Methods for identifying modulators of N-type ion channel inactivation Number:7,049,083 from the United States Patent and Trademark Office (PTO) owispatent

Title: Methods for identifying modulators of N-type ion channel inactivation

Abstract: Methods and compositions for identifying compounds which disrupt the functional interaction of an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein are disclosed. Compounds that disrupt the functional or binding interaction of these two regions have significant modulatory effects on ion channel activity, and thus are likely to be useful for treating and/or preventing a wide variety of diseases and pathological conditions associated with ion channel dysfunction. Such conditions include, for example, neurological disorders, cardiac diseases, metabolic diseases, tumor-driven diseases, and autoimmune diseases.

Patent Number: 7,049,083 Issued on 05/23/2006 to Young,   et al.

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Inventors:	Young; Kathleen H. (Newtown, PA); Rhodes; Kenneth J. (Neshanic Station, NJ)
Assignee:	Wyeth (Madison, NJ)
Appl. No.:	051843
Filed:	January 17, 2002

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Current U.S. Class:	435/7.2 ; 435/173.1; 435/252.3; 435/254.2; 435/320.1; 435/325
Current International Class:	C12N 15/63 (20060101); C12N 1/15 (20060101); C12N 1/16 (20060101); C12N 15/00 (20060101); G01N 33/566 (20060101)
Field of Search:	435/7.2,320.1,325,173.1,254.2,252.3

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References Cited [Referenced By]

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U.S. Patent Documents

	5776689		July 1998		Karin et al.
	5856155		January 1999		Li
	6080557		June 2000		Sims et al.

Foreign Patent Documents

	WO 95/34646		Dec., 1995		WO
	WO 97/31112		Aug., 1997		WO

Other References

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Primary Examiner: Murphy; Joseph
Attorney, Agent or Firm: Nixon Peabody LLP Dyke; Raymond Van

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Parent Case Text

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This is a continuation-in-part of application(s) Ser. No. 09/295,999 filed on Apr. 21, 1999, now abandoned the entire disclosure of which is hereby incorporated by reference. The present invention relates to methods and compositions for identifying compounds which modulate N-type inactivation of voltage-gated ion channels.

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Claims

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What is claimed is:

1. A method for identifying compounds which inhibit N-type inactivation of a voltage-gated ion channel, comprising: a) administering a compound to a modified host cell comprising: i) a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage to either 1) an S4 S5 cytoplasmic loop of an .alpha.-subunit of a voltage-gated ion channel; or 2) an amino-terminal inactivation region of an ion channel protein; ii) a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to said S4 S5 cytoplasmic loop if said DNA-binding domain is in polypeptide linkage to said amino-terminal inactivation region or to said amino-terminal inactivation region if said DNA-binding domain is in polypeptide linkage to said S4 S5 cytoplasmic loop; and iii) a reporter gene whose transcription is dependent upon the first hybrid protein and the second hybrid protein being bound to each other, thereby reconstituting a transcriptional activator; b) incubating the modified host cell for a suitable period; c) determining whether the administration of the compound inhibits expression of the reporter gene; and d) identifying a compound which inhibits expression of the reporter gene as an inhibitor of N-type inactivation of said voltage-gated ion channel.

2. A method for identifying an agent capable of modulating inactivation of an ion channel, said method comprising detecting binding of first protein to a second protein in the presence or absence of a molecule of interest, wherein said first protein comprises an S4 S5 cytoplasmic loop of a voltage-gated ion channel, and said second protein comprises an amino-terminal inactivation region of an ion channel subunit, and wherein said first protein binds to said second protein in the absence of said molecule of interest, and a decrease in said binding in the presence of said molecule of interest as compared to that in the absence of said molecule of interest is indicative that said molecule of interest is capable of modulating inactivation of said voltage-gated ion channel.

3. The method of claim 2, wherein said S4 S5 cytoplasmic loop is an S4 S5 cytoplasmic loop of a potassium channel .alpha.-subunit, and said amino-terminal inactivation region is an amino-terminal inactivation region of a potassium channel .alpha.- or .beta.-subunit.

4. The method of claim 3, wherein said first protein further comprises a DNA-binding or transcription activation domain of a transcriptional activator, and said second protein further comprises: a DNA-binding domain if said first protein comprises the transcription activation domain of said transcriptional activator, or a transcription activation domain if said first protein comprises the DNA-binding domain of said transcriptional activator, wherein binding of said first protein to said second protein forms a transcriptional activator.

5. The method of claim 4, comprising expressing said first protein and said second protein in a host cell in the presence or absence of said molecule of interest, wherein binding of said first protein to said second protein activates expression of a reporter gene in said host cell.

6. The method of claim 5, wherein said host cell is yeast.

7. The method of claim 5, wherein said S4 S5 cytoplasmic loop is an S4 S5 cytoplasmic loop of a potassium channel .alpha.-subunit selected from the group consisting of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6 and Kv3.4, and said amino-terminal inactivation region is an amino-terminal inactivation region of a potassium channel .alpha.- or .beta.-subunit selected from the group consisting of Kv.beta.1, Kv.beta.1.2, Kv.beta.1.3, Kv.beta.3., Kv.beta.3.4, and Kv1.4.

8. The method of claim 5, wherein said S4 S5 cytoplasmic loop is an S4 S5 cytoplasmic loop of potassium channel Kv1.1 or Kv1.4, and said amino-terminal inactivation region is an amino-terminal inactivation region of potassium channel Kv.beta.1.

9. The method of claim 5, wherein said first portein consists essentially of said S4 S5 cytoplasmic loop and the DNA-binding or transcription activation domain of said transcriptional activator.

10. The method of claim 5, wherein said S4 S5 cytoplasmic loop comprises SEQ ID NO:1 or SEQ ID NO:2, and said amino-terminal inactivation region comprises SEQ ID NO:5 or SEQ ID NO:6.

11. The method of claim 3, wherein said first protein further comprises a first polypeptide selected from a peptide binding pair, and said second protein further comprises a second polypeptide selected from said peptide binding pair, and wherein binding of the first polypeptide to the second polypeptide in a host cell is capable of producing a detectable event or a selectable phenotype in said cell.

12. The method of claim 11, wherein said cell is yeast.

13. The method of claim 11, wherein said S4 S5 cytoplasmic loop is an S4 S5 cytoplasmic loop of a potassium channel .alpha.-subunit selected from the group consisting of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6 and Kv3.4, and said amino-terminal inactivation region is an amino-terminal inactivation region of a potassium channel .alpha.- or .beta.-subunit selected from the group consisting of Kv.beta.1, Kv.beta.1.2, Kv.beta.1.3, Kv.beta.3, Kv3.4 and Kv1.4.

14. The method of claim 11, wherein said S4 S5 cytoplasmic loop is an S4 S5 cytoplasmic loop of potassium channel Kv1.1 or Kv1.4, and said amino-terminal inactivation region is an amino-terminal inactivation region of potassium channel Kv.beta.1.

15. The method of claim 3, wherein one protein selected from said first and second proteins further comprises a cell compartment localization domain capable of recruiting said one protein to a cell compartment of a host cell, and the other protein selected from said first and second proteins comprises an effector protein, and wherein recruitment of said one protein to the cell compartment and binding of said first protein to said second protein produce a detectable event or a selectable phenotype in said host cell.

16. An agent identified according to claim 2, wherein said agent inhibits binding between said S4 S5 cytoplasmic loop and said amino-terminal inactivation region.

17. A method for identifying an agent capable of modulating an interaction between an S4 S5 cytoplasmic loop of a potassium channel .alpha.-subunit and an amino-terminal inactivation region of a potassium channel .alpha.- or .beta.-subunit, said method comprising: expressing a first protein and a second protein in a host cell, wherein said first protein comprises (1) said S4 S5 cytoplasmic loop and (2) a DNA-binding or transcription activation domain of a transcriptional activator, wherein said second protein comprises (1) said amino-terminal inactivation region and (2) a DNA-binding domain if said first protein comprises the transcription activation domain of said transcriptional activator or a transcription activation domain if said first protein comprises the DNA-binding domain of said transcriptional activator, and wherein said S4 S5 cytoplasmic loop is capable of binding to said amino-terminal inactivation region in the absence of a molecule of interest, and binding of said first protein to said second protein forms a transcriptional activator capable of activating expression of a reporter gene in said host cell; contacting the molecule of interest wit said host cell; and detecting any change in said expression of the reporter gene, wherein a decrease in said expression is suggestive that the molecule of interest is capable of modulating the interaction between said S4 S5 cytoplasmic region and said amino-terminal inactivation region.

18. A method for identifying an agent capable of modulating an interaction between an S4 S5 cytoplasmic loop of a potassium channel .alpha.-subunit, and amino-terminal inactivation region of a potassium channel .alpha.- or .beta.-subunit, said method comprising: expressing a first protein and a second protein in a host cell, wherein said first protein comprises said S4 S5 cytoplasmic loop and a first polypeptide, and said second protein comprises said amino-terminal inactivation region and a second polypeptide, and wherein said S4 S5 cytoplasmic loop binds to said amino-terminal inactivation region in the absence of a molecule of interest, and interaction or close proximity between said first polypeptide and said second polypeptide is capable of producing a detectable event or a selectable phenotype in said host cell; contacting the molecule of interest wit said host cell; and detecting any change in said detectable event or selectable phenotype, wherein a change in said event or phenotype is suggestive that the molecule of interest is capable of modulating the interaction between said S4 S5 cytoplasmic region and said amino-terminal inactivation region.

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Description

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BACKGROUND OF THE INVENTION

Ion channels are transmembrane proteins that regulate entry of various ions into cells from the extracellular matrix. Ion channels are physiologically important, playing essential roles in regulating intracellular levels of various ions and in generating action potentials in nerve and muscle cells. Hille, B., Ionic Channels of Excitable Membranes (Sinauer, Sunderland, Mass., 1992). Passage of ions through ion channels is characterized by selective filtering and by a gating-type mechanism which produces a rapid increase in permeability. Angelides, K. J. and T. J. Nuttov, J. Biol. Chem. 258:11858 11867 (1981). Ion channels may be either voltage-gated, implying that current is gated (or regulated) by membrane potential (voltage), or chemically-gated (e.g., acetylcholine receptors and .gamma.-aminobutyric acid receptors), implying that current is gated primarily by binding of a chemical rather than by the membrane potential. Butterworth, J. F. and G. R. Strichartz, Anesthesiology 72:711 734 (1980). An important characteristic of certain voltage-gated channels is inactivation: soon after opening they close spontaneously, forming an inactive channel that will not reopen until the membrane is repolarized. Miller, C., Science 252:1092 1096 (1991). Rapidly inactivating ("A-type") voltage-gated ion channels control the rate at which excitable cells reach the threshold for firing action potentials and thus are key regulators of neuronal excitability. B. Hille, supra.

Many voltage-gated ion channels that generate action potentials have been cloned and sequenced, and all have a remarkably similar structure. A typical potassium channel contains four copies of an approximately 600-amino-acid polypeptide, each of which has six membrane-spanning .alpha.-helices. Heginbotham, L., et al., Science 258:1152 (1992). Sodium and calcium channels are single polypeptides of about 2000 amino acids that contain four homologous domains, each comprised of six transmembrane domains which are similar in sequence and structure to a potassium channel protein. These domains are connected and flanked by shorter stretches of nonhomologous residues. Jessell, T. M. and E. R. Kandel, Neuron 10(Supp):1 3 (1993). It is believed that the .alpha.-helical structures provide conformational flexibility for the ion channel which is functionally responsible for the channels gating mechanism. See Heinemann, S., et al., J. Physiol. 88:173 180 (1994).

In addition to affecting action potentials, ion channels facilitate other important physiological functions such as cardiac pacemaking, neuron bursting, and possibly learning and memory. Crow, T., Trends Neurosci. 11:136 142 (1988); Hodgkin, A. L. and Huxley, A. F., J. Physiol. 117:500 544 (1952). In addition to their involvement in normal cellular homeostasis, ion channels are associated with a variety of disease states and immune responses. Diseases believed to be associated with dysfunction of ion channels include neurological disorders, metabolic diseases, cardiac diseases, tumor-driven diseases, and autoimmune diseases.

Due to the importance of ion channels in both normal cellular homeostasis and disease, considerable research effort has focused on ion channels, and particularly on identifying compounds which affect their function. Thus, several techniques have been developed to evaluate the gating mechanism of ion channels and the mode of action of channel-drug interaction. Electrophysiological recording has been used to define the roles of ion currents, and especially potassium and sodium currents, in generating action potentials in excised nerves. Hodgkin, A. L. and A. F. Huxley, supra. This technique, however, is not suitable for mass screening of compounds due to its technical complexity and the requirement of a high degree of sophistication to generate reproducible results. Radioligand binding assays have been used to characterize the site of action of various classes of ion channel blockers. However, the availability of radiolabelled ligands, the level of nonspecific binding, and the physico-chemical property of the molecules have limited the application of this technique. Strichartz, et al., Ann. Rev. Neurosci. 10:239 67 (1987). Fluorescent-labelled neurotoxin probes have also been used to map the molecular structure of the functional site of the channel, but have not gained general popularity for broader use. Angelides, K. A. and T. J. Nuttov, J. Biol. Chem. 256:11958 11967 (1983).

Recently, a modified yeast "two-hybrid" system has been developed to identify compounds that bind to either the NH.sub.2-terminal multimerization domain (commonly referred to as the "NAB" or "T1" domain) on the .alpha.-subunit of a Shaker-like potassium channel or to the "core" domain of the .beta.-subunit of the potassium channel, thereby preventing the .alpha./.beta. subunit interaction. See U.S. Pat. No. 5,856,155 (M. Li), issued Jan. 5, 1999; and PCT App. No. PCT/US97/02292, published Aug. 28, 1997 (WO 97/31112). Unfortunately, significant inherent limitations in this system may prevent or limit its practical application. Such limitations include, for example, the extraordinarily tight nature of the .alpha.-NAB/.beta.-core interaction (which survives such harsh treatments as detergent extraction and affinity chromatography), the limited applicability to potassium channels whose activity requires interaction between the NAB domain of the .alpha.-subunit and the core domain of the .beta.-subunit, and, most importantly, the potentially significant inhibitory effect such compounds would have on potassium channel surface expression. [Regarding the tight association of .alpha.- and .beta.-subunits, see Parcej, D. N., and J. O. Dolly, Biochem. J. 257:899 903 (1989) and Muniz, Z. M., et al., Biochemistry 31:12297 12303 (1992).] With respect to the latter limitation, .beta.-subunits have been shown to promote N-linked glycosylation and surface expression of .alpha.-subunits. Shi, G., et al. Neuron 16:843 852 (1996). Thus, one would expect compounds that bind to the core domain of the .beta.-subunit to block these chaperone-like effects, thereby reducing, if not preventing, the biosynthesis of functional potassium channels. By affecting the abundance or distribution of potassium channels in excitable membranes, such compounds would essentially act as ion channel blockers, and thus would likely have adverse neurophysiological effects. Finally, any compound that can effectively block the strong .alpha.-NAB/.beta.-core binding interaction (i.e., compounds identified using this modified yeast two-hybrid system) must themselves have extremely high binding affinity for potassium channel subunits, and thus would likely be toxic to a mammalian host.

In view of the complexity of ion channel pharmacology and its attractiveness as a target site for the discovery of novel therapeutic compounds, there exists a need for an alternative technique which will enable the large-scale screening of compounds for ion channel modulatory activity in a simple and reliable manner. The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides several novel methods and compositions for identifying compounds which affect binding between two key regulatory regions of voltage-gated ion channels. More specifically, the invention relates to methods and compositions for identifying compounds which affect the binding of an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein. Compounds that disrupt or facilitate the functional or binding interaction of these two key regulatory regions have significant modulatory effects on ion channel activity, and thus are likely to be useful for treating and/or preventing a wide variety of diseases and pathological conditions associated with ion channel dysfunction. Such conditions include, for example, neurological disorders, cardiac diseases, metabolic diseases, tumor-driven diseases, and autoimmune diseases. Many of these compounds are expected to have potent anticonvulsant and neuroprotective properties which will prove especially useful for the prevention and/or treatment of neurodegenerative disorders such as epilepsy, stroke, cerebral ischemia, cerebral palsy, hypoglycemia, Alzheimer's disease, Huntington's disease, asphyxia and anoxia, as well as for the treatment of neuropathic pain, spinal cord trauma, and traumatic brain injury.

In one aspect, the invention provides methods of evaluating and screening candidate compounds for the ability to affect binding of an intracellular receptor region of an .alpha.-subunit and an amino-terminal inactivation region of an ion channel protein. The methods comprise contacting the compound with the intracellular receptor region and the amino-terminal inactivation region, and determining the ability of the compound to interfere with or facilitate the functional interaction or binding of these two regions. A decrease in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound inhibits functional interaction or direct binding between these two regulatory regions. Similarly, an increase in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound facilitates functional interaction between these two regulatory regions.

In another aspect, the invention provides methods for evaluating or screening candidate compounds comprising adding a candidate compound to a modified host cell and comparing the expression of a reporter gene in the presence and absence of the compound. A decrease (or increase) in expression of the reporter gene is an indication that the compound inhibits (or promotes) functional or binding interaction between the intracellular receptor region and the amino-terminal inactivation region.

In yet another aspect, the invention provides modified host cells and methods for evaluating or screening candidate compounds for ion channel modulatory activity. The modified host cells contain a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage to either (i) an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel or (ii) an amino-terminal inactivation region of an ion channel protein, and a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to the intracellular receptor region if the DNA-binding domain is in polypeptide linkage to the amino-terminal inactivation region or to the amino-terminal inactivation region if the DNA-binding domain is in polypeptide linkage to the intracellular receptor region. The modified host cell may optionally comprise a reporter gene whose expression is inhibited in the presence of an inhibitor of N-type inactivation.

In still another aspect, the modified host cell contains a first hybrid protein comprising an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel in polypeptide linkage to a first peptide of a peptide binding pair, and a second hybrid protein comprising an amino-terminal inactivation region of an ion channel protein in polypeptide linkage to a second peptide of the peptide binding pair, wherein binding interaction between the two peptides causes activation of a signal transduction pathway in the modified host cell. Activation of the signal transduction pathway does not occur in the presence of a molecule which inhibits binding of the intracellular receptor region and the amino-terminal inactivation region of an ion channel protein.

In other aspects, the invention provides polynucleotides, expression vectors, and host cells transfected or transformed with expression vectors containing nucleotide sequences which encode an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein, or biologically active fragments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid comparison of the intracellular receptor regions ("loops") of the human Kv1.2 ("hKv1.2"), human Kv1.3 ("hKv1.3"), human Kv1.4 ("hKv1.4"), human Kv1.5 ("hKv1.5"), human Kv1.6 ("hKv1.6") and human Kv3.4 ("hKv3.4") are shown in alignment with human Kv1.1 ("hKv1.1"). The black boxes indicate sequence identity; shaded boxes indicate conservative ammo acid substitutions. The intracellular receptor regions of hKv1.1, hKv1.2, hKv1.3, hKv1.4, hKv1.5, hKv1.6, and hKv3.4 are designated as hKv1.1 LOOP (SEQ ID NO:1), hKv1.2LOOP (SEQ ID NO:1), hKv1.3 LOOP (SEQ ID NO:1), hKv1.4 LOOP (SEQ ID NO:2), hKv1.5 LOOP (SEQ ID NO:24), hKv1.6 LOOP (SEQ ID NO:24), and hKv3.4 LOOP (SEQ ID NO:25), respectively. paragraph beginning at page 6, line 5, amended paragraph:

FIG. 2. Amino acid comparison of the amino-terminal inactivation regions ("N") of the human Kv.beta.1b ("hKv.beta.1b"; also known as "hKv.beta.1.2"), human Kv.beta.1c ("hKv.beta.1c"; also known as "hKv.beta.1.3"), human Kv.beta.3 ("hKv.beta.3"), human Kv1.4 ("hKv1.4"), and human Kv3.4 ("hKv3.4") are shown in alignment with human Kv.beta.1 ("hKv.beta.1"). The black boxes indicate sequence identity; shaded boxes indicate conservative amino acid substitutions. The amino-terminal inactivation regions of hKv.sym.1, hKv.beta.1b, hKv.beta.3, hKv3.4, hKv.beta.1c, and hKv1.4 are designated as hKv.beta.1N (SEQ ID NO:5), hKv.beta.1bN (SEQ ID NO:26), hKv.beta.3N (SEQ ID NO:26), hKv3.4N (SEQ ID NO:27), hKv.beta.1CN (SEQ ID NO:28), and hKv1.4N (SEQ ID NO:6), respectively.

FIG. 3. (FIG. 3a) Electrophysiological current recordings of inactivating channels expressed in Xenopus oocytes. Xenopus oocytes were injected with 0.5 ng of hKv1.1:10 ng of hKv.beta.1 mRNA transcribed in vitro using standard procedures (Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual). Cells were challenged with families of voltage pulses of 200 ms duration ranging from -60 mV to 50 mV once every 2 min. Cells were exposed to each dose of "Wy-8340" (C.sub.10H.sub.15NO; 6-aminothymol or 4-amino-2-isopropyl-5-methylphenol) for 6 min and cumulative dose-response curves were performed. Relative inactivation was calculated by measuring the amplitude of the peak and steady-state currents, setting the inactivation (without compound) for each cell to 100% (i.e., maximum) and measuring the percent disinactivation with each dose of compound. (FIG. 3b) Concentration-response curves showing the effect of Wy-8340 on inactivation of hKv1.1 and hkv.beta.1 channels expressed in Xenopus Oocytes.

FIG. 4. Protection against pentelenetetrazol-induced seizures in the mouse. Adult male mice were treated with valproic acid (FIG. 4a), Compound A (FIG. 4b), and Compound B (FIG. 4c) at doses from 30 178 mg/kg i.p. (n=8/dose). Thirty minutes later, these animals were challenged with pentelenetetrazol (85 mg/kg, SC) and observed for onset of seizures during a 30 minute test period. The number of animals protected from seizures was plotted versus dose of test compound and ED50s were estimated from this dose-response data.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence containing the intracellular receptor region of the .alpha.-subunit of the hKv1.1 protein.

SEQ ID NO:2 is an amino acid sequence containing the intracellular receptor region of the .alpha.-subunit of the hKv1.4 protein.

SEQ ID NO:3 is a nucleotide sequence containing the nucleotide sequence encoding the intracellular receptor region of the .alpha.-subunit of the hKv1.1 protein.

SEQ ID NO:4 is a nucleotide sequence containing the nucleotide sequence encoding the intracellular receptor region of the .alpha.-subunit of the hKv1.4 protein.

SEQ ID NO:5 is an amino acid sequence containing the amino-terminal inactivation region of the hKv.beta.1 protein.

SEQ ID NO:6 is an amino acid sequence containing the amino-terminal inactivation region of the hKv1.4 protein.

SEQ ID NO:7 is a nucleotide sequence containing the nucleotide sequence encoding the amino-terminal inactivation region of the hKv.beta.1 protein.

SEQ ID NO:8 is a nucleotide sequence containing the nucleotide sequence encoding the amino-terminal inactivation region of the hKv1.4 protein.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

As used herein, the term "ion channel protein" refers generally to voltage-gated ion channels, including the pore-forming .alpha.-subunit proteins (".alpha.-subunits") and the cytoplasmic.beta.subunit proteins (also known in the art as "auxiliary subunits"or ".beta.-subunits").

The term "intracellular receptor region" means a portion of an .alpha.-subunit of a voltage-gated ion channel which can form a specific binding interaction with an amino-terminal portion (i.e., an amino-terminal inactivation region) of an ion channel protein. The term "S4 S5 cytoplasmic receptor domain" refers to the stretch of hydrophilic amino acid residues between the membrane-spanning segments S4 and S5 (also known as "H4") of a pore-forming .alpha.-subunit.

The term "amino-terminal inactivation region" means a portion of an ion channel protein which can form a specific binding interaction with an intracellular receptor region of an .alpha.-subunit. The amino-terminal inactivation region (also known in the art as the "inactivation gate," "inactivating ball," or "ball peptide") is a globular domain on the amino-terminus of an ion channel protein, including, for example, the globular domain on the amino-terminus of an .alpha.-subunit (i.e., linked to the first membrane-spanning segment of an .alpha.-subunit) or the amino-terminus of .beta.-subunit.

As used herein, the term "biologically active fragment" means a portion of an intracellular receptor region or an amino-terminal inactivation region capable of binding to an amino-terminal inactivation region or an intracellular receptor region, respectively. The term "fragment," as applied in this context, will typically be at least about 6 amino acids, usually at least about 8 contiguous amino acids, preferably at least about 10 contiguous amino acids, more preferably at least about 12 contiguous amino acids, and most preferably at least about 14 or more contiguous amino acids in length. Such fragments can be generated by methods known to those skilled in the art