WO2005029039A2

WO2005029039A2 - Non-radioactive inositol phosphate assay for phospholipase c-coupled drug target screening

Info

Publication number: WO2005029039A2
Application number: PCT/US2004/030749
Authority: WO
Inventors: Paul O. Neilsen; Edson Assis; Angie M. Branch; Beth E. Drees
Original assignee: Echelon Biosciences Incorporated
Priority date: 2003-09-19
Filing date: 2004-09-20
Publication date: 2005-03-31
Also published as: WO2005029039A3

Abstract

A non-radioactive assay which is amenable to high throughput (HT) screening for antagonists of PLC- activated Gαq receptors and/or tyrosine kinase coupled receptors comprising the steps of measuring longer-lived Ins(1,4,5)P3 metabolites (IPn s), i.e., Ins(1,3,4,5)P4 and Ins(1,4)P2, as a means to report the PLC activity in cells or cell lysates.

Description

Non-radioactive Inositol Phosphate Assay for Phospholipase C-C'oupled Drug Target Screening

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to assays for drug discovery. More particularly, this invention relates to materials and methods for a cell-based, non- radioactive, high-throughput functional assay for receptors coupled naturally or artificially (directly or indirectly) to Phospholipase C.

Background of the Invention The importance of receptors that activate phospholipase C (PLC), including both G-Protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), in biological processes, and by extension, as drug targets is well established. The significance of these classes of receptors and their associated cell biology is evidenced by the current annual publication rate of over 2,000 papers in journals indexed by PubMed. Furthermore, it is estimated that 40-60 % of current drugs target GPCRs, with over $60 billion in USD in sales for this class of drugs in the year 2000. In addition to established drugs which target known RTKs and GPCRs, there remain hundreds of orphan receptors with unidentified ligands, some of which have already been validated as drug targets. One estimate states that GPCRs represent a full 15% and protein kinases 22% of the draggable genome respectively; and implies that antagonizing these classes of receptors will remain a primary focus of pharmaceutical and biotech research and development well into the future. Hopkins, A. L., and Groom, C. R., Nat Rev Drug Discov, 1, 727 (2002) As an example, GPCRs are next discussed in detail, but it is recognized that similar rationales exist for targeting RTKs and any signaling pathway that activates PLC enzymes. It is estimated that there are between 800 and 1 ,000 non-olfactory GPCR proteins '_*•- encoded by the human genome which can be classified into four subfamilies based on the G-proteins to which they couple and the corresponding second messenger system effected. See Table 1 and Nambi, P., and Aiyar, ASSAY and Drug Development Technologies, 7, 305 (2003) All members of the Gα_q subfamily activate the beta isoform of phosphoinositide-specific phospholipase C (PLC). Rhee, S. G., Annu Rev Biochem, 70, 281 (2001) PLC enzymes cleave a rare phospholipid (phosphatidylinositol(4,5) bisphosphate, aka PI(4,5)P₂) found on the inner leaflet of the plasma membrane to produce two second messengers, diacylglycerol and inositol( 1,4,5) trisphosphate(Ins(l,4,5)P₃), see FIG. 1. Diacylglycerol activates protein kinase C and Ins(l,4,5)P₃ binds to receptors on the endoplasmic reticulum that promote calcium release into the cytoplasm. Cytoplasmic calcium is a promiscuous second messenger with many effects on cells including transcription factor activation in the nucleus. Even though Gα_q seems to be the primary activator of PLC-β, there are at least two additional ways for GPCRs that don't normally couple to Gα_q to activate PLC-β: one via certain Gβγ subunils, and the other through chimeric and promiscuous Gα_15/ι₆ proteins. Park, D., et al., JBiol Chem, 268, 4573 (1993); Liu, A. M., et al., J Biomol Screen, 8, 39 (2003); Szekeres, P. G., Receptors Channels, 8, 297 (2002) Table 1. Subclassification of G-Protein Coupled Receptors and Relevance to Current Assay

