# Patent

## Title
Methods of screening for Janus kinase 3 interacting compounds

## Abstract

The present invention provides a method for screening compounds for their ability to inhibit autophosphorylation of Janus kinase 3 in the absence of any additional substrate. The present invention also provides a method for screening compounds that bind to Janus kinase 3 domains other than the kinase domain, to identify synthetic or natural compounds including biomolecules, that modulate Janus kinase 3 activity. This invention also describes a multi-component screening kit composed of purified recombinant Janus kinase 3 proteins and recombinant phosphorylated Janus kinase 3 fusion proteins including, one or more phosphorylated or non-phosphorylated domain-deleted Janus kinase 3 fusion proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part under 35 U.S.C. §120 of pending non-provisional application U.S. Ser. No. 14/483,622, filed Sep. 11, 2014, which claims benefit of priority under 35 U.S.C. §119(e) of provisional application U.S. Ser. No. 61/960,652, filed Sep. 23, 2013, now abandoned, the entirety of both of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is in the field of pre-clinical therapeutics. More specifically, the present invention is directed to a method of screening synthetic and natural compounds for Janus Kinase 3 (Jak3) modulating activity.
Description of the Related Art
Janus Kinases (Jaks) are a family of non-receptor tyrosine kinase with four members; Jak1, Jak2, Jak3, and Tyk2, that transduce cytokine-mediated signals through interactions with the common γ chain of the immune receptors for IL-2, 1L-5, IL-7, IL-9, and IL-15 (1). Several studies have shown that deficiency or over-activation of Jak3 in particular, results in a multitude of disorders including, autoimmunity (2), arthritis (3) allergy (4,5), cancer (6-8), diabetes (9,10), immune deficiencies (11-13), transplant rejection (14-17) and neuromuscular diseases (18). Therefore, synthetic compounds and biomolecules that modulate Jak3 functions, without compromising normal immune functions are valuable in the treatment of these diseases.
While widely expressed in different organs in both humans and mice, Jak3 shows significant expression in the cells of the epithelial and hematologic lineages (1,19-20). In agreement with this distribution, abnormal Jak3 activation is associated with human hematologic and epithelial malignancies (21, 22).
Pharmacological inhibitors of Jak3 have shown utility in autoimmune disorders, acute lymphoblastic leukemia, Type 1 diabetes, rheumatoid arthritis, allergy and asthma. While, many of these have demonstrated promise in clinical trials, there is a need to develop sensitive, selective and reproducible tools for identifying synthetic and natural compounds that modulate Jak3 function.
The N-terminal region of Jak3 (about 550 amino acids) is known to interact not only with the cytoplasmic tail of the cytokine receptors discussed above, but also to cytoskeletal and adapter proteins. This region further contains a four-point-one ezrin/radixin/moesin (FERM) domain (23) that is implicated in cell-cell communication and cell adhesion (24), important contributors to cell motility. Thus, targeting the FERM domain is expected to inhibit kinase activity and in addition, disrupt Jak3/cytokine receptor engagement. The present invention offers a high-throughput screening method to rapidly identify compounds in tandem, for their ability to interact with the FERM domain and/or the kinase domain, with desired end use as therapeutics in the management of Jak3-associated diseases.
Currently available methods for identifying Jak3 regulators employ the kinase domain of Jak3 in two popular formats viz; (a) Caliper format and (b) Perkin Elmer format. Caliper's mobility shift assay (Caliper Life Science, Hopkinton, Mass.) uses a nanofluid-based technology, which involves electrophoretic separation of fluorescently labeled phosphorylated and non-phosphorylated substrates on a microchip, followed by fluorimetric quantification. Additionally, the reactions are run on a chip or in micro-plate wells with the microchip being used solely to separate the substrate from the phosphorylated product. The main limitation of this technology is use of external peptide substrate and the need to screen an efficient substrate that can fit into this technology. Screening of these substrates introduces yet another layer of complexity, in addition to being expensive on account of instrument and manpower costs.
Perkin Elmer's (Waltham, Mass.) Streptavidin-coated Flashplate radiometric assay differs from Caliper's assay in the use of a radiolabeled external substrate. This method uses one of three ways to assess phosphorylation: (1) measuring ATP depletion (easy-Lite-Kinase), (2) direct measurement of phosphate incorporation in the substrate using 33P-labeled ATP (FLASHPLATE), or (3) capture and measurement of phosphorylated substrate (ALPHASCREEN, Lance, DELFIA).
Another technology marketed by Dynamii Pharmaceticals (DynamixFit) also uses the kinase-only domain of Jak3, but rather than using the classical “lock and key” enzyme model, it utilizes an “induced fit” model, which is based on the concept that, enzymes being flexible proteins, have 3D active-site structures that are continuously reshaped by interactions with their substrates (or inhibitors).
Overall, there is a deficiency in the art for optimal screening tools, due to limitations imposed by use of kinase-only domains and external substrates, that is further complicated due to sensitivity, need for radioactive detection, absence of a user-friendly interface and poor cost-effectiveness. Thus, there is a recognized need for improved methods to screen Jak3 regulators. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention is directed to a method of screening synthetic and natural compounds that inhibit Jak3 autophosphorylation, in the absence of an external substrate.
This invention is also directed to a method of screening synthetic and natural compounds that bind Jak3 or its truncated, domain-deleted mutants.
This invention also describes development of a screening kit comprising a recombinant Jak3 protein, to identify compounds that inhibit Jak3 autophosphorylation.
