vacuum manifold. Relative quantification of the ligand in each sample was performed by UHPLC-MS-MS using the area ratio of the ligand to the internal standard.

Development of UHPLC-MS/MS method

[00122] UHPLC-QQQ analysis was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA).

[00123] Chromatographic separation was performed on a C18 column (Poroshell 120 EC-C18, Agilent). The injection volume was 20 μl (full loop injection). The mobile phase consisted of two solutions, solvent A (0.1% formic acid and 6 mM ammonium acetate in water) and solvent B (0.1% formic acid and 6 mM ammonium acetate in acetonitrile), the column was thermostatted in an oven at 35°C, and the flow rate was 650 μl/min.

[00124] Chromatographic gradient used for a C18 column; initial composition B was 0% for 0.3 min and increased to 80% from 0.3 to 0.9 min, then 100% was reached from 1 min to 1.3 min, followed by re-equilibration to the initial state for 0.3 min.

[00125] For MS analysis, data were acquired using electrospray ionization (ESI) in positive ion mode with the ion spray voltage set to 5500 V. Source desolvation was performed using the following set parameters: temperature (TEM) at 600 °C, gas ion source 1 (GS1) at 40 psi (276 kPa), gas ion source 2 (GS2) at 50 psi (345 kPa), and curtain gas (CUR) at 50 psi (345 kPa). Specific MRM method parameters allowing ligand quantification and monitoring are described in Table 8. Raw data were processed in Sciex Analyst and the individual AUC (area under the curve) for each analyte in each sample was determined using MultiQuant software.

Q1, mass (Yes) Q3, mass (Yes) Time (ms) ID DP
(volt) EP
(volt) CE
(volt) CXP
(volt) 305,200 263,100 150 CPX 100 10 32 11 352,200 113,000 150 PIRENZEPINE 80 10 27 18 384,142 95,000 150 PRAZOSIN 196 10 77 14 235,100 203,100 150 RX821002 20 10 23 9 288,100 179,115 150 SCH23390 10 10 31 8 248,100 147,100 150 8-OH-DPAT 40 10 28 7 377,200 209,200 150 EMD281014 20 10 31 9 330,100 192,200 150 PAROXETINE 40 10 29 8 485,500 165,100 150 D600 60 10 37 22 222,100 178,100 150 MK801 50 10 54 8 328,100 212,200 150 NALOXONE 60 10 53 9

DP: declustering potential, EP: input potential, CE: collision energy and CXP: collision cell output voltage.

Table 8

Linking via MS experiments

Competitive mass binding assays:

[00126] Ligand displacement assays were performed using rat cortical membrane preparations naturally containing the following receptors: A1 (adenosine), M1 (muscarinic), Alpha1ns (adrenergic), Alpha2ns (adrenergic), D1 (dopamine), 5HT1a (serotonin), 5HT2a (serotonin), 5H-trans (serotonin), Ca ²⁺ channel (verapamil site), glutamate (non-selective) rat ion channel, and opioid non-selective receptors

Receptor Specific ligand/concentration used Inhibitor A1-adenosine CPX/1 nM NECA M1--muscarinic PIRENZEPINE/1 nM atropine Alpha1 ns--adrenergic PRAZOSIN/1 nM WB 4101 Alpha2 ns-- adrenergic RX821002/1nM Yohimbine D1-dopamine SCH23390/1nM Butaclamol 5HT1a -- serotonin 8-OH-DPAT/5 nM serotonin 5HT2a--serotonin EMD281014/1nM serotonin 5H-trans-serotonin PAROXETINE/1 nM Zimelidine Cave (Ca channel) D600/1 nM D888 PCP--opioid receptor sigma-type MK801/ 5nM SKF10047 Opioid NS NALOXONE/1 nM DAMGO

Table 9

[00127] Ligand displacement assays were performed using 8 concentrations of inhibitor (see Table 9) (ranging from 0.1 nM to 100 μM) and a mixture of one concentration of each specific ligand (see Table 9). They were co-incubated with 200 μg/well rat cortical membrane in assay buffer (50 mM Tris-HCl, 5 mM EDTA/Tris, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 0.1% BSA), in triplicate. Incubation was stopped by filtration after incubation for 60 min at 22°C. The remaining amount of each specific ligand (see Table 9) was determined by UHPLC-MS/MS.

EXAMPLE 5: Multiplexing of different heterologous tissues - membranes ex vivo : rat cortex, rat cerebellum and rat ventricular tissue

[00128] This example shows multiplexing of the MS competitive binding assay as described, but with different tissues in the same experiment.