Ins( l,4,5)P₃ formed by phosphoinositide-specific PLC enzymes has three immediate known fates in cells: it is phosphorylated by Ins(l,4,5)P₃ 3-kinase, it is dephosphorylated by Ins(l,4,5)P₃ 5-phosphatase, or it binds to Ins(l,4,5)P receptors and Ins(l,4,5)P binding proteins (See FIG.l) Similar to other second messengers, cells maintain low levels of Ins(l,4,5)P₃ in the nonstimulated state and upon activation, Ins(l,4,5)P₃ levels rise rapidly 5-10 fold to approximately 3 μM in cells. In some experimental systems, Ins(l,4,5)P 5-Phosphatase and additional inositol phosphatase enzymes seem to dominate Ins(l,4,5)P₃ metabolism and recycle inositol polyphosphates from Ins(l,4,5)P₃ to Ins(l,4)P₂ to Ins(4)P, and finally back to the general inosjtol pool for incorporation into phosphatidylinositol. However, in a computer model simulating PLC- β-coupled GPCR activation by glutamate, Ins(l,4,5)P₃ was predominantly metabolized by 3-kinase generating 60 μM intracellular Ins(l,3,4,5)P . Mishra, J. and Bhalla, Biophys J, 83, 1298 (2002) It was estimated that levels of Ins(l,3,4,5)P₄ rise 70-fpld after GPCR 7- *^"*' activation and remain elevated for more than 5 minutes. A second simulation study confirmed the first and found that Ins(l,3,4,5)P4 levels exceeded 12 μM in cells and peaked between 1 and 2 minutes. Nalaskowski,'M. M. and Mayr, Curr Mol Med, 4, 277 (2004) An important parameter for both of these models was the phosphorylation and activation of Ins(l,4,5)P₃ 3-kinase by calcium and calmodulin dependant kinase II (CaMKII) as well as the inhibition of Ins(l,4,5)P₃ 5-phosphatase by this same mechanism. Communi, D.et al., JBiol Chem, 276, 38738 (2001) Inhibition of major Ins(l,4,5)P.j metabolizing enzymes (i.e. 5-phosphatase and 3- kinase) will alter the profile of TP_n metabolites in activated cells which will be useful if a particular metabolite is found to be a better species for measurement. Several inhibiting agents have been reported in the literature (see FIG. 1). First, lower inositol phosphatases are inhibited by Li⁺ ion, and Ins(l,4)P₂ and Ins(4)P levels are both elevated and protracted in cells pretreated with Li⁺ before receptor stimulation. York, J. D., et al., Proc Nαtl Acαd Sci USA, 90, 5833 (1993) This indicates pne or both phosphates activities are inhibited, but not the activity of Ins(l,4,5)P₃ 5-Phosphatase. This view is supported by the observation that in Li⁺ treated bovine tracheal smooth muscle cells stimulated with carbachol, greater than 85% of Ins(l,4,5)P₃ is metabolized by 5-phosphatase to Ins(l,4)P₂. Lynch, B. J., et al., J Pharmacol Exp Ther, 280, 974 (1997) A second strategy to encourage buildup of Ins(H,4)P₂ is to inhibit Ins(l,4,5)P 3-Kinase which should theoretically decrease the 3-phosphorylated inositols, sustain increased Ins(l,4,5)P₃ levels, and further encourage metabolism via the 5-phosphatase pathway. This was observed in adrenal glomerulpsa cells treated with 2 mM strontium ion, Sr²⁺, before stimulation with angiotensin II resulting in significantly higher (and longer sustained) Ins(l,4,5)P₃ levels. Van der Wal, J., et al., J Biol Chem, 276, 15337 (2001) Thus treatment of cells with Li and/or inhibition of Ins(l,4,5)P₃ 3-Kinase are established methods for increasing the amount and duration of Ins(l,4)P₂ after cell activation, making this secondary metabolite an attractive analyte for measuring receptor activation. In order to encourage buildup of Ins(l,3,4,5)P₄ one needs only inhibit", polyphosphate 5-phosphatase which dephosphorylates both Ins(l,3,4,5)P₄ an<3

Ins(l,4,5)P₃. Inhibition of this single enzyme activity would theoretically result in sustained cellular levels of Ins(l,3,4,5)P₄ by protecting both this metabolite directly and its immediate precursor from degradation. This was verified by antisense targeting of Type I inositol polyphosphate 5-phosphatase in rat kidney cells which resulted in approximately a 45% reduction in 5-phosphatase activity and a 2-4 fold increased in basal

Ins(l,4,5)P₃ and Ins(l,3,4,5)P₄ levels. Speed, C. J., et al., Embo J, 15, 4852 (1996) A more phamacological-like inhibitor of Ins(l,4,5)P₃ 5'-phosphatase, spermine, inhibited the tlrrombin stimulated increase in cytoskeletal actin due to decreased amounts of Ins(l,4)P₂ which is consistent with inhibiting 5-Phosphatase activity in these cells (even though

Ins(l,3,4,5)P₄ levels were not directly measured in this study). Huang, C. and Liang, N.

C, Cell Biol hit, 18, 797 (1994) The indirect inhibition of polyphosphate 5-phosphatase was reported by Verjans and colleagues. Verjans, B.,et al., Eur J Biochem, 196, 43 (1991)

They found that Ins(l,3,4,5)P₄ levels peak at 20 seconds in thyroid cells treated with carbachol, and pretreatment of the cells with Staurosporine, an inhibitor of protein kinase

C, resulted in increased levels of both [ns(l',4,5)P₃ and Ins(l,3,4,5)P₄ consistent with inhibition of 5-phosphatase. Altogether, these results demonstrate the feasibility of using established inhibitors of the two main [ns(l,4,5)P₃ metabolic pathways to manipulate levels of secondary inositol phosphate metabolites in GPCR activated cells. Contemporary drag discovery in the GPCR area often begins by characterizing potential small molecule inhibitors that disrupt receptor activation by a known agonist in living cells via some sort of "functional assay". Current screening for antagonists to PLC- activated, Gα_q coupled receptors often measures downstream Ca²⁺ spikes or ¹ 1 involves loading cells with radioactive inositol followed by laborious detection methods.

Both Ca 2+ and the immediate inositol product, In ^! s(l ,4,5)P₃, decay very rapidly in cells making adaptation to high throughput (HT) assays problematic if not impossible. This patent describes a simple, HT amenable, homogenous assay that quantifies longer-lived metabolites of Ins(l,4,5)P₃. A summary of the state of the art and current limitations in measuring PLC-activated GPCRs follows. See also Table 2. There are currently two non-radioactive methods on the market to measure Ins(l,4,5)P₃ produced by cells: an AlphaScreen assay by PerkinElmer Life Sciences, and a fluorescent polarization assay sold by DiscoveRx. These assays measure Ins(l,4,5)P₃ itself, thus they have a limited signal and screening window due to a very short half-life of Ins(l,4,5)P₃. While these assays have been hailed as an alternative to Ca²-⁺ imaging by FLIPR® (see below), they have been only modestly received in the market mainly due to the limitations outlined in this application. Radioactive assays measure incorporation of a radioactive tracer into the aqueous