This invention further describes development of a screening kit comprising, a plurality of recombinant, Jak3 proteins and Jak3 fusion proteins including, truncated Jak3 fusion proteins. This kit further comprises instructions to identify compounds that inhibit Jak3 autophosphorylation, and to identify compounds that bind full-length Jak3 and/or truncated Jak3.
Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E show the cDNA sequences for WT and domain deleted mutants of Jak3, which were cloned (26) in a pGEX-4T vector. FIG. 1A shows the cDNA sequence encoding the WT-Jak3 (full length) (SEQ ID NO: 1). FIG. 1B shows the cDNA sequence encoding the GST-WT-Jak3 fusion protein (SEQ ID NO: 2). FIG. 1C shows the cDNA sequence encoding the GST-Jak3 fusion protein lacking the JH1 domain (SEQ ID NO: 3). FIG. 1D shows the cDNA sequence encoding GST-Jak3 fusion protein lacking the JH1 and JH2 domains (SEQ ID NO: 4). FIG. 1E shows the cDNA sequence encoding the GST-Jak3 fusion protein lacking the JH1, JH2, JH3, and JH4 domains (SEQ ID NO: 5).
FIGS. 2A-2E show the schematic representation and the amino acid sequences for WT and domain-deleted mutants of Jak3, expressed in a E. coli BL21 and where indicated, their phosphorylated forms expressed in a E. coli TKX1 protein expression system. FIG. 2A shows the schematic representation and the amino acid sequence of the WT-Jak3 (full length) recombinant protein (SEQ ID NO: 6). FIG. 2B shows the schematic representation and the amino acid sequence of the non-phosphorylated GST-WT-Jak3 fusion protein (SEQ ID NO: 7) and phosphorylated GST-WT-Jak3 fusion protein (SEQ ID NO: 11) Underlined amino acid sequences 603-703 and 1202-1251 correspond to the regions in the SH2 and JH1 domain sequences in which phosphorylation occurs, respectively. FIG. 2C shows the schematic representation and the amino acid sequence of the GST-Jak3 fusion protein lacking the JH1 domain (SEQ ID NO: 8) and phosphorylated GST-Jak3 fusion protein lacking the JH1 domain (SEQ ID NO: 12). FIG. 2D shows the schematic representation and the amino acid sequence of the GST-truncated Jak3 fusion protein lacking the JH1 and JH2 domains (SEQ ID NO: 9) and phosphorylated GST-truncated Jak3 fusion protein lacking the JH1 and JH2 domains (SEQ ID NO: 13). Underlined amino acids 603-703 corresponds to the SH2 domain sequence in which phosphorylation occurs in FIGS. 2D-2E. FIG. 2E shows the schematic representation and the amino acid sequence of the GST-truncated Jak3 fusion protein lacking the JH1, JH2, JH3 and JH4 domains (SEQ ID NO: 10).
FIGS. 3A-3B show expression and purification of recombinant Jak3 proteins and recombinant Glutathione-S-transferase (GST)-Jak3 fusion proteins. FIG. 3A Immunoblotting (IB) analysis of purified recombinant proteins expressed in BL21 and TKX1 cells. A phospho-tyrosine specific antibody (pY20) was used to visualize phosphorylated Jak3 and a Jak3 antibody was used to visualize both non-phosphorylated and phosphorylated Jak3. These data demonstrate that only the TKX1 expressed protein is phosphorylated. FIG. 3B shows western blot analyses of the purified recombinant fusion proteins expressed in bacteria. Anti-GST antibody was used as the primary antibody and HRP-conjugated secondary antibody was used to visualize the fusion proteins. Arrows indicate recombinant proteins.
FIG. 4 shows time-dependent changes in tyrosine autophosphorylation of recombinant wild-type Jak3 in the presence or absence (control) of ATP using a 96-well ELISA multiplate coated with GST-Jak3 and the phospho-tyrosine specific antibody, pY20 as the primary binding partner. Measurements were made for a times ranging from 0 to 200 minutes.
FIG. 5 shows inhibition of GST-WT-Jak3 autophosphorylation in the presence or absence of different concentrations of the Jak3 inhibitor, CP-690505 ranging from 0 nM to 500 nM. pY20 antibody was used as the primary binding partner.
FIGS. 6A-6B shows binding profile for Jak3-Villin interactions by multiplate ELISA assay and the calculation of binding parameters. FIG. 6A shows binding of Jak3 to the cytoskeletal protein, Villin, measured in 96-well ELISA multiplates pre-coated with phospho-Villin (P-Villin), and using increasing concentrations of GST-P-Jak3 fusion protein ranging from 0 μM to 600 μM. GST is the reporter protein and the primary binding partner is a GST antibody. FIG. 6B represents the Hill equation plot of the data from FIG. 6A, and shows the relation between the log Y/(1−Y) and log (Jak3), where Y is the fractional saturation of absorbance.
FIGS. 7A-7B shows binding profile for Jak3-p52ShcA interactions by multiplate ELISA assay and the calculation of binding parameters. FIG. 7A shows binding of Jak3 with the adapter protein, p52ShcA, measured in 96-well ELISA multiplate pre-coated with phospho-p52ShcA, and using increasing concentrations of GST-P-Jak3 fusion protein, ranging from 0 μM to 600 μM. GST is the reporter protein and the primary binding partner is a GST antibody. FIG. 7B shows a Hill equation plot of the data from FIG. 7A, and shows the relation between the log Y/(1−Y) and log (Jak3), where Y is the fractional saturation of absorbance.
FIGS. 8A-8B shows binding of recombinant truncated-Jak3 proteins with Villin by multiplate ELISA assay and the calculation of binding parameters. FIG. 8A shows direct interactions between Villin and, recombinant, GST-Jak3 or its indicated truncated-Jak3 mutants, or GST-P-Jak3 or its truncated mutants. Absorbance values for wells containing P-Villin contacted with GST alone were used as control, and data presented on the ordinate axis represent absorbance values after subtraction of control values. FIG. 8B shows binding of GST-Jak3G257 to the cytoskeletal protein, Villin, measured in 96-well ELISA multiplates pre-coated with phospho-Villin (P-Villin), and using increasing concentrations of GST-Jak3-G257 ranging from 0 μM to 20 μM.
FIG. 9 shows a working model for Jak3 binding with Villin. Tyrosine phosphorylation of the SH2 domain of Jak3 disrupts the interactions between the Jak3-FERM domain and Jak3-SH2 domain, making the F3 subdomain of the FERM domain available to interact with phosphorylated Villin.



