WO2004074836A2 - Methods for investigating the specificity of compounds that interact with receptors - Google Patents

Abstract

A method for investigating molecular interactions, comprises: (a) contacting molecules to be assessed with ligands each having a chemical or biochemical tag; (b) if desired, removing any non-interacting molecules and ligands; and (c) contacting the molecule-Iigand complexes formed in step (a) with a substance that interacts with the tag, thereby allowing the complexes to be purified; and (d) releasing the components of the complexes.

Description

METHODS FOR INVESTIGATING THE SPECIFICITY OF COMPOUNDS THAT INTERACT WITH RECEPTORS Field of the Invention

The invention relates to methods for defining the specificity of compounds that bind to receptors, including proteins occurring in cells and tissues, and using these methods for purification of the receptors and discovering compounds that bind to the receptors. Background of the Invention

Receptors are macromolecules, and macromolecular assemblies, that bind chemical agents. In biology, the binding between a receptor and certain chemical agents may lead to the transmission of a signal that alters biological activity. In pharmacology, the binding between a receptor and other chemical agents may enhance or inhibit the transmission of such biological signals. The definition of the interaction between specific chemical agents and specific receptors can be exploited in numerous ways. One of these applications may lead to the purification of either receptor or corresponding ligand. Another relates to the identification and redesign of chemical agents that bind to receptors, a process known as drug discovery. Summary of the Invention The present invention is based at least in part on one or more of the following principles:

1. Interaction of a receptor with a chemical agent may result in the establishment of an equilibrium between the "free" receptor and receptor which is "bound" to its corresponding ligand. 2. If either the receptor or ligand exhibits a property that can be identified and/or quantified, then the existence of a relationship between the ligand and its receptor can be established experimentally. 3. This relationship may represent a degree of complementarity between the ligand and its corresponding binding site, which in turn can lead to the identification of features of the binding site, and the ligands which bind to it, that may be useful in drug design and drug discovery. According to the present invention, a library of chemical agents is used, each bearing a feature that is recognisable using a range of biophysical techniques. This feature is chemically a part of the compounds, such as a chemical "tag". These recognizable chemical agents can then be used in screening, e.g. to identify ligand-interacting molecules from a large collection of molecules, most of which do not interact with the ligand. The invention also provides a way of purifying the ligand-receptor complex, using the recognizable features of the ligand, or introducing additional recognizable features in the receptor. Consequences of the invention are that specific ligands can be used to identify receptors, purify receptors, as diagnostics, as drugs, and in drug and biotherapeutic discovery. Brief Description of the Drawings

In the accompanying, illustrative drawings:

Figure 1 is a schematic representation of application of the invention to screening for receptor-interacting molecules in a "tagged" library, and its application to purification and site exploration within individual receptor molecules; and

Figure 2 schematically shows reactions occurring in the Example, below, in which streptavidin-alkaline phosphatase is captured by immobilised streptavidin.

Description of Preferred Embodiments The implementation and use of the invention can readily be understood and achieved by one of ordinary skill in the art, having appreciated the nature of the invention. Preferred aspects of the invention are defined in the claims.

By way of example, a suitable procedure comprises:

1. The design and synthesis of a set of compounds (a chemical "library") that contains a specific identifiable feature or "tag". An example of such a tag might be the chemical compound biotin, or the protein GST.

2. The addition of the tagged chemical library to a set of molecules within which are to be found specific receptors, and the establishment of receptor-ligand complexes. These molecules, to be assessed, may be immobilised (e.g. in the form of bead, plate, chip or slide). In the assay, target receptors, or beads or other products containing target receptors, pure or partically purified, or their inverse (non-target receptors for discriminatory assays), are contacted with the chemical library. 3. The identification and purification of the receptor-ligand complexes by adsorption on to a matrix that recognises the tag. In the case of biotin, this might be immobilised streptavidin.

4. The identification of the individual components of the isolated receptor ligand complexes by techniques such as SDS-PAGE and mass spectrometry.

5. The establishment of an assay to detect compounds that displace the tagged ligand from its corresponding receptor, measuring the effectiveness of the compounds by the release from the complex of the tagged ligand. In the case of biotinylated compounds, this would involve the detection and quantitation of the associated biotin.

In one embodiment of the invention, proteins obtained from cells, which may be recombinant, constitute the molecules to be addressed. Thus, for example, proteins are labelled, and information on the interaction between these labelled proteins and labelled chemical ligands can be deconvoluted for therapeutic discovery and ligand-screening purposes.

In a more specific example, human protein kinases can be purified from transfected mammalian cells. The proteins can then be immobilised on a column, and tagged chemical libraries passed over them. Bound ligands can then be eluted. The structures of the liberated chemical ligands can be determined by mass spectrometry, if required.

In another example, immobilised proteins can be complexed with tagged chemical ligands (e.g. biotinylated amines), and the tagged ligands within the receptor-ligand complex subsequently detected by radiolabelled or enzymatically labelled detection systems (e.g. radioactive streptavidin or alkaline phosphatase-conjugated streptavidin). Further chemical or biological ligands can also be used to displace the tagged ligands, thereby displacing the associated radioactivity or enzymatic activity. Residual immobilised radioactivity or enzymatic activity can be measured as an endpoint of the assay, or the displaced radioactivity or enzymatic activity can be measured directly.