(Ins(l,4,5)P₃) or membrane (GTPγS) phase following activation of G Protein-coupled effectors. These assays are attractive because they measure proximal events in the signaling pathway closely related to receptor activation thus decreasing the opportunity for interference. However, both assays require loading cells with large amounts of ' I radioactive precursors to ensure labeling, phase separations, and often inefficient sampling-handling steps. Furthermore, because cells process the radioactive molecules via multiple pathways, non-specific background radiation can sometimes be high resulting in a low signal to noise ratio. Recently, the assay signal has been increased significantly by using immobilized metal ion affinity chromatography to assay for total inositol phosphates. Liu, J. J., et al., Anal Biochem, 318, 91 (2003) Further progress has allowed miniaturization and some automation, but there will always be the cost and trouble of working with and disposing of radioactive nuclides. i Measuring intracellular calcium as the readout for GPCR screening is popular because the signal is generated in cells with an acceptable signal to background ratio.

However, because Ca²⁺ is a downstream second messenger, assays that measure Ca²⁺ may not faithfully reflect receptor¹ activation and are¹ more prone to interference. This was shown in an elegant study revealing heterogeneity to different GPCR agonists by monitoring PtdIns(4,5)P₂ substrate hydrolysis by PLC-linked GPCRs not seen by monitoring Ca²⁺. Van der Wal, J., et al., JBiol Chem, 276, 15337 (2001) In addition, there are multiple routes of Ca²⁺ entry into the cytoplasm, so a calcium signal does not l ' always correspond to PLC activation of GPCRs. Berridge, M. J., et al., Nat Rev Mol Cell

Biol, 4, 511 (2003) Because of this, current screening programs usually include a second radioactive assay to confirm hits from the Ca²⁺ screen. And commonly used Ca²⁺ assays require expensive image-based readers making the technology unavailable to many non- pharmaceutical researchers. Direct Cat imaging using calcium binding dyes and the fluorometric imaging plate reader (FLIPR®) is probably the most popular assay for

GPCR receptor activation. A similar system widely used is based on the apoprotein aequorin which generates a luminescent signal in the presence of Ca²⁺. Additional problems with the aequorin method include the- need for expression of the aequorin enzyme in cells and reading of the signal generated within seconds after addition of ligand and drug which makes the method difficult to automate. Transcription of several genes havq been coupled to GPCR activation, including, β-galactosidase, receptor selection and amplification technology (R-SAT), β-lactamase, luciferase, and aequorin. Because the readout (transcription) is so far downstream from receptor activation, these methods are prori'e to interference and frequently generate high numbers of false-positives in a screen. GPCRs are frequently internalized following ligand binding via recruitment of Arrestin proteins from the cytoplasm. This process offers the opportunity to follow one of two translocation events: green fluorescent protein (GFP)-tagged GPCRs away from the plasma membrane or GFP-Arrestin to the plasma membrane. Both events have been used to assay for GPCR activation. One drawback is that the GFP-chimeric proteins must be engineered with the possibility of G'rFP-induced artifacts and cell toxicity. Also, specialized instrumentation and software is required to image and quantitate cellular relocalization making these assays difficult to perform in a high-throughput manner.

Table 2. Comparison of Current PLC-linked GPCR Screening Assays and the Assays of the Present Invention

GFP-Chimera Not Always Low'ito Some Norak Transfluor technology Translocation Moderate configurations requires expensive image-based i.e. β-Arrestin reader, difficult to quantitate. One advantage is it also reports receptor desensitization.

Present Yes _. High Yes Currently would measure Ga_q and invention Got, ₂/ι ₃ Could be engineered to be "universal reporter" for all GPCR subclasses, RTKs and other pathways that produce inositol I phosphate species.

* HTS Compatible includes homogeneous (pipett e and read in one plate), minimum incubation time, stability of signal (as plates wait on stacker to be read), ability to miniaturize (1-30 μL reactions), and repeatability.

The continued research and release of new products in this area are evidence that interest remains high for such assays, and acknowledges there is room for improvement upon existing methods. Therefore, there is a need to develop a direct, non-radioactive, yet HT compatible assay which can be used for screening antagonists to PLC- activated GPCR and/or RTKs. Table 2 summarizes the comparison of the current PLC-linked GPCR screening assays and the assays disclosed in the present invention.

SUMMARY OF THE INVENTION It has been recognized that it would be advantageous to develop a direct, non- radioactive, homogenous assay amenable to high throughput screening which can be used to screening for antagonists of PLC-activated GPCR and/or RTKs. The present invention provides a non-radioactive assay which is amenable to high throughput HT) screening for antagonists of PLC- activated GPCR and/or RTKs by measuring longer-lived Iris(l,4,5)P₃ metabolites (IP_ns), i.e., Ins(l,3,4,5)P₄ and hιs(l,4)P₂₎ to report PLC activity in cells or cell lysates. One aspect of the present invention relates to identifying, producing and characterizing particular inositol phosphate binding proteins (TJPBPs) and biotinylated or fluorescent inositol phosphate tracers to provide the desired inositol phosphate binding profile. Another aspect of the present invention relates to optimizing a prototype competitive assay, simulating receptor activation by spiking IP_ns into cell lysates, and using the simulated assay to demonstrate robustness of the assay sufficient for HT screening. One embodiment of the present invention provides an assay which can be used as a screening tool by activating specific types of GPCRs in human embryonic kidney (HEK-293) cells which general es a competitive signal lasting 1 to 20 minutes after receptor activation. Additional feature's and advantages of the invention will be apparent from the detailed description which follows, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, features of the invention. i¹,