DETAILED DESCRIPTION OF THE PRESENT INVENTION
For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected herein. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.
The articles “a” and “an” when used in conjunction with the term “comprising” in the claims and/or the specification, may refer to “one”, but it is also consistent with the meaning of one or more“, at least one”, and “one or more than one”. Some embodiments of the invention may consist of or consist essentially of one or more elements, components, method steps, and/or methods of the invention. It is contemplated that any composition, component or method described herein can be implemented with respect to any other composition, component or method described herein.
The term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included.
The term “including” is used herein to mean “including, but not limited to”. “Including” and “including but not limited to” are used interchangeably.
Provided in the present invention are screening tools to identify not only Jak3 inhibiting compounds but also compounds that interact with Jak3 without interfering with Jak3 activation. Such interacting compounds have implications in treatment of diseases where normal kinase functions of Jak3 are desired, but the disease causing functions of Jak3 are undesirable. For example, biomolecules contribution to cell motility, such as the cytoskeletal proteins Villin and gelsolin, were determined, using this invention, to interact with Jak3 domains other than the kinase domain. Similarly, this invention was used to identify interactions between adapter protein, p52ShcA and Jak3, such interactions positively contributing to cell migration. Screening for compounds that interact with Jak3 therefore finds utility in cancer treatment, since these compounds may be employed to interfere with cancer cell migration, which is central to metastatic spread of the disease, the primary cause of fatalities in cancer. Similarly, screening for Jak3 interacting compounds also finds utility in preventing spurious immune cell migration in allergy and autoimmune disease,
The present invention is directed to a method for screening for a compound that inhibits Jak3 protein, comprising the steps of; selecting a potential inhibitory compound; contacting, a phosphate donor with a recombinant Jak3 protein and the potential inhibitory compound in a first sample; contacting said phosphate donor with the recombinant Jak3 protein in a second control sample; and measuring a level of autophosphorylation of said Jak3 protein in the first sample and in the second sample; wherein a reduction in the level of Jak3 autophosphorylation in the first sample compared to the level in the second control sample indicates that the compound inhibits Jak3 protein.
Potential inhibitory compounds that may be selected for screening include, synthetic compounds, their derivatives and analogs; chemical libraries; synthetic peptides, natural peptides, their derivatives and analogs; peptide libraries; proteins, modified proteins, antibodies or their fragments thereof; synthetic and natural lipids including, fatty acids, phospholipids, sterols, their derivatives and analogs; synthetic and natural sugars, including, monosaccharides, disaccharides, their derivatives and analogs.
In one embodiment, the method of screening compounds that inhibit Jak3 autophosphorylation encompasses use of a recombinant full length Jak3 protein. This Jak3 protein may be produced in a bacterial, yeast, insect or mammalian host cell that is transduced with the appropriate vector comprising the cDNA sequence of said Jak3 protein, using methods well known in the art.
In a second embodiment, the method of screening compounds that inhibit Jak3 autophosphorylation comprises use of a recombinant full length Jak3-fusion protein that is attached at the N-terminus to a reporter molecule.
In a preferred embodiment, the reporter molecule attached to the N-terminus of the Jak3 proteins discussed supra may be a peptide such a poly(histidine)x-tag, where x=6 or 10 histidine molecules, attached either directly to the N-terminus sequence of said Jak3, or attached via a linker amino acid sequence, to the N-terminus of Jak3. Methods for attaching the poly(histidine) tag nucleotide sequence, with or without the linker amino acid nucleotide sequence, upstream to the nucleotide sequence of said Jak3 protein(s), and methods to package these sequences in vectors so as to express fusion proteins in high yield, are well known in the art (25).
Alternatively, the reporter molecule may be a FLAG-tag, 3xFLAG-tag, a human influenza hemagglutinin (HA)-tag or a myc-tag, all of which are also well known in the art and may be attached upstream to the Jak3 sequence as discussed supra. In a preferred embodiment, the reporter molecule is glutathione-S-transferase (GST).
The method of screening for compounds inhibiting Jak3 autophosphorylation encompasses use of a high-energy phosphate donor. Such phosphate donors include, nucleotide triphosphates, nucleotide diphosphates, nucleotide monophosphates, their derivatives and analogs. In a preferred embodiment, the phosphate donor is a nucleotide triphosphate, wherein the nucleoside is adenosine or guanosine, and in a more preferred embodiment, the phosphate donor is adenosine triphosphate. In other embodiments, the phosphate donor may be generated in situ using a high-energy phosphate regenerating system such as an ATP-regenerating system, which is well known in the art.