[00129] The results are shown in Table 10 below. Various tissues were used in this example. Illustrative tissue sources for the target receptor molecules are cerebral cortex, cerebellum, and ventricular membrane (rat or human). The binding assays shown in column 1 were A1, M1, etc. In each case, a known ligand (shown as [ligand] in column 2) was added and the extent of binding to the tissues of interest was measured. The known (marker) ligands were as used in Example 4. A calibration curve was generated. As shown below, SNR refers to the signal to noise ratio and %CV refers to the percent coefficient of variation.

Binding analysis Rat cortex, 180 mcg Cerebellum, 180 mcg Ventricular (membrane), 180 mcg A1 [ligand]: nM 0,1 1 5 SNR: 7 4.5 1.5 %CV 5.30 3.7 51.6 M1 [ligand]: nM 5 SNR: 17.5 %CV 6.5 Alpha 1 ns [ligand]: nM 0,1 0,1 0,1 SNR: 53.7 13.5 28.1 %CV 12 25.7 13.1 Alpha 2 ns [ligand]: nM 0,1 1 SNR: 51.3 11.2 %CV 4.2 6.5 D1 [ligand]: nM 1 SNR: 46.6 %CV 7.7 5HT1a [ligand]: nM 1 SNR: 4.1 %CV 32.7 5HT2a [ligand]: nM 0,1 0,1 1 SNR: 14.2 2.5 2,3 %CV 12.2 80.4 113.4 5HT-trans [ligand]: nM 0,1 0,1 0,1 SNR: 5 2.5 2.8 %CV 6.9 29.4 127.5 CAVE [ligand]: nM 1 0,1 SNR: 2.4 3.2 %CV 7 31.7 PCP [ligand]: nM 10 50 SNR: 2,2 2.4 %CV 26.9 27.4 OPIOID NS [ligand]: nM 1 SNR: 9.8 %CV 8.7

Table 10

EXAMPLE 6: Single-well multiplexing mass analysis of 20 ligand binding in rat membrane mixtures ex vivo or in mixtures of recombinant membranes

[00130] In this example, different tissues and/or receptor molecules are combined in the same well in a single reaction. Rat cortex, cerebellum, and ventricular membrane are added to a single well, and a series of reactions are performed using ligands as shown in Example 5.

Materials and methods:

Obtaining membranes ex vivo

[00131] Rat cortices from male Wistar rats are collected and transferred to 50 mM Tris-HCl (pH 7.4) and homogenized with polyton. The homogenate is centrifuged at 50,000 g for 15 min at 4°C. The resulting pellet is washed in lysis buffer containing 50 mM Tris-HCl (pH 7.4) containing 1 μg/ml leupeptin and 1 μM pepstatin and centrifuged at 50,000 g for 15 min at 4°C. The pellet is finally resuspended in a smaller volume of lysis buffer and the final protein concentration is determined according to the Bradford method using bovine serum albumin as a standard.

[00132] Preparation of rat cerebellum, liver and ventricular membranes was carried out as described above.

Obtaining a recombinant membrane

Cell culture and expression

[00133] Stable transfection of a human cell line is accomplished using a suitable expression vector containing coding sequences for the receptor of interest. Individual colonies of stably transfected cells are then cultured in selective media using a specific antibiotic. Final selection of clones is based on the binding affinity of the clones for the specific ligand.

Isolation of membranes

[00134] A dried cell pellet from a clone of human cells stably expressing the receptor of interest was resuspended in lysis buffer (50 mM Tris-HCl, 5 mM Tris-EDTA, 20 mM NaCl, 1.5 mM CaCl2, 5 mM MgCl2, 10 μg/ml trypsin inhibitor, 1 μg/ml leupeptin, 75 μg/ml PMSF). Cells were lysed using an ultrasonic probe (Sonifier 250, Branson). The cell lysate was centrifuged at 50,000 x g for 15 minutes at 4°C. The membrane pellet is resuspended in lysis buffer containing 10% (v/v) glycerol, and the final protein concentration is determined according to the Bradford method using bovine serum albumin as a standard.

Filtration and elution of samples

[00135] The incubation is stopped by filtration after transferring the binding sample (200 μl aliquots per well) to 96-well glass filter plates and then rapidly filtered under vacuum, the membrane fraction bound to the filters is washed several times with wash buffer (50 mM Tris-HCl and 150 mM NaCl) in a vacuum manifold. The membrane filters are pretreated for 1 hour with 50 mM Tris/HCl and 0.3% polyethyleneimine (PEI) solution.

[00136] The filters are dried for one hour at 50°C and cooled to room temperature before elution of the specific ligands using acetonitrile (containing 100 pM antipyrine as an internal standard) via a vacuum manifold. Relative quantification of the ligand in each sample is performed by UHPLC-MS-MS using the area ratio of the ligand to the internal standard.

Development of UHPLC-MS/MS method

[00137] UHPLC-QQQ analysis was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA).