Also, tagged chemical ligands can be used as "adaptors" for direct purification of complexed receptors. In this procedure, a mixture of proteins can be complexed with, say, biotinylated ligands (the "adaptors") and the complex captured by affinity chromatography on immobilised streptavidin. In another example, hexa-his-modified adaptors could be captured on nickel affinity chelate columns for complex purification. It will be evident that affinity ligands can be designed for specific purposes. By way of example, these include

1. High affinity probe / spacer / high affinity capture ligands, e.g. biotin- spacer-biotin; drug-spacer-biotin; useful for drug profiling

2. Low specificity probe / spacer / high affinity capture ligands, e.g. benzamidine / spacer / biotin; useful for protein family purification

3. High affinity / variable spacer / high affinity capture ligands, e.g. drug / variable spacer / biotin; useful to explore active sites and optimize purification processes

4. Probe / multivalent spacer / high affinity capture ligands, e.g. benzamidine / multi-antennary spacer / high affinity capture Ligand; useful amongst other applications, for efficient purification of several different recombinant proteins from over-expressing cultures in one step.

The invention has significant advantages over conventional purification procedures: 1. Purification can be achieved and assessed in a flexible fashion. A large series of ligands can be assembled, possibly as a combinatorial library, bound to a generic tether, which is capable of facile and cost- effective separation. Purification would not require the synthesis and use of multiple affinity adsorbents. Rather, a single, inexpensive adsorbent, produced cost-effectively in large quantities, could be used to review the entire series of ligands.. 2. Separation may be achieved after solution phase binding, which represents a more natural or physiological state compared with direct binding to immobilised ligands bound to solid matrices, which are more complex in their potential molecular interactions. For example, target protein binding to the matrix during the affinity capture step is avoided using initial solution phase binding. 3. Accessibility of active sites within target proteins can vary depending on their location within the protein. Affinity chromatography requires the use of spacer molecules to offset ligands from the adsorbent, normally present as a heterogeneous macromolecular structure. In the novel procedure, the spacers used to present a ligand to its corresponding target are homogeneous - each ligand bears an exact, chemically- defined spacer whose presentation to interacting molecules is both reproducible and homogeneous, devoid of the additional steric hindrance, produced by the adsorbent matrix, that is necessarily present in solid-phase supports.

As indicated in Fig. 1 , the interactions leading to binding of a ligand to a target molecule may be followed by elution and analysis of the bound ligands and/or receptors. These interactions can be developed into formats in which the interaction between tagged ligands and receptors can be used as an assay to discover drugs and biotherapeutics.

The following Examples illustrate the invention. The procedure is illustrated schematically in Fig. 2. Reagents and chemicals

Buffer reagents were molecular biology grade, purchased from Sigma- Aldrich (UK). In addition to these reagents, the following were also from Sigma-Aldrich: streptavidin-alkaline phosphatase conjugate (S2890 reconstituted in 0.5mls water before use); bovine thrombin (T9000) and p- nitrophenyl phosphate (N4645). Dynabeads (M280 streptavidin, 112.05) were obtained from Dynal Biotech Asa, Oslo, Norway. Electrophoresis gels and reagents were from Invitrogen Ltd, Paisley, Scotland.

N-(5-Aminopentyl)biotinamide, sulfo-succinimidyl-6-(biotinamido) hexanoate and sulfo-succinimidyl-6-(biotinamido)-6-hexanamido hexanoate were purchased from Pierce Biotechnology Inc. (USA). All other materials were purchased from Sigma-Aldrich. Synthesis of Compound A, i.e. N-(N-(N-Biotinamido-6-hexanamido)-6- hexanamido)-5-aminopentylbiotinamide

N-(5-Aminopentyl)biotinamide (4.9 mg, 14.93 mmol.) and sulfo- succinimidyl-6-(biotinamido)-6-hexanamido hexanoate (10 mg, 14.93 mmol.) were stirred in dry N,N-dimethylformamide (500 ml) for 18 hours at room temperature. The solvent was removed in vacuo and the residue washed with water (2 x 500 ml). The resulting solid was dried in vacuo to give the title compound 9.6 mg (96%) as a colourless solid. Compound A:

Synthesis of ompound , .e. -( -B ot nami o-6-hexanamido)-5- aminopentylbiotinamide

N-(5-Aminopentyl)biotinamide (5.9 mg, 17.97 mmol.) and sulfo- succinimidyl-6-(biotinamido) hexanoate (10 mg, 17.97 mmol.) were stirred in dry N,N-dimethylformamide (500 ml) for 18 hours at room temperature. The solvent was removed in vacuo and the residue washed with water (2 x 500 ml). The resulting solid was dried in vacuo to give the title compound 7.2 mg (72%) as a colourless solid. Compound B:

Preparation of protein-ligand conjugates

Ten micrograms of streptavidin-alkaline phosphatase conjugate were incubated with ten microlitres of DMSO (control) or compounds A or B dissolved in DMSO at 10 milligrams per ml. The total volume was made up to 250 microlitres with buffer (50mM Tris CI, 0.5M NaCI pH 7.4). After incubation at 4°C on a rotating mixer for one hour, the samples were applied to a column of G25 (HiTrap desalting, Amersham Biosciences, UK) equilibrated in 50mM tris CI, 0.5M NaCI pH 7.4 in order to remove unbound compound or DMSO. Fractions of approximately 20 microlitres were collected, and the peak of streptavidin-alkaline phosphatase conjugate identified by transferring 5 microlitre samples to 100 microlitres of p-nitrophenyl phosphate (one milligram per ml) in carbonate buffer pH 9.4. A yellow colour developed within less than one minute, and indicated the presence of the alkaline phosphatase. Peak fractions were pooled prior to adsorption to magnetic beads bearing streptavidin.

Adsorption of biotin-protein conjugates to streptavidin coupled to magnetic beads

Three hundred and eighty microlitres of protein-compound samples were mixed with 100 microlitres of streptavidin-labelled Dynal beads (prewashed with one millilitre of 50mM tris CI, 0.5M NaCI pH 7.4), and incubated at 4°C on a rotating mixer for thirty minutes. The beads were recovered on a magnetic tube rack (Dynal) and the unbound fraction collected. The beads were then washed three times with one millilitre of 50mM tris CI, 0.5M NaCI pH 7.4, and then split into two aliquots. One was washed once in carbonate buffer pH 9.4, then incubated with two hundred microlitres of p-nitrophenyl phosphate (one milligram per ml). After two minutes, the reaction was stopped with an equal volume of two molar sodium carbonate, and the OD405 read on a Spectramax Plus spectrophotometer (Molecular Devices Inc). The other aliquot of beads was incubated with fifty microlitres of

NuPage electrophoresis sample buffer at 90°C for five minutes prior to removal of the beads on the magnetic rack. Analysis of protein fractions by SDS-PAGE

Ten microlitres of each protein sample were incubated with electrophoresis sample buffer under reducing conditions at 70°C for ten minutes. The total sample was applied to a 12% NuPage BisTris gel (1.0 mm thick) and run at two hundred volts for one hour. Molecular weight standards (five microlitres Mark 12 standard, Invitrogen, catalogue number LC5677) were run in parallel. The gel was rinsed briefly in ultrapure water, and stained with silver using the SilverQuest kit from Invitrogen (catalogue number LC6070). The gel was then imaged on a flatbed scanner. Analysis of protein binding to streptavidin-coated Dynabeads

Streptavidin-agarose conjugate was incubated separately with compound A, compound B, or DMSO control to allow binding of the biotin- containing ligands to the protein in solution. After removal of excess compound by gel filtration on G25 columns, the protein samples were mixed with magnetic streptavidin beads, and the unbound material retained for analysis. After washing to remove all unbound material, the beads were divided into two aliquots. One was incubated with alkaline phosphatase substrate to reveal bound streptavidin-alkaline phosphatase conjugate.

The results are tabulated below:

It can be seen that both compounds A and B in solution enabled capture of the alkaline phosphatase-conjugated streptavidin, with insignificant non-specific capture in the control where the biotinylated ligand was absent.

These results were confirmed by eluting protein from the second aliquot of magnetic beads using strong denaturing conditions (SDS sample buffer). A 12% gel was run with unbound and SDS-eluted samples, and stained with silver.

Unbound protein profiles were similar in all three cases, but bands corresponding to the streptavidin-alkaline phosphatase conjugate were only present in the SDS-eluted fractions from biotin-ligand-treated samples. The higher intensity of bands in the compound B lane relative to compound A is consistent with the alkaline phosphatase assay data tabulated above.

Claims

1. A method for investigating molecular interactions, which comprises:

(a) contacting molecules to be assessed with ligands each having a chemical or biochemical tag; (b) if desired, removing any non-interacting molecules and ligands; and (c) contacting the molecule-Iigand complexes formed in step (a) with a substance that interacts with the tag, thereby allowing the complexes to be purified; and (d) releasing the components of the complexes.

2. A method according to claim 1, which comprises displacing ligands from the molecule-Iigand complexes.

3. A method according to claim 1 or claim 2, wherein the tag is capable of binding said substance.

4. A method according to claim 3, wherein said substance is a stationary phase.

5. A method according to any preceding claim, wherein each ligand is chemically bound to its tag via a soluble linker.

6. A method according to any preceding claim, which comprises releasing the ligands from the molecule-Iigand complex.

7. A method according to any preceding claim, wherein the molecules to be assessed are immobilised.

8. A method according to any preceding claim, wherein the molecules to be assessed are biological macromolecules such as proteins.

9. A method according to any preceding claim, which additionally comprises screening the released components.

10. A method according to any preceding claim, which comprises identifying and/or quantitating the screened ligands.

11. A method according to any preceding claim, which additionally comprises identifying receptors that interact with the ligands.

12. A method according to any preceding claim, for the identification of a therapeutic or diagnostic agent.