The method further encompasses measuring the level of Jak3 autophosphorylation in the test sample. In one embodiment, this includes use of primary binding partners such as, peptides, or, antibodies or their fragments thereof, which selectively bind to the phosphotyrosine moiety, or bind selectively to phosphorylated Jak3 but not to non-phosphorylated Jak3. In a more preferred embodiment, such a binding partner would be an immunologic binding partner including, a monoclonal antibody having specificity to phosphorylated-Jak3.
The measurement step further encompasses methods and reagents for quantitating binding of the primary binding partner to autophosphorylated Jak3. These include, radiometric, preferably, fluorimetric and more preferably, colorimetric methods.
In one embodiment of the quantitation method, a radioisotope is conjugated directly to the primary binding partner. Alternately, the radioisotope is conjugated to a secondary molecule such as a secondary antibody, which binds specifically to the primary binding partner. Examples of radioisotopes that may be used for this purpose include, gamma emitters like 125Iodine, beta emitters like 14Carbon and alpha emitters like 3H (tritium).
In a preferred embodiment of the quantitating step, a fluorophore is conjugated directly to the primary binding partner, or to a secondary molecule such as a secondary antibody, which binds specifically to the primary binding partner.
Alternatively, quantitation by use of the fluorimetric method is by a proximity assay such as, Förster resonance energy transfer (FRET), in which the primary and the secondary binding partners are each conjugated with a donor or acceptor fluorophore that constitute a resonance energy transfer system. Examples of such fluorophore pairs known in the art include, FITC/Rhodamine, Alexa488/Rhodamine and Cy3/Cy5. In yet another modification of the FRET proximity assay, the donor/acceptor pairs are selected such that binding of the secondary binding partner to the primary binding partner results in a reduction in fluorescence quantum yields due to quenching. Examples of such reagents include the fluorophore/quencher pairs; Alexa488/Dabcyl and Cy3/DDQ II.
A more preferred embodiment of the method of quantitation is the colorimetric assay, which uses an enzyme-linked antibody to drive a colorimetric reaction. Such enzymes are known in the art to include, horseradish peroxidase and alkaline phosphate that catalyze the release of colored dyes, concentration of the dye directly correlating with the level of phosphorylated Jak3.
In all of the above instances, data analysis includes comparison of the test sample with control samples. Examples of control samples include, contacting the recombinant Jak3 protein with the phosphate donor in the absence of the potential inhibitory compound (positive control sample) and contacting the potential inhibitory compound with the phosphate donor in the absence of recombinant Jak3 (negative control sample).
This invention is also directed to a method for screening for a compound that binds Jak3 protein comprising the steps of selecting a potential binding compound; contacting a recombinant Jak3 fusion protein comprising a reporter protein with the potential binding compound in a first sample; contacting said reporter protein with the potential binding compound in a second control sample; and, measuring a level of binding in the first sample and in the second control sample; wherein an increase in the level of binding of the potential binding compound in the first sample compared to the level of binding in the second control sample indicates that the compound binds Jak3 protein.
Potential binding compounds that may be selected for screening include, synthetic compounds, their derivatives and analogs; chemical libraries; synthetic peptides, natural peptides, their derivatives and analogs; peptide libraries; proteins, modified proteins, antibodies or their fragments thereof; synthetic and natural lipids including, fatty acids, phospholipids, sterols, their derivatives and analogs; synthetic and natural sugars, including, monosaccharides, disaccharides, their derivatives and analogs.
In one embodiment, the method of screening compounds that bind Jak3 encompasses use of a recombinant full length Jak3 protein. This Jak3 protein may be produced in a bacterial, yeast, insect or mammalian host cell that is transduced with the appropriate vector comprising the cDNA sequence of said Jak3 protein, using methods well known in the art.
The method of screening also encompasses use of recombinant Jak3 protein(s) truncated to contain only one of the domains, JH1 (C-terminus) to JH7 (N-terminus), or sequentially truncated to exclude domains JH1 to JH6 (truncation from the C-terminus) or JH7 to JH2 (truncation from the N-terminus). Such truncated mutant Jak3 proteins may be produced in a bacterial, yeast, insect or mammalian host cell that is transduced with the appropriate vector comprising the cDNA sequence of said truncated Jak3 protein(s), using methods well known in the art.
In a second embodiment, the method of screening compounds that bind Jak3 comprises use of a recombinant full length Jak3-fusion protein that is attached at the N-terminus to a reporter molecule. The method also comprises use of recombinant truncated-Jak3 fusion protein(s) that is attached at the N-terminus to a reporter molecule. In this case, the Jak3 is truncated to contain only one of the domains, JH1 (C-terminus) to JH7 (N-terminus), or truncated to exclude one or a multiplicity of domains JH1 to JH6 (truncation from the C-terminus) or JH7 to JH2 (truncation from the N-terminus). Both the recombinant full length Jak3-fusion protein and the truncated mutant Jak3-fusion proteins may be produced in a bacterial, yeast, insect or mammalian host cell that is transduced with the appropriate vector comprising the cDNA sequence of said truncated Jak3 protein(s), using methods well known in the art.