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a diagram of Phospholipase C activation and Ins(l,4,5)P₃ metabolism, including important enzymes and inhibitors; FIG. 2 illustrates an assay scheme of one embodiment of the present invention; FIG. 3 compares assay improvements (new) made since the provisional filing (previous); FIG. 4 illustrates binding specificities of three anti-PJP (Phosphoinositide) antibody clones to phosphoinostides on PIP- Array; I FIG. 5 demonstrates that Grpl-PH protein binds specifically to biotinylated Ptdlns(3,4,5)P₃; FIG. 6 demonstrates that Grpl-PH and Btk-PH proteins have low nanomolar affinity for displacement by Ins(l,3,4,5)P₄, and that Ins(l,3,4,5)P₄ competes specifically, compared to other inositol phosphates, with biotinylated Ins(l,3,4,5)P₄ tracer for binding to Btk-PH protein; FIG. 7 shows robustness of simulated receptor activation in cell lysates; FIG. 8 indicates that activating cells produces a competitive signal in the assay which compares favorably with the current state of the art, Ca²⁺ assay; FIG. 9 shows that the competitive signal after receptor activation in one cell I system can be measured for up to 20 minutes; FIG. 10 shows activation of a specific receptor in an engineered cell line (PAF-R HEK-293 cells) generates measurable Ins(l,3,4,5)P₄; FIG. 11 demonstrates the concentration of Ins(l,3,4,5)P generated in PAF-R cells as a function of time calculated from the standard curve; FIG. 12 establishes that activation of a second specific receptor in an engineered cell line (mGluR5 HEK-293 cells) generates measurable Ins(l ,3,4,5)P₄; and FIG. 13 shows that activation of mGluR5 receptor in an engineered cell line generates measurable Ins(l,3,4,5)P₄ that is detected in a second assay format: fluorescence polarization.

DETAILED DESCRIPTION Reference will now be made to the exemplary embodiments illustrated in the i drawings, and specific language will be used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alterations and further modifications of the inventive features illustrated herein, and additional applications of the principles of the inventions as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention. In a broad sense, the present invention involves a non-radioactive assay which is amenable to high throughput(HT) screening for antagonists of PLC- activated GPCR and/or tyrosine kinase receptors (RTK) comprising the steps of measuring longer-lived Ins(l,4,5)P₃ metabolites (IP_ns), i.e., Ins(l,3,4,5)P₄ and Ins(l,4)P_2> as a means to report the PLC activity in cells or cell lysates. i More particularly, the present invention involves the use of materials and procedures for measuring stable metabolites of the second messenger Ins(l,4,5)P₃, i.e., Ins(l,3,4,5)P₄ and Ins(l,4)P₂, produced in cells by activation of PLC when coupled to signaling receptors, i.e. GPCRs and RTKs. These receptors represent major classes of drag targets and non-radioactive HT assays are needed to implement screening programs i I of large (10,000 to 10,000,000) compound libraries. Not only are IP_ns longer-lived than the parent molecule, but also reach much higher concentrations in cells upon receptor activation. J. Mishra and U. S. Bhalla, Biop ys J S3 (1298-316, 2002). The concentration of IPpS in cells can be further increased by adding the appropriate inhibitors to cells just prior to the assay (see background section and FIG.l). Despite being further downstream from receptor activation than Ins(l,4,5)P₃ itself, IP_nS still faithfully report receptor activation because resting levels are low and only appreciably increase via receptor activation (unlike Ca²⁺ which can also come from extracellular, non-signaling receptor sources). Thus, the essence of the present invention is that by measuring longer-lived Ins(l,4,5)P₃ metabolites (IP_ns), i.e., Ins(l,3,4,5)P₄ and Ins(l,4)P₂, instead of measuring the short-lived primary second messenger itself, i.e. Ins(l,4,5)P₃, the present assays open a window of time and increased sensitivity which allows for improved assay robustness enough to make it HT compatible. One aspect of the present invention relates to characterization of inositol phosphate binding proteins(IPBPs) both with polyselective and specific binding properties. PLC coupled receptor activation was simulated and a non-specific activator was used to demonstrate the feasibility of measuring longer-lived inositol phosphate metabolites as a read out of receptor activation._' Two cell-lines containing different subtypes of GPCRs were activated to demonstrate the generality of the assay. Finally, cell activation was measured in two different formats to demonstrate that measuring longer-lived inositol phosphates is compatible with multiple assay modalities and I technologies. , FIG.2 shows the assay scheme for one embodiment of the present invention; and

FIG. 3 compares this embodiment with another embodiment of the present invention which is disclosed in provisional patent application Serial No. 60/504,446, hereby fully incorporated by reference. Specifically, it relates to a 40 or 50 μL reaction which is compatible with either 96-well or 384- well plate densities and could be miniaturized by a I factor of 5 or more to 8 to 10 μL or less thus making it compatible for a 1,536-well plate.