[00138] Chromatographic separation is performed on a C18 column (Poroshell 120 EC-C18, Agilent). The injection volume is 20 μl (full loop injection). The mobile phase consists of two solutions, solvent A (0.1% formic acid and 6 mM ammonium acetate in water) and solvent B (0.1% formic acid and 6 mM ammonium acetate in acetonitrile), the column is thermostatted in an oven at 35°C, and the flow rate is 650 μl/min.

[00139] Chromatographic gradient used for a C18 column; initial composition B is 0% for 0.3 min and increases to 80% from 0.3 to 0.9 min, then 100% is reached from 1 min to 1.3 min, followed by re-equilibration to the initial state for 0.3 min.

[00140] For MS analysis, data were acquired using electrospray ionization (ESI) in positive ion mode with the ion spray voltage set to 5500 V. Source desolvation was performed using the following set parameters: temperature (TEM) at 600 °C, gas ion source 1 (GS1) at 40 psi (276 kPa), gas ion source 2 (GS2) at 50 psi (345 kPa), and curtain gas (CUR) at 50 psi (345 kPa). Specific MRM method parameters allowing ligand quantification and monitoring are described in Table 11. Raw data were processed in Sciex Analyst and individual AUC (area under the curve) for each analyte in each sample was determined using MultiQuant software.

Q1, mass (Yes) Q3, mass (Yes) Time (ms) ID DP
(volt) EP
(volt) CE
(volt) CXP
(volt) 305,200 263,100 50 CPX 100 10 32 11 408.1 219.2 50 CGS 21680 131 10 35 10 300.2 270.2 50 AB-MECA 175 10 19 13 352,200 113,000 50 PIRENZEPINE 80 10 27 18 479.3 240.1 50 AF-DX384 120 10 28 9 384,142 95,000 50 PRAZOSIN 196 10 77 14 235,100 203,100 50 RX821002 20 10 23 9 288,100 179,115 50 SCH23390 10 10 31 8 356.2 325.2 50 Methylspiperone 90 10 15 13 248,100 147,100 50 8-OH-DPAT 40 10 28 7 377,200 209,200 50 EMD281014 20 10 31 9 330,100 192,200 50 PAROXETINE 40 10 29 8 485,500 165,100 50 D600 60 10 37 22 222,100 178,100 50 MK801 50 10 54 8 328,100 212,100 50 NALOXONE 60 10 53 9 1052.5 958.2 50 CGP 42112A 50 10 17 10 1060.6 938.5 50 Bradykinin 75 10 22 16 352,200 113,000 50 CP 55,940 80 10 27 18 1064.2 1001.2 50 CCK8 76 10 23 8 112.1 95.1 50 Histamine 159 10 24 11 497.3 434.3 50 LTD4 125 10 26 17

DP: declustering potential, EP: input potential, CE: collision energy and CXP: collision cell output voltage.

Table 11

Linking via MS experiments

Competitive mass binding assays:

[00141] Ligand displacement assays are performed using mixtures of 4 different ex vivo membranes from rat cortex, cerebellum, ventricular, and liver membrane preparations. Equal amounts of each tissue membrane preparation are mixed (50 μg).

[00142] In addition, ligand displacement assays were also performed using a mixture of 20 different recombinant membranes (see Table 12), equal amounts (10 μg) of each membrane preparation were mixed.

Receptor Specific ligand Inhibitor A1 CPX NECA A2A (h) CGS 21680 NECA A3(h) AB-MECA IB-MECA M1 PIRENZEPINE atropine M2 (h) AF-DX384 4-DAMP Alpha1 ns PRAZOSIN WB 4101 Alpha2 ns RX821002 Yohimbine D1 SCH23390 Butaclamol D2S (h) Methylspiperone Butaclamol 5HT1a 8-OH-DPAT serotonin 5HT2a EMD281014 serotonin 5H-trans PAROXETINE Zimelidine Cave D600 D888 PCP MK801 SKF10047 Opioid NS NALOXONE DAMGO AT2 (h) CGP 42112A Angiotensin II B2(h) Bradykinin HOE 140 CB1(h) CP 55,940 WIN 55,212-2 CCK1 (CCKA) CCK-8S SIB-CCK8 H4 (h) Histamine PDGF-BB CysLT1(LTD4)(h) LTD4 MK571

Table 12

[00143] Competitive mass binding assays were performed using 8 concentrations of inhibitors (see Table 12) (ranging from 0.1 nM to 100 μM) and a mixture of one concentration of each specific ligand (see Table 12), in which each ligand was present at a final concentration of 5 nM. They were co-incubated at 200 μg/well of either the ex vivo membrane mixture or the recombinant membrane mixture in assay buffer (50 mM Tris-HCl, 5 mM EDTA/Tris, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 0.1% BSA), in triplicate. The incubation was stopped by filtration after incubation for 60 minutes at 22°C. The remaining amount of each specific ligand (see table) is determined by UHPLC-MS/MS.

EXAMPLE 7: Single-well multiplexing for security testing

[00144] In this example, a combination of different tissue types are combined in individual wells as shown in Table 13 below:

Receptor Textile Known Ligand/Substrate GPCR, adenosine A1 receptor Widely distributed throughout the body. Adenosine cyclooxygenase 2 (COX2) synoviocytes, endothelial cells, chondrocytes, osteoblasts and monocytes/macrophages stimulated using cytokines Arachidonic acid Monoamine oxidase (MAO) Hypothalamus and hippocampal uncus Serotonin, melatonin, norepinephrine, adrenaline Dopamine transporter Brain (substantia nigra) dopamine

Table 13

[00145] The above-mentioned target receptor molecules can be obtained from the listed tissues or produced in a cloned cell.

[00146] The materials and methods are carried out as described above.

EXAMPLE 8A, 8B: Pharmacological determination of K _on and K _off

[00147] Figure 5 is a schematic workflow for using MS to determine the binding kinetics of a test compound to its cognate receptor molecule. As shown, a material containing target molecules (e.g., rat cortex) is incubated with a ligand and a test compound (imipramine and serotonin in this figure). Bound ligands are isolated by methods as described above and the amount of each ligand is determined. As shown in Figure 5, a sample containing at least one target receptor molecule (e.g., rat cortex) is first incubated 501 with a ligand and a test compound, e.g., imipramine (5 nM) and serotonin (10 μM), respectively, in a buffer containing Tris/HCl, NaCl, KCl, and BSA. After incubation, the bound receptor-ligand complex is separated 502 by methods as described in the present invention. The separated ligand-receptor complex is then processed to separate the ligand from the bound receptor target molecule, for example, by using acetonitrile and a glass filter allowing only unbound ligands to pass through (503). After separation, the bound ligand molecules from each well are quantified in a multiplate reader 504 505 by liquid chromatography/ESI-MS/MS using a calibration curve to determine K _on and K _off .

[00148] As shown in Fig. 5, buffer, ligand (imipramine), and non-specific binder serotonin are incubated in separate wells. Separation of the complexed target molecule in rat cortex, the serotonin transporter (5-HT), is performed as before. The complex is separated using acrylonitrile, and the separated serotonin is measured by MS to determine K _on and K _off .

[00149] Figures 6A, 6B, and 6C are a series of graphs showing the results of the MS method for determining the binding kinetics, K _on (Figure 6A), and the dissociation kinetics, K _off (Figures 6B and 6C). Figure 6B shows the dissociation kinetics of GABAB1b/2 from CGP54626, at a concentration of 1 nM, by a displacement method by adding 10 μM CPG52432. Data points represent specific binding (mean +/- SD, n=2). Figure 6C shows the dissociation kinetics of GABA _B1b/2 from CGP54626, at a concentration of 5 nM, by a dilution method. Data points represent specific binding (mean +/- SD, n=2).

[00150] Fig. 6A shows the determination of the binding kinetics curve and K _on by measuring specific binding at different time intervals. Fig. 6B shows the dissociation kinetics curve and K _off obtained by measuring the decrease in specific binding of the ligand to the target receptor molecule over time. Fig. 6C shows the dissociation kinetics as measured by dilution.

Example 8A: K _on / _Koff GABA 1b

Cell culture and GABA expression _{B 1 b /2}

[00151] Stable transfection of the CHO-S cell line was performed using the pCi/neo vector (Promega) containing coding sequences for the human GABA receptor B 2-unit 1b (NM_021903) as well as GABA 2 (NM_005458). Single colonies of stably transfected cells were further cultured in selective media using geneticin. Final clone selection was based on the binding affinity of the clones for ^3H [CGP54626].

Isolation of membranes

[00152] The dried cell pellet of the clone from CHO-S cells stably expressing GABAB1b/2 was resuspended in lysis buffer (50 mM Tris-HCl, 5 mM Tris-EDTA, 20 mM NaCl, 1.5 mM CaCl2, 5 mM MgCl2, ₁₀ μg/ml trypsin inhibitor, 1 μg/ml leupeptin, 75 μg/ml PMSF). The cells were lysed using an ultrasonic probe (Sonifier 250, Branson). The cell lysate was centrifuged at 50,000 xg for 15 min at 4°C. The membrane pellet was resuspended in lysis buffer containing 10% (v/v) glycerol and the final protein concentration was determined according to the Bradford method using bovine serum albumin as standard.

Filtration and elution of samples

[00153] The incubation was stopped by filtration after transferring the binding sample (200 μl aliquots per well) to 96-well glass filter plates and then rapidly filtered under vacuum, the membrane fraction bound to the filters was washed several times with wash buffer (50 mM Tris-HCl and 150 mM NaCl) in a vacuum manifold. The membrane filters were pretreated for 1 hour with 50 mM Tris/HCl and 0.3% polyethyleneimine (PEI) solution.

[00154] The filters were dried for one hour at 50°C and cooled to room temperature prior to elution of CGP54626 using acetonitrile (containing 100 pM antipyrine as an internal standard) via a vacuum manifold. Relative quantification of ligand in each sample was performed by UHPLC-MS-MS using the area ratio of ligand to internal standard.

Development of UHPLC-MS/MS method

[00155] UHPLC-QQQ analysis was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA).

[00156] Chromatographic separation was performed on a C18 column (Poroshell 120 EC-C18, Agilent). The injection volume was 20 μl (full loop injection). The mobile phase consisted of two solutions, solvent A (0.1% formic acid and 6 mM ammonium acetate in water) and solvent B (0.1% formic acid and 6 mM ammonium acetate in acetonitrile), the column was thermostatted in an oven at 35°C, and the flow rate was 650 μl/min.

[00157] Chromatographic gradient used for a C18 column; initial composition B was 0% for 0.3 min and increased to 80% from 0.3 to 0.9 min, then 100% was reached from 1 min to 1.3 min, followed by re-equilibration to the initial state for 0.3 min.

[00158] For MS analysis, data were acquired using electrospray ionization (ESI) in positive ion mode, with the ion spray voltage set to 5500 V. Source desolvation was performed using the following set parameters: temperature (TEM) at 600 °C, gas ion source 1 (GS1) at 40 psi (276 kPa), gas ion source 2 (GS2) at 50 psi (345 kPa), and curtain gas (CUR) at 50 psi (345 kPa). Specific parameters of the MRM method allowing quantification and monitoring of ligands (CGP54626) are described in Table 14. Raw data were processed in Sciex Analyst and individual AUC (area under the curve) for each analyte in each sample was determined using MultiQuant software.

Q1, mass (Yes) Q3, mass (Yes) Time (ms) ID DP
(volt) EP
(volt) CE
(volt) CXP
(volt) 408.1 236.0 150 CGP54626-1 131 10 27 10 408.1 219.2 150 CGP54626-2 131 10 35 10

DP: declustering potential, EP: input potential, CE: collision energy and CXP: collision cell output voltage.

Table 14

Linking via MS experiments

Determination of the optimal concentration of receptor and ligand

[00159] Membrane preparations containing GABA _B1b/2 and CGP54626 were incubated in triplicate in assay buffer (50 mM Tris-HCl, 2.5 mM CaCl2, 10 μg/ml trypsin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) in polypropylene 96-well deep-well plates at 22°C. First, 6 concentrations (0.1, 0.5, 1, 3, 5, 10, 25 and 50 nM) of CGP54626 (Tocris, ref: 1088) were co-incubated for 60 min at 22°C, with 3 concentrations (45, 100 and 180 μg/well) of recombinant GABA _B1b/2 receptor.

[00160] Non-specific binding was determined by co-incubation with 10 μM CGP52432.

[00161] The incubation was stopped by filtration after the entire volume of the binding reaction mixture had been transferred to a filter plate. The remaining amount of CGP54626 was determined by UHPLC-MS/MS.

For saturation analyses:

[00162] Membrane aliquots containing 10-180 μg of GABA _B1b/2 protein were incubated in triplicate in the presence of 1 nM CGP54626 in a total volume of 200 μl of assay buffer. Incubation was stopped by filtration after incubation for 60 min at 22°C.

[00163] Non-specific binding was determined by co-incubation with 10 μM CGP52432

[00164] The incubation was stopped by filtration after the entire volume of the binding reaction mixture had been transferred to a filter plate. The remaining amount of CGP54626 was determined by UHPLC-MS/MS.

Mass binding assays (K _on ):

[00165] Aliquots of membrane containing 22.5 μg/100 μl of GABAB1b/2 membrane protein were incubated in a total volume of 2000 μl of assay buffer at 22°C with 1 nM CGP54626. At each time point, 200 μl of the reaction mixture was removed and the incubation was stopped by filtration. The remaining amount of CGP54626 was determined by UHPLC-MS/MS. See Fig. 6A.

[00166] Non-specific binding was determined by co-incubation with 10 μM CGP52432.

Competitive mass binding assays:

[00167] Ligand displacement assays were performed using eight concentrations of the competitive ligand, CGP52432 (ranging from 1 nM to 30 μM), GABA (ranging from 10 nM to 1 mM), and baclofen (ranging from 10 nM to 1 mM). They were coincubated with 45 μg/well of GABAB1b/2 membrane protein and 1 nM CGP54626 in assay buffer, in triplicate. Incubation was stopped by filtration after incubation for 60 min at 22°C. The remaining amount of CGP54626 was determined by UHPLC-MS/MS.

Binding-dissociation mass assays - displacement.

[00168] Aliquots of membrane containing 22.5 μg/100 μl of GABA _B1b/2 membrane protein were incubated in a total volume of 2000 μl of assay buffer at 22°C using 1 nM CGP54626. The reaction was allowed to equilibrate for 60 min before dissociation was initiated by the addition of 10 μM CPG52432. Dissociation was stopped at specified time intervals (1-80 min) by filtration of 200 μl of the reaction mixture. Samples for each time point were prepared in duplicate. The remaining amount of CGP54626 was determined by UHPLC-MS/MS. See Fig. 6B.

Binding-dissociation mass assays - dilution method

[00169] To determine the K _off constant by dilution, 112.5 μg/100 μl GABA _B1b/2 membrane protein was incubated with 5 nM CGP54626 at 22°C for 60 min. A 22 μl aliquot was removed and added to 2178 μl assay buffer, resulting in a 1:100 dilution. Dissociation was stopped by filtration at specified time intervals (1-80 min). Samples for each time point were prepared in duplicate. The remaining amount of CGP54626 was determined by UHPLC-MS. See Fig. 6C.

Example 8B: Binding experiments by mass spectrometry multiplexing k determination _on /k _off or in one membrane ex vivo , or in membrane mixtures ex vivo , alternatively, in mixtures of recombinant membranes

Obtaining membrane mixtures

[00170] K _on and K _off determinations are performed either on rat cortex membrane or using mixtures of 4 different membranes ex vivo from rat cortex, cerebellum, ventricular and liver membrane preparations. Equal amounts of each tissue membrane preparation are mixed (50 μg). In addition, K _on and K _off determinations are also performed using a mixture of 20 different recombinant membranes (see Table 15), equal amounts of each membrane preparation are mixed (10 μg).

Receptor Specific ligand Inhibitor A1 CPX NECA A2A (h) CGS 21680 NECA A3(h) AB-MECA IB-MECA M1 PIRENZEPINE atropine M2 (h) AF-DX384 4-DAMP Alpha1 ns PRAZOSIN WB 4101 Alpha2 ns RX821002 Yohimbine D1 SCH23390 Butaclamol D2S (h) Methylspiperone Butaclamol 5HT1a 8-OH-DPAT serotonin 5HT2a EMD281014 serotonin 5H-trans PAROXETINE Zimelidine Cave D600 D888 PCP MK801 SKF10047 Opioid NS NALOXONE DAMGO AT2 (h) CGP 42112A Angiotensin II B2(h) Bradykinin HOE 140 CB1(h) CP 55,940 WIN 55,212-2 CCK1 (CCKA) CCK-8S SIB-CCK8 H4 (h) Histamine PDGF-BB CysLT1(LTD4)(h) LTD4 MK571

Table 15

Mass binding assays (K _on ):

[00171] Aliquots of membrane containing 22.5 μg/100 μl of each membrane protein mixture are incubated in a total volume of 2000 μl of assay buffer at 22°C with a mixture of specific ligands (see Table 15), each at a final concentration of 1 nM. At each time point, 200 μl of the reaction mixture is removed and the incubation is stopped by filtration. The remaining amount of each specific ligand (see Table) is determined by UHPLC-MS/MS.

[00172] Non-specific binding is determined by co-incubation with a mixture of specific inhibitors (see table), each at a final concentration of 10 μM.

Binding-dissociation mass assays - displacement

[00173] Aliquots of membrane containing 22.5 μg/100 μl of each membrane protein mixture were incubated in a total volume of 2000 μl of assay buffer at 22°C with a mixture of specific ligands (see Table 15), each at a final concentration of 1 nM. The reaction was allowed to reach equilibrium for 60 min before dissociation was initiated by the addition of a mixture of specific inhibitors (see Table), each at a final concentration of 10 μM. Dissociation was stopped at specified time intervals (1-80 min) by filtration of 200 μl of the reaction mixture. Samples for each time point were prepared in duplicate. The remaining amount of each specific ligand was determined by UHPLC-MS/MS.

Binding-dissociation mass assays - dilution method

[00174] To determine the K _off constant by dilution, 112.5 μg/100 μl of each membrane protein mixture was incubated with a mixture of specific ligands (see Table), each at a final concentration of 1 nM, and incubated at 22°C for 60 minutes. A 22 μl aliquot was removed and added to 2178 μl of assay buffer, resulting in a 1:100 dilution. Dissociation was stopped by filtration at specified time intervals (1-80 minutes). Samples for each time point were prepared in duplicate. The remaining amount of each specific ligand was determined by UHPLC-MS/MS.

CONCLUSION

[00175] The foregoing specific description is intended to exemplify and illustrate the invention and is not to be construed as limiting the scope of the invention, which is limited by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials that can be used to carry out specific aspects of the invention that may not be explicitly taught but that may be understood by those skilled in the art. The contents of such patents or publications are hereby incorporated by reference to the same extent as if each were specifically and individually indicated by reference and contained in this specification, as necessary for the purpose of describing and enabling the practice of the method or material referenced.

[00176] The foregoing merely illustrates the principles of the present description. It is understood that one skilled in the art is able to devise various arrangements which, although not explicitly described or shown in the present description, embody the principles of the invention and are included within the spirit and scope thereof. Furthermore, all examples and conventions cited in the present description are principally intended to facilitate the reader's understanding of the contribution of the principles of the invention and the concepts of the present inventors to the advancement of the art, and should be construed as not limiting such specifically recited examples and conventions. Furthermore, all statements in the present description reciting principles, aspects and embodiments of the invention, as well as specific examples thereof, are intended to include both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents that will be developed in the future, i.e., any developed elements that perform the same function, regardless of structure. The scope of the present invention is therefore not intended to be limited to the illustrative embodiments shown and described herein.

Claims (56)

1. A multiplex method for quantitatively assessing the binding of a test compound to a predetermined target molecule and to non-target molecules, comprising the steps of: (a) obtaining a mixture of target molecules from a crude extract from at least one of (i) healthy or unhealthy human or non-human tissue, and (ii) a synthetic protein preparation, wherein said target molecules are present in a mixture of target molecules that do not naturally exist in the same tissue preparation; (b) incubating said mixture of target molecules in a plurality of mixtures of ligands and test compounds, wherein said target molecules bind to different ligands; (c) removing unbound ligands from said plurality of target molecule mixtures; (d) isolating ligands bound to said target molecules in said mixture of target molecules; (e) determining the amount of said ligand bound by the target molecule by measuring the ligands obtained in step (d) at specified times in the reaction mixture using mass spectrometry and a calibration curve; (f) determining the affinity of the test compound for said target molecules in said mixture of said target molecules using the data obtained in step (e); (g) measuring the binding of said test compound to a predetermined target molecule and comparing said binding to the binding of said test compound to non-target molecules; and (h) calculating K _on and K _off of said test compound for said target molecule using the data obtained in step (e). 2. The method according to claim 1, wherein said mixture of target molecules further comprises a heterologous mixture of said target molecules, wherein said heterologous mixture of said target molecules is either (a) a mixture of different target molecules not naturally present in a single tissue preparation; or (b) a mixture of different target molecules present in a single tissue, wherein each target molecule has a biological function that is different from any other target molecule present in said mixture of different target molecules. 3. The method according to claim 1 or 2, wherein said mixture of target molecules comprises targets that are human target molecules. 4. The method according to any one of claims 1-3, wherein step (c) comprises removing unbound ligands from said plurality of mixtures using a glass filter. 5. The method of any one of claims 1-4, wherein step (d) comprises eluting bound ligands from the filter using a solvent, then concentrating samples from the filter. 6. The method according to any one of claims 1 to 5, wherein step (e) using mass spectroscopy comprises the use of liquid chromatography/electrospray ionization tandem mass spectrometry. 7. The method according to claim 1, where K _off is determined by means of a displacement method. 8. The method according to claim 1, wherein K _off is determined by means of a dilution method. 9. The method according to any one of claims 1-8, wherein said target molecules are selected from at least one of the group consisting of: Na+ channels, alpha-1-beta-adrenergic receptors, alpha-2-beta-adrenergic receptors, adenosine A1 receptor, muscarinic receptor M1, serotonin receptor 5-HT _2A , adrenergic receptor Alpha 1 ns, adrenergic Alpha 2 ns, dopamine receptor D1 and serotonin receptor 5H-trans. 10. The method according to any one of claims 1-9, wherein the ligands are at least one ligand selected from the group consisting of: CPX, pirenzepine, prazosin, RX821002, SCH233900, 8-OH-DPAT, EMD281014, paroxetine, D600, MK801 and naloxone. 11. A multiplex method for quantitatively assessing the binding affinity of at least two different test compounds (test compound C1-C _n ) for at least two different target receptor molecules (receptor RT1 for C1, RT _n for C _n ), based on competitive binding between the test compounds and known binders for RT1 and RT _{n ,} known binder B1 for RT1 and B _n for RT _n , respectively, comprising: (a) obtaining a mixture from a crude extract containing (i) test compounds C1-C _n ; (ii) known B1-B _n binders; and (iii) RT1-RT _n target receptor molecules, wherein RT1-RT _n do not naturally exist in the same tissue preparation; (b) ensuring the possibility of forming complexes in the said mixture between the test compounds C1-C _n , RT1-RT _n and B1-B _n ; (c) separating test compounds that do not form complexes with their target molecules from said complexes; (d) isolating known binders from the complexes obtained in step (c) and passing the isolated binders through a mass spectrometer to measure the binding of test compounds using mass spectroscopy; and (e) determining the relative affinities of the compounds C1-C _n for RT1-RT _n , respectively, where C _n , B _n and RT _n represent series of members where n is between 2 and 40. 12. The method according to claim 11, wherein said target receptor molecules are obtained from membranes ex vivo from at least two of the membrane preparations: cortex membrane preparations, cerebellar membrane preparations, ventricular membrane preparations, and liver membrane preparations. 13. The method according to any one of claims 11 or 12, wherein said step of obtaining RT1-RT _n receptor target molecules comprises obtaining human receptor target molecules. 14. The method according to any one of claims 11-13, wherein step (c) comprises separation using a glass filter and washing. 15. The method of any one of claims 11-14, wherein step (d) comprises eluting the bound ligand from the filter using a solvent, then concentrating the samples from the filter. 16. The method according to any one of claims 11-15, wherein said mass spectroscopy comprises the use of liquid chromatography/electrospray ionization tandem mass spectrometry. 17. The method according to any one of claims 11-16, further comprising the step of determining K _on and K _off of the test compound for the target molecule. 18. A multiplex method for quantitatively assessing the binding affinity of a test compound to a target molecule, comprising the steps of: (a) obtaining at least three target molecules from the crude extract as shown in the scheme below Target molecule Adenosine A1 receptor Muscarinic acetylcholine receptor Serotonin 5-HT _2A Alpha-1A-adrenergic receptor Alpha-2A adrenergic receptor Dopamine receptor D1 Transporter 5HT 5HT1a receptor 5HT2a receptor Ca channel Cave PCP receptor Opioid receptor (b) incubating said target molecules in a plurality of mixtures of ligands and test compounds, wherein said target molecules bind to different ligands, (c) removal of unbound ligands from mixtures; (d) isolating the ligands bound to said target molecules after incubation; (e) determining the amount of each ligand present on said target molecules by measuring the ligands obtained in step (d) at specified times in the reaction mixture using mass spectrometry and a calibration curve obtained using known concentrations of the ligand; (f) calculating the affinity of the test compound for the target molecule from the data obtained in step (e); and (g) calculating K _on and K _off of said test compound for said target molecule using the data obtained in step (e). 19. The method of claim 18, wherein the same test compound is used with each target molecule. 20. The method according to claim 18, comprising the use of the following target molecules and ligands: Target molecule Ligand Adenosine A1 receptor CPX Muscarinic acetylcholine receptor M1 pirenzepine Serotonin 5-HT _2A EMD281014 Alpha-1A-adrenergic receptor Prazosin Alpha-2A adrenergic receptor RX82102 Dopamine receptor D1 SCH23390 Transporter 5HT paroxetine 5HT1a receptor 8-OH-DPAT 5HT2a receptor EMD281014 Ca channel Cave D600 PCP receptor MK801 Opioid receptor naloxone 21. A multiplex method for determining the K _on and/or K _off values of a test compound for a target molecule, comprising the steps of: (a) obtaining a mixture of target molecules from a crude extract from at least one of (i) healthy or unhealthy human or non-human tissue, and (ii) a synthetic protein preparation, wherein said target molecules are present in a mixture of target molecules that do not naturally exist in the same tissue preparation; (b) incubating said target molecules in a plurality of mixtures of ligands and test compounds, wherein said target molecules bind to different ligands; (c) removal of unbound ligands from mixtures of target molecules; (d) isolating ligands bound to said target molecules; (e) determining the amount of said ligand bound by the target molecule by measuring the ligands obtained in step (d) at specified time points in the reaction mixture using mass spectrometry and a calibration curve; and (f) calculating K _on or K _off of the test compound for the target molecule using the data obtained in step (e). 22. The method according to claim 21, wherein K _on and K _off are determined in membrane mixtures containing different membranes ex vivo , with at least two of the membrane preparations: cortex membrane preparations, cerebellar membrane preparations, ventricular membrane preparations and liver membrane preparations. 23. The method of claim 22, wherein said membrane mixtures comprise at least two of the receptors A1, A2A, A3, M1, M2, Alpha 1ns, Alpha 2ns, D1, D2S, 5HT1a, 5HT2a, 5HT-trans, Cave, PCP, opioid ns, AT2, B2, CB1, CCK1, H4, and CysLT1. 24. The method according to claim 23, wherein the membrane mixtures contain all of the listed receptors. 25. The method according to any one of paragraphs 21-24, wherein K _off is determined by means of a displacement method. 26. The method according to any one of paragraphs 21-25, wherein K _off is determined by means of a dilution method.