In a third embodiment, the method of screening compounds that bind Jak3 comprises use of a recombinant phosphorylated full length Jak3-fusion protein that is attached at the N-terminus to a reporter molecule. The method also comprises use of a recombinant phosphorylated truncated-Jak3 fusion protein(s) that is attached at the N-terminus to a reporter molecule. In this case, the Jak3 is truncated to contain only one of the domains, JH1 (C-terminus) to JH7 (N-terminus), or truncated to exclude one or a multiplicity of domains JH1 to JH6 (truncation from the C-terminus) or JH7 to JH2 (truncation from the N-terminus). Both the recombinant phosphorylated Jak3-fusion protein and the phosphorylated truncated mutant Jak3-fusion proteins may be produced in a bacterial, yeast, insect or mammalian expression system and preferably in a TKX1 bacterial expression system. Such expression hosts will be transduced with the appropriate vector comprising the cDNA sequence of said truncated Jak3 protein(s), using methods well known in the art.
In a preferred embodiment, the reporter molecule attached to the N-terminus of the Jak3 proteins discussed supra may be a peptide such a poly(histidine)x-tag, where x=6 or 10 histidine molecules, attached either directly to the N-terminus sequence of said Jak3, or attached via a linker amino acid sequence, to the N-terminus of Jak3. Methods for attaching the poly(histidine) tag nucleotide sequence, with or without the linker amino acid nucleotide sequence, upstream to the nucleotide sequence of said Jak3 protein(s), and methods to package these sequences in vectors, so as to express fusion proteins in high yield, are well known in the art (25). Alternatively, the reporter molecule may be a FLAG-tag, 3xFLAG-tag, a human influenza hemagglutinin (HA)-tag or a myc-tag, all of which are also well known in the art and may be attached upstream to the Jak3 nucleotide sequence as discussed supra.
In a more preferred embodiment, the reporter molecule may be a bioluminescent protein, including Renilla luciferase, Nano-lantern, Yellow fluorescent protein or, a florescent protein including, green fluorescent protein, red fluorescent protein, cyan fluorescent protein or yellow fluorescent protein. In a most preferred embodiment, the reporter molecule is glutathione-S-transferase (GST).
The method of screening for compounds that bind Jak3 or its truncated mutants or phosphorylated Jak3 or phoshorylated truncated Jak3 discussed supra also encompasses measuring the level of Jak3 binding in the test sample. In one embodiment, this includes, measurement of fluorescence quantum yields using a fluorescent protein reporter. Such reporters may include, green fluorescent protein, red fluorescent protein, cyan fluorescent protein or yellow fluorescent protein. In a second embodiment, measurement is by luminescence using a bioluminescent protein reporter. Such reporters include, Renilla luciferase, Nano-lantern and Yellow fluorescent protein among others. In a third, preferred embodiment, primary binding partners including an immunologic binding partner may be used. These include, antibodies or their fragments thereof, which selectively bind to the reporter molecule. Examples of such reporter molecules include, poly(histidine)-tag, HA-tag, FLAG-tag, myc-tag and GST-tag.
In a preferred embodiment, the immunologic binding partner is a polyclonal antibody, and in a more preferred embodiment, the immunologic binding partner is a monoclonal antibody. In a most preferred embodiment the reporter is GST and the primary binding partner is a monoclonal GST antibody.
For the methods not employing fluorescent or luminescent reporters, the measurement step further encompasses methods and reagents for quantitating binding of said primary binding partner to the reporter-Jak3 or the reporter-truncated Jak3 fusion proteins. These include, preferably, radiometric, more preferably, fluorimetric and most preferably, colorimetric methods.
In one embodiment of the quantitation method, a radioisotope is conjugated directly to the said primary binding partner. Alternately, the radioisotope is conjugated to a secondary molecule such as a secondary antibody, which binds specifically to the primary binding partner. Examples of radioisotopes that may be used for this purpose include, gamma emitters like 125Iodine, beta emitters like 14Carbon and alpha emitters like 3H (tritium).
In a more preferred embodiment of the quantitating step, a fluorophore is conjugated directly to the said, primary binding partner, or to a secondary molecule such as a secondary antibody, which binds specifically to the primary binding partner.
Alternatively, quantitation by use of the fluorimetric method is by a proximity assay such as, Förster resonance energy transfer (FRET), in which the primary and the secondary binding partners are each conjugated with a donor or acceptor fluorophore that constitute a resonance energy transfer system. Examples of such fluorophore pairs known in the art include, FITC/Rhodamine, Alexa488/Rhodamine and Cy3/Cy5. In yet another modification of the FRET proximity assay, the donor/acceptor pairs are selected such that binding of the immunologic binding partner to the reporter-Jak3 or the reporter-truncated Jak3 fusion protein, results in a reduction in fluorescence quantum yields due to quenching. Examples of such reagents include the fluorophore/quencher; Alexa488/Dabcyl and Cy3/DDQ II.
A most preferred embodiment of the method of quantitation is the colorimetric assay, which uses an enzyme-linked antibody to drive a colorimetric reaction. Such enzymes are known in the art to include, horseradish peroxidase and alkaline phosphate that catalyze the release of colored dyes, concentration of the dye directly correlating with the level of bound Jak3 or bound truncated Jak3.
In all of the above instances, data analysis includes comparison of the test sample with control samples. Examples of control samples include, contacting the reporter-Jak3 or the reporter-truncated Jak3 fusion protein with the cytoskeletal protein Villin (positive control sample) whereas the negative control sample may, for example, when doing protein-protein interactions, BSA alone or GST alone.
This invention is also directed to a screening kit comprising a recombinant Jak3 protein and instructions for using this to screen for a compound that inhibits Jak3 autophosphorylation. In a preferred embodiment, the recombinant Jak3 protein has a sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7. The instructions provide methods to perform the Jak3 autophosphorylation assay in the presence of the potential inhibitory compound, and these are discussed supra and further illustrated by means of Examples provided below. All of the reagents including, primary binding partners, secondary binding partners, colorimetric dyes and buffers are known in the art and availably commercially to one of skill in this art.
This invention is further directed to a screening kit comprising a multiplicity of recombinant Jak3 proteins and instructions to, screen for a compound that inhibits Jak3 autophosphorylation and screen for a compound or biomolecule that binds Jak3 or truncated Jak3 or phosphorylated Jak3 or phoshorylated truncated Jak3. In a preferred embodiment, the recombinant Jak3 protein has a sequence shown in SEQ ID NO: 6, recombinant reporter-Jak3 fusion protein has a sequence shown in SEQ ID NO: 7, recombinant reporter-truncated-Jak3 fusion proteins discussed supra have sequences shown in SEQ ID NO: 8-10, the recombinant phosphorylated reporter-Jak3 fusion protein has a sequence shown in SEQ ID NO: 11, the recombinant phosphorylated reporter-truncated-Jak3 fusion proteins discussed supra have sequences shown in SEQ ID NO: 12-13, The instructions provide methods to perform, the Jak3 autophosphorylation assay in the presence of the potential inhibitory compound, and the non-phosphorylated and phosphorylated Jak3 or truncated-Jak3 binding assay in the presence of the potential binding compound, and these are discussed supra and further illustrated by means of Examples provided below. All of the reagents including, primary binding partners, secondary binding partners, colorimetric dyes and buffers are known in the art and availably commercially to one of skill in this art.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
EXAMPLE 1
Materials
ELISA multiplates, horseradish peroxidase (HRP)-conjugated secondary antibody, and 3,3,5,5-tetramethylbenzidine substrate kit were purchased from Thermo Scientific (Waltham, Mass.). Adenosine triphosphate, β-D-1-thiogalactopyranoside, indoleacrylic acid and buffers were purchased from Sigma-Aldrich (S. Louis Mo.), pY20 monoclonal antibody to phosphorylated tyrosine was purchased from MP Biomedical (Santa Ana, Calif.), pGEX-4T vector was purchased from GE Healthcare. pGEX2T was purchased from Addgene. BL21 and TKX1 competent cells were from Agilent Technologies (Santa Clara Calif.).
EXAMPLE 2
Expression and Purification of Recombinant Proteins and Recombinant Phosphorylated Proteins
GST-tagged wild type and truncated, mutant Jak3 cDNAs having SEQ ID NO: 1 (FIG. 1A), SEQ ID NO: 2 (FIG. 1B), SEQ ID NO: 3 (FIG. 1C), SEQ ID NO: 4 (FIG. 1D) and SEQ ID NO: 5 (FIG. 1E) were cloned (26) in a pGEX-4T vector. Proteins and fusion proteins having amino acid sequences having SEQ ID NO: 6 (FIG. 2A), SEQ ID NO: 7 (FIG. 2B), SEQ ID NO: 8 (FIG. 2C), SEQ ID NO: 9 (FIG. 2D) and SEQ ID NO: 10 (FIG. 2E) were expressed using an E. coli, BL21 protein expression system, by induction using Isopropyl β-D-1-thiogalactopyranoside. Recombinant phosphorylated Jak3 (P-Jak3) proteins having SEQ ID NO: 11 (FIG. 2B), SEQ ID NO: 12 (FIG. 2C), SEQ ID NO: 13 (FIG. 2D) were obtained by expression in a E. coli, TKX1 protein expression system, using a two-step induction method with β-D-1-thiogalactopyranoside and indoleacrylic acid as described (27). FIG. 3A shows by immunoblotting that while recombinant Jak3 protein is expressed in both BL21 and TKX1 cells, only the protein expressed in the later is phosphorylated.
Recombinant cytoskeletal protein, Villin was cloned in the prokaryotic expression vector pGEX2T, expressed using a BL21 expressing system and purified as reported before (28). Phosphorylated (P) Villin was generated by cloning and expression in a E. coli, TKX1 protein expression system, as described supra.
Recombinant adapter protein, p52ShcA was cloned in the pGEX2T vector and the protein expressed using an E. coli, BL21 protein expression system as described supra. The phosphorylated form of this protein, P-p52ShcA was generated by cloning and expression in a E. coli, TKX1 protein expression system, as described supra.
FIG. 3B shows immunoblotting (IB) analyses of the purified recombinant wild type and fusion proteins expressed in E. coli BL21, using GST antibody. Arrows indicate recombinant proteins.
EXAMPLE 3
Kinetics of Jak3 Autophosphorylation
To determine whether the non-phosphorylated form of Jak3 was functionally active, a phosphorylation assay was performed in the presence of a phosphate donor, to test the ability of Jak3 to phosphorylate itself. Jak3 was contacted with the phosphate donor, adenosine triphosphate (ATP) for different times ranging from 0 min to 200 minutes. The level of phosphorylated Jak3 (P-Jak3) was then measured by multiplate ELISA, using the phospho-tyrosine specific antibody, pY20, as the primary binding partner. FIG. 4 shows that Jak3 is rapidly autophosphorylated in a time dependent manner by the addition of ATP, with a half time maximum (t1/2) value of 135 s.
Screening of CP-690505 as a Potential Inhibitor of Jak3 Autophosphorylation
To test the utility of this invention to screen for inhibitors of Jak3 autophosphorylation, in the absence of additional substrates, recombinant non-phosphorylated Jak3 purified from BL21 cells were contacted with CP-690505, in the absence (control sample) or presence (test sample) of the phosphate donor, ATP. The level of P-Jak3 was determined by an ELISA-based multiplate assay, using the phospho-tyrosine specific pY20 antibody as primary binding partner, horse radish peroxidase conjugated antibody as the secondary binding partner and 3,3′,5,5′-tetramethylbenzidine as the colorimetric substrate. Raw absorbance data for the control samples were subtracted from the absorbance for the test samples to obtain the corrected absorbance values. FIG. 5 shows a CP-690505 concentration dependent decrease in the level of P-Jak3, with an IC50 value of 128 nM, in agreement with an established function for CP-690505 as an inhibitor of Jak3 phosphorylation (29).
EXAMPLE 4
P-Jak3 Binds to the Cytoskeletal Protein P-Villin
To determine the ability of Jak3 to interact with natural compounds including biomolecules, binding studies were performed in a multiplate using the cytoskeletal protein, Villin, known in the art to be a substrate for P-Jak3. Multiplates pre-coated with P-Villin were contacted with increasing concentrations of GST-P-Jak3 fusion protein (test samples) or GST alone (control sample). Binding levels were measured using GST antibody as the primary binding partner, horse radish peroxidase conjugated antibody as the secondary binding partner and 3,3′,5,5′-tetramethylbenzidine as the colorimetric substrate. Raw absorbance data for the control samples were subtracted from the absorbance for the test samples to obtain the corrected absorbance values. FIG. 6A shows an increase in the level of binding as measured by the corrected absorbance, as a function of P-Jak3 concentration ranging from 0-400 nM, with a dissociation constant (Kd) of 23 nM. FIG. 6B shows a rearranged Hill equation plot (30), which shows the relationship between log Y/(1−Y) and log (Jak3), where Y is the fractional saturation of absorbance. The Hill coefficient (h) is determined to be 3.7, suggestive of positive cooperative binding.
P-Jak3 Interacts to the Adapter Protein P-p52ShcA
To further establish the utility of this screening method, the ability of Jak3 to bind the phosphorylated adapter molecule, P-p52ShcA was determined by performing the multiplate assay as described supra with the exception that the P-Villin was replaced with P-p52ShcA in the pre-coating step. FIG. 7A shows an increase in the level of binding of P-Jak3 with P-p52ShcA, as a function of P-Jak3 concentration (0-600 nM). The dissociation constant (Kd) of this binding was found to be 220 μM, which is about 10 fold weaker compared to P-Jak3 binding with P-Villin (FIG. 6A). Analysis of these data using a rearranged Hill equation plot as described supra, revealed a Hill coefficient (h) value of 1.08 (FIG. 7B) that is suggestive of non-cooperative binding, which is also distinct from the data obtained using P-Villin as the binding compound.
The use of internal controls in the screening method of this invention is advantageous, as it eliminates ambiguity in interpreting raw absorbance values, allowing the end user to compare binding parameters for a plurality of compounds screened on different multiplates and in different multiplate formats. Moreover, the data discussed above underscore the advantages of using this screening method as a high-throughput analysis tool for scoring a plurality of potential binding compounds, based on their binding affinities and Hill parameters, allowing the end user to rapidly select compounds for any desired application including, preclinical, clinical and, research and development.
EXAMPLE 5
FERM Domain of Jak3 is Sufficient for Interactions Between Jak3 and Villin
To determine whether the kinase domains of Jak3 are critical for Villin binding, recombinant GST-truncated-Jak3 fusion proteins shown in FIGS. 2C-2E were expressed in BL21 (for non-phosphorylated proteins) or TKX1 (for phosphorylated proteins) cells, and purified. Multiplates pre-coated with P-Villin were contacted with these GST-tagged recombinant truncated-Jak3 or with GST-tagged recombinant WT-Jak3, and binding measured as described supra. FIG. 8A shows that, among the various proteins tested, Jak3-G257 (SEQ ID NO. 10), which contains the FERM domain alone, but not the inhibitory SH2 domain, bound P-Villin at approximately the same level as P-WT-Jak3. The dissociation constant (Kd) value for Jak3-G257 was found to be 40 nM (FIG. 8B) compared to a value of 23 nM for P-WT-Jak3 (FIG. 6A)
FIG. 8A also shows that the recombinant non-phosphorylated Jak3 proteins, Jak-V484 (SEQ ID NO. 9), Jak3-T788 (SEQ ID NO. 8) and WT-Jak3 (SEQ ID NO. 7) displayed relatively lower level of binding, though the level of binding is above the GST-alone controls (subtracted values are shown in FIG. 8A). P-Jak-V484 bound P-Villin at approximately the same level as P-WT-Jak3 and Jak3-G257 because the inhibitory property of SH2 domain being annulled due to phosphorylation (FIG. 9).
The following references are cited herein:

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The present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention. The terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee.

## Claims

1. A method for screening for a compound that inhibits Janus kinase 3 (Jak3) protein, comprising the steps of:
selecting a potential inhibitory compound;
contacting, a phosphate donor with a recombinant Jak3 protein and the potential inhibitory compound in a first sample;
contacting said phosphate donor with the recombinant Jak3 protein in a second control sample wherein the recombinant Jak3 protein has a sequence of SEQ ID NO. 6 or SEQ ID No. 7;
measuring a level of autophosphorylation of said Jak3 protein in the first sample and in the second sample; wherein a reduction in the level of Jak3 autophosphorylation in the first sample compared to the level in the second control sample indicates that the compound inhibits Jak3 protein.

2. The method of claim 1, wherein the recombinant Jak3 protein is a reporter-Jak3 fusion protein.

3. The method of claim 2, wherein the reporter protein is Glutathione-S-transferase (GST).

4. The method of claim 1, wherein the phosphate donor is adenosine triphosphate.

5. The method of claim 1, wherein the level of Jak3 phosphorylation is measured by ELISA.

6. The method of claim 5, wherein the primary antibody for ELISA is a monoclonal phosphorylated-Jak3 specific antibody.

7. The method of claim 1 wherein the compound is a biomolecule.

8. A method for screening for a compound that binds Jak3 protein comprising the steps of:
selecting a potential binding compound;
contacting a recombinant Jak3 fusion protein or a recombinant phosphorylated Jak3 fusion protein comprising a reporter protein with the potential binding compound in a first sample;
contacting said reporter protein with the potential binding compound in a second control sample wherein the recombinant Jak3 fusion protein has a sequence of SEQ ID NO. 6 or SEQ ID No. 7;
measuring a level of binding in the first sample and in the second control sample; wherein an increase in the level of binding of the potential binding compound in the first sample compared to the level of binding in the second control sample indicates that the compound binds Jak3 protein.

9. The method of claim 8, wherein the reporter protein is Glutathione-S-transferase (GST).

10. The method of claim 8, wherein the recombinant Jak3 fusion protein or the recombinant phosphorylated Jak3 fusion protein comprises a truncated Jak3 protein sequence.

11. The method of claim 10, wherein the recombinant truncated Jak3 fusion protein has a sequence shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

12. The method of claim 10, wherein the recombinant phosphorylated truncated-Jak3 fusion protein has a sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13.

13. The method of claim 8, wherein the level of binding is measured by ELISA.

14. The method of claim 13, wherein the primary antibody for ELISA is a monoclonal GST specific antibody.

15. The method of claim 8 wherein the compound is a biomolecule.