Five to thirty thousand cells expressing either a native or engineered receptor of interest, i.e., a PLC-coupled receptor, are solubilized in an isotonic buffer at pH 7.4 and then added to the wells of a microplate. Inliibitors or potential inhibitors for signaling receptor or PLC activity to be screened from a compound library are then added to the wells. A known activator of the receptor of interest, i.e., a PLC-coupled receptor, may be optionally added to increase the concentration of the Ins(l,4,5)P₃ metabolites (IP_ns), i.e., i Ins(l,3,4,5)P₄ and Ins(l,4)P₂. After 1 to 5 minutes, cell activation is stopped by lysing the cells with 5 μL of 0.2 to 0.3 N perchloric acid. Perchloric acid acidifies the assay mix ¹ I causing at least partial lysis of the cells within 5 minutes. This is sufficient to make I inositol phosphates available for binding, but complete lysis and hence better competition requires from 15 to 30 minutes. The reaction mixture is then brought to a neutral pH by adding 5 μL of an alkaline TBS buffer. ^he Inositol phosphate binding protein (IPBP) is then added directly to the wells and allowed to bind cell-derived -inositol phosphates for 5 to 15 minutes before addition of the appropriate inositol phosphate tracer and detection mix. For a fluorescent polarization (FP) assay using a fluorescent inositol phosphate tracer, this is all that is needed. The assay can then be read with an FP plate reader after 30 minutes to 4 hours or more of incubation. According to another embodiment of the present invention, AlphaScreen donor and acceptor beads are added simultaneously with a biotinylated inositol phoβphate tracer in neutral PBS or similar media. The plate is protected from light and after 1 to 24 hours, of incubation at room temperature, preferably at 27 ^°C, the plate is read on an AlphaScreen™ compatible plate reader. According to the present invention, the assay can be applied to any eukaryotic or bacterial cell naturally containing or artificially engineered to express a receptor or receptors coupled to PLC or any inositol phosphate producing activity. For example,

Chinese Hamster Ovary cells, SF9 insect cells, yeast cells, E. coli, or the like. Preferably, the assay is applied to Human embryonic kidney (HΕK-293) cells. Suitable receptors for the present invention can be any receptor which naturally or is artificially engineered to activate any PLC or enzyme activity which produces inositol phosphates. This activity might be intracellular or be an extracellular PLC enzyme or inositol phosphate producing activity in the cell lysate or be completely free of cells in a purified or partially purified in vitro form. For example, epidermal growth factor (ΕGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), or an antigen receptor which activates PLC-γ; ion channels and Ca²⁺ which activates PLC-δ; or Ras which activates PLC-ε; or type I inositol 5-phosphatase or Ins(l,4,5)P₃ 3-kinase, or the like. Preferably, the receptors are Gαq-protein coupled receptors (GPCR) which activate phospholipase C beta (PLC-β). The suitable agonist, partial agonist, or super agonist for the present invention can be a chemical or biological natural or artificial substance which causes direct or indirect activation of any PLC or inositol phosphate producing activity. A partial or super agonisi is any chemical or biological natural or artificial substance causing a partial/attenuated or ! ' i a prolonged/increased agonist action. Preferably, the agonist is acetylcholine chloride (or i , i carbachol), platelet activating factor, or glutamic acid. Suitable inhibitors for the present invention can be any chemical or biological natural or artificial substance or combination of such that interferes blocks or decreases action of so stated agonist (See Sigma Cell Signaling and Neuroscience Catalog, 2002-

2003 edition, pp 630-650). Preferably, the inl ibitor is a member of known inhibitors specific for the class of receptor under investigation or any variety of a chemical compound library with molecules of undetermined inhibitor properties. One such inhibitor specific for muscarinic GPCRs is atropine or its derivatives. The inositol phosphates suitable for the present invention can be any secondary inositol (phytic acid), inositol phosphaite metabolite, inositol polyphosphate, or inositol pyrophosphate produced by any receptor, enzyme, or activity as previously defined.

Production may be either in cells, tissues, extracts of these materials, or completely in vitro. For example Ins(l,4)₂ or Ins(l,3,4,5,6)P5. Preferably, the inositol phosphate is

Ins(l,3,4,5)P₄ I The inositol phosphate detector suitable for the present invention can be any natural or engineered protein, antibody, receptor, polynucleotide, carbohydrate, or like which binds to or recognizes or interacts with any isomer of inositol, inositol phosphate or inositol polyphosphate species as defined. Preferably, the inositol phosphate detector is a pleckstrin homology (PH) domain of Grp 1. The assay as currently embodied uses a known phosphoinositide-binding protein, Grpl and either biotinylated or fluorescent analogs of phosphatidylinositol(3,4,5)P₃ or Ins(l,3,4,5)P₄ as the binding partner. Other similar IPBPs, mutants, and anti- phosphoinositide antibodies with specificity for Ins(4)P, Ins(l,4)P₂, or Ins(l,3,4,5)P₄ can also be used. It is recognized that the pattern of long-lived metabolites in cells after receptor activation will be different by using inhibitors of inositol phosphate kinases and inositol phosphate phosphatases. The present invention can be configured as any type of assay that measures or quantifies any inosilol phosphate produced as defined. These may or may not require labeled tracer inositol phosphate, i.e. a sandwich assay where two detectors of like or different composition binds or captures inositol phosphate in order to report the quantity of inositol phosphate in a 'sample. A competitive assay using non-radioactively labeled tracer is preferred in the present invention. One skilled in the art could develop appropriate fluorescent, biotinylated, digoxigen, or like inositol phosphate labeled tracer. These tracers could be used in many assay formats such as, enzyme-linked immuno absorbance (ELISA), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET), AlphaScreen™, electromagnetic, electrochemiluminescence, or the like. The following Examples are provided to further aid in understanding the invention, and pre-suppose an understanding of conventional methods well-known to those persons having ordinary skill in the art to which the examples pertain. Such methods are described in detail in numerous publications including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989),

Ausubel et al. (Eds.), Current Protocols iri Molecular Biology, John Wiley & Sons, Inc.

(1994); and Ausubel et al.|(Eds.), Short Protocols in Molecular Biology, 4th ed., John

Wiley & Sons, Inc. (1999). The particular materials and conditions described hereunder are intended to exemplify particular aspects of the invention and should not be construed to limit the reasonable scope thereof.

Example 1 IPBP Discovery Several approaches to generate proteins which bind to phosphoinositides and inositol phosphotates were used in the present invention. These include 1) the generation of anti-PIP-(Phosphoinositide) antibodies^ 2) cloning and/or and expression of known inositoTphosphate/phosphoinositide-binding proteins, and 3) generating novel inositol phosphate/phosphoinositide binding proteins tlirough random, directed, and phage display mutagenisis. In order to develop an assay to measure inositol phosphate metabolites, anti-PIP antibodies and mutants were screened. It was found that two antibodies with specificity for PI(3)P as well as one antibody that reςάgnizes all eight PIP headgroups, analyzed by binding to dilutions of all eight Phosphoinositid'es spotted on a nitrocellulose membrane (i.e. Echelon's PIP-Array™ Assay, FIG.4). Briefly, the membrane is blocked with 0.5% milk then overlain with the protein or antibody of interest. After a one hour to overnight incubation on a rotary shaker, the membrane is washed with TBS containing 0.1% Tween-20. A secondary antibody, coupled to horseradish peroxidase and specific for the protein or antibody added previously, is then overlain on the membrane for one hour then washed as before. Finally, a chemiluminescent peroxidase substrate is placed on the membrane and the spots visualized by exposure¹ to film or an imager, such as the Kodak

Image Station 440cf or BioRad Chemidoc. S. Dowler, et al, Sci STKE 2002 PL6, 2002. Particularly, the antibody 'clone that recognizes all phosphoinositide headgroups can be used as the IPBP partner in the IPn assay of the present invention. In addition, promising proteins, mutant proteins or antibodies that show binding selectivity may function as IPBPs which recognize biotinylated and non-biotinylated inositol phosphates with headgroups corresponding to Ins(l,4)P₂, Ins(l,4,5)P₃, and Ins(l,3,4,5)P₄. The binding specificity and selectivity for the three major metabolites after receptor activation namely. Ins(l,4)P₂, ]ns(l ,4,5)P₃, and Ins(l,3,4,5)P_4jhave been carefully characterized (See Table

3). Example 2 IPBP Characterization It was discovered that Grpl-PH and Btk-PH domains bind specifically to biotinylated PtdIns(3,4,5)P₃ and biotinylated Ins(l,3,4,5)P₄. The binding activity of several biotinylated Ptdlns analogs to a GST-fusion protein containing the PH domains of

Grpl and Blk have been tested in several assay modalities with the luminescent

AlphaScreen binding assay described. Briefly, a 10 to 250 nM final concentration of protein in 5 μL of Alpha assay buffer (10 mM Tris pH 8.0, 150 mM NaCl, Phosphate buffered saline, pH 7.0-7.6, or similar, ) was added to triplicate wells of a 384-well white micro titer plate containing 5 μL of biotinylated Inositol(l,3,4,5)P₄ or Ptdlns(3,4,5)₃ (also at 10 to 250 nM final concentrations) ar L5 μL of Alpha assay buffer. To this complex is added both AlphaScreen donor and acceptor beads in 10 μL of Alpha assay buffer (25 μL total per well). The plate was protected from light and incubated for 1 or 2 hours at room temperature (22-28 °C). A strong binding interaction was indicated when the protein and biotinylated analog concentrations were 50 to 250 nM (FIG 5). As shown in FIG. 6, upper panel, both Grpl and Btk-PH- domains were sensitively inhibited with nanomolar concentrations of Ins(l,3,4,5)P₄ from binding to a biotinylated PtdIns(3,4,5)P₃ tracer. In this experiment the half-maximum inhibitory concentration (IC₅₀)was 20 nM for Grpl and 40 nM for Btk indicating that these binding partners should be sensitive enough to detect Ins(l,3,4,5)P generated by activated cells. FIG. 6, lower panel, illustrates the specificity of Btk -PH inhibition by Ins(l,3,4,5)P₄ compared to Ins(l,4)P₂ or Ins(l,4,5)P₃ binding to Biotinylated PtdIns(3,4,5)P₃ with Ins(l,3,4,5)P₄ recognized at more than a 350 times lower concentration than Ins(l,4,5)P₃. Similar results were obtained with Grp-1. Together these results demonstrate sensitive and specific binding of two PH domain proteins with three major' inositol phosphate metabolites. Three additional proteins were tested in a similar fashion for competitive displacement by the three primary inositol i phosphate metabolites with the goal being to identify binding proteins sensitive and specific for Ins(l,3,4,5)P₄ or Ins(l,4)P₂. Multiple experiments were performed, and the

IC₅₀ values indicated are average values obtained from at least two separate experiments.

These results are summarized in Table 3. Table 3. Summary of the Characterization ©f Several Inositol Phosphate Binding Proteins.

NC = No competition

Example 3 Characterization of the Assay Components It was found that all of the proteinsi tested could have utility in the assay of the present invention due to IC₅₀ values for at least one of the metabolites being close to the levels achieved in cells after receptor activation. To demonstrate proof of concept for the assay, either Grpl-PH or Btk-PH and either biotinylated PtdIns(3,4,5)P₃ or biotinylated Ins(l,3,4,5)P₄ were chosen for the remaining experiments. These binding partners were chosen due to availability' and stability of the components, the high affinity and specificity of their interactions, and the likelihood that Ins(l,3,4,5)P₄ reaches comparatively high concentrations in activated cells. i First, receptor activation was simulated by spiking 5 μL of several concentrations of Ins(l,3,4,5)P₄ into the wells of a micro titer plate containing 5-30 million wild-type HEK-293 cells in 10 μL of HEPES buffer at a pH of 7.0 to 7.4. After adding Ins(l,3,4,5)P₄, the cells were then lysed by addition of 10 μL of 1.05% (-0.2- N) trichloroacetic acid and incubated for 30 minutes. The solution was neutralized, and competitive binding initiated by adding 60 μL of GST-Grpl-PH or GST-Btk-PH in 50 mM TRIS at a pH of 8.5. After a 60 minute incubation period, biotinylated Ins(l,3,4,5)P₄ tracer was added, and receptor/donor AlphaScreen beads in 10 μL TRIS at a pH of 7.5 were then processed and read as outlined. It was found that 0.2 to 20 μM Ins(l,3,4,5)P₄ spiked into an HEK-293 cell extract competed for about at least 90% of the signal generated by biotinylated Ins(l,3,4,5)P₄ binding to each IPBP (data not shown). The competitive IC₅₀ values were essentially identical to the values we observed in vitro without cells (see Table 3). This result demonstrates that the components are stable to the conditions of the assay and the sensitive binding and competition reactions retain their properties in the complex milieu of a cell lysate. This spiking experiment was repeated in! 30 wells of a 96-well plate using either a 10 μM concentration of Ins(l,3,4,5)P₄ , no competitive Ins(l,3,4,5)P₄ or no IPBP in order to determine the repeatability or robustness! of the assay. The measure of robustness is called the Z-factor which was 0.65 for this assay (FIG. 7). Generally, if an assay has a value below 0.5, the assay is not compatible for high- throughput methods and the closer to 1, the more repeatable the assay is. These two results together demonstrate that in simulated receptor activation conditions, the assay of the present invention is both sensitive enough to detect physiological levels of inositol phosphates and robust enough to be used with automated robotic steps in high-throughput manner, i.e., at least a 384- well plate density.

Example 4 Validating the Assay with Endogenous Receptors in Native Cell Line To prove the assay would work in cells, wild-type human embryonic kidney 293 (HEK-293) cells were stimulated with the muscarinic receptor agonist, carbachol, and it was found that with 10 μM carbachol a robust Ca²⁺ signal was generated which remained elevated for just over 3 minutes (FIG. 8, Top panel). This shows that the cell assay system used to validate the present invention is functional using a well-established Ca fluorescence assay. HEK-293 cells were then activated with several concentrations of carbachol for two minutes and it was found using the inositol phosphate assay of the present invention that 10 μM carbachol produced an inositol phosphate signal strong enough to compete for more than 90%. of the signal given by Grpl-PH binding to biotinylated Ins(l,3,4,5)P₄ (FIG. 8, Bottom panel). This establishes that 10,000 HEK-293 cells with wild-type expression levels of native GPCRs produce enough inositol phosphates to compete for the Grpl-PH/biotinylated PtdIns(3,4,5)P₃ binding partners in this assay and this level of inositol phosphates is present at least two minutes after activating the receptor — much longer than the 10-30 seconds of Ins(l,4,5)P₃ as in the existing art. Finally, a time course experiment was performed to see how long an assay window was available to assay for these long-lived metabolites and it was found that almost 50 % of the signal was still present 20 minutes after addition of carbachol (FIG.

9). This provides an ample window for an automated liquid handling system to activate and then stop the cells in all of the wells of a 96; 384, or 1536-well micro titer plate.

Example 5 An Assay Using Two Classes of Specific Receptors in Engineered Cell-lines The validated assay (new 40 μL format)¹ was used to test activation of two cell lines engineered to express different classes of GPCRs. First, Grp- PH domain protein and biotinylated Ins(l,3,4j5)P were used as binding partners to measure cell-derived I

Ins(l,3,4,5)P₄ at 0 to 4 minutes after addition of the GPCR ligand, platelet activating factor (PAF), to triplicate wells according to the procedure in FIG. 2. FIG. 10 (top panel) shows the standard curve generated by adding increasing concentrations of Ins(l,3,4,5)P which generates a competitive signal. The competitive Ins(l,3,4,5)P₄ signal generated from activation of these cells is shown as Alpha luminescent values lower than the "no competitor" control in the lower panel. FIG. 11 shows, the concentration of Ins(l,3,4,5)P₄ generated, as calculated from non-linear regression of the standard curve. Note that the levels of Ins(l,3,4,5)P₄ remain elevated for the entire time course establishing a lengthened window of time for activating and stopping the cell activation portion of the assay. This' prolonged activation time allows for automation of the assay i I i for HT screening. FIG. 12 shows two panels from a similar experiment performed with a cell-line expressing a different class of PLC-coupled GPCR receptor, the metabatropic

Glutamate 5 receptor. A similar standard curve was observed (top panel), but different kinetics of Ins(l,3,4,5)P₄ generation were obtained (bottom panel). This cell-line showed accumulation of almost 800 nM Ins(l,3,4,5)P₄ at 0.5 minutes with some of the secondary metabolite persisting for 3 minutes. This experiment demonstrates that measuring secondary inositol phosphate metabolites reports GPCR activation for multiple receptor classes, and establishes the generality of the assay principle for at least two different classes of GPCR receptors. One skilled in the art could imagine that for some receptor-coupled cell systems, the 5-phosphatase activity would dominate and more Ins(l,4)P₂ secondary metabolite would be generated compared to Ins(l,3,4,5)P by the 3-kinase enzyme. Perhaps this explains the different Ins(l ,3,4,5)P₄ kinetics between platelet activating factor receptor and metabatropic glutamate receptor in FIG. 10 and FIG. 12. In such a case, one could use the LL5α or Tubby proteins shown, in Table 3 or the antibody shown in FIG 4. panel

C to assay for competitive binding to cell-derived Ins(l,4)P2 as a measure of receptor activation in cells.

Example 6 Receptor Activation Assessed by Fluorescence Polarization A similar cell activation experiment with glutamate-receptor cell-line was I performed, but this time reading the competitive inhibition of fluorescent polarization (milliP units). Five nM fluorescently labeled BODIPY-TMR- Ins(l,3,4,5)P₄ probe and 50 nM Grpl-PH domain were the binding partners used. Again, a competitive standard curve was generated by adding defined amounts of Ins(l,3,4,5)P₄ to triplicate wells (FIG 13, upper panel). A robust competitive signal was generated and persisted for the 3- minute duration of the time course (FIG 13. lower panel). This demonstrates that multiple assay formats can be used to measure long-lived secondary inositol phosphate metabolites after PLC-coupled receptor activation. It is to be understood that the above-referenced arrangements are only illustrative I of application of the principles of the present invention. Numerous modifications and alternative arrangements can be devised without departing from the spirit and scope of the present invention. While the present invention has been shown, in the drawings and is fully described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications can be made without departing from the principles and concepts of the invention as set forth in the claims.

Claims

CLAIMSWe claim:

1. A process of monitoring a receptor coupled signaling enzymes having inositol phosphate producing activity, comprising: , measuring an inositol phosphate metabolite in cells or cell lysates by using a non- radioactive assay which is amenable to high throughput(HT) screening.

2. The process according to Claim 1, wherein said inositol phosphate producing activity is inositol (1,4,5,) triphosphate(Ins(l,4,5)P₃) producing activity and said inositol phosphate metabolite is inositol(l, 3,4,5) tetrakisphosphate (Ins(l,3,4,5)P₄) or inositol(l ,4) bisphosphate (Ins(l,4)P₂)l

3. The process according to Claim 2, wherein said non-radioactive assay comprises the steps of:

(a) mixing a predetermined amount of eukaryotic or bacterial cells capable of expressing a receptor of interest with a predetermined amount of an agonist of said receptor to form a reaction mixture;

(b) incubating said reaction mixture to allow formation of inositol phosphate metabolites;

(c) lysing said cells to form a cell lysate; _ι

(d) contacting said cell lysate with an inositol phosphate metabolite detector, a non- radioactively labeled inositol phosphate metabolite tracer and a detection mix; and

(e) quantifying said inositol phosphate metabolite by measuring non-radioactive signals released from said non-radioactively labeled inositol phosphate metabolite tracer and a detection mix.

4. The process according to Claim 1, wherein said receptor is a member selected from the group consisting of G-protein coupled receptors (GPCRs) which activates phosphoinositide-specific phospholipase C beta (PLC-β), PLC- activated Gα_q receptors, receptor tyrosine kinases (RTKs); a receptor capable of activating a member selected from the group consisting of epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and nerve growth factor (NGF), an antigen receptor which activate PLC-γ, ion channels and Ca 2+ wh ^'ich ^' activates PLC ' -δ, and Ras which activates PLC-ε, type I inositol 5-phosphatase and Ins(l,4,5)P₃ 3-kinase.

5. The process according to Claim 3, wherein sajd inositol phosphate metabolite detector is a natural or engineered protein, antibody, receptor, polynucleotide, carbohydrate, or a synthetically-prepared small molecule artificial receptor which specifically binds to or recognizes or interacts with said inositol phosphate metabolite.

6. A non-radioactive assay which is amenable to high throughput (HT) screening for antagonists or agonists of la receptor coupled signaling enzymes having inositol phosphate producing activity comprising the steps of :

(a) mixing a compound to be screened to a predetermined amount of eukaryotic or bacterial cells capable of expressing a receptor of interest, followed by adding a predetermined amount of an agonist of said receptor to form a reaction mixture;

(c) lysing said cells to form a cell lysale;

(d) contacting said cell lysate with an inositol phosphate metabolite detector, a non- I I radioactive ly labeled inositol phosphate metabolite tracer and a detection mix;

(e) quantifying said inositol phosphate metabolite by measuring non-radioactive signals released from said non-radioactively labeled inositol phosphate metabolite tracer and a detection mix; and

(f) screening for a compound which has an inhibitory or an enhancing effect on said inositol phosphate producing activity.

7. The assay according to claim 6, wherein said receptor is a member selected from the group consisting of G-protein coupled receptors (GPCRs) which activates phosphoinositide-specific phospholipase C beta (PLC-β), PLC- activated Gα_q receptors I and receptor tyrosine kinases (RTKs); said agonist is acetylcholine chloride or carbachol, platelet activating factor, or glutamic acid; said inositol phosphate detector is a phosphoinositide-binding protein or pleckstrin homology (PH) domain of a general receptor for phosphoinositides (Grpl), and said non-radioactively labeled inositol phosphate metabolite is biotinylated or fluorensenated posphatidylinositol (3,4,5)P₃, or biotinylated or fluorensenated Ins(l,3,4,5)P₄.

8. The assay according to claim 6, wherein said assay is configured as a HT type of assay selected from the group consisting of enzyme-linked immuno absorbance (ELISA), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), time- resolved fluorescence resonance energy transfer (TR-FRET), AlphaScreen™, electromagnetic and electrochemiluminescence.

9. An antagonist of a receptor coupled phosphoinositide-specific phospholipase C (PLC) or other receptor coupled signaling enzymes having inositol phosphate producing activity, which is obtained by the screening assay according to Claim 6.

10. An agonist of a receptor coupled phosphoinositide-specific phospholipase C PLC) or other receplor coupled signaling enzymes having inositol phosphate producing activity, which is obtained by the screening assay according to Claim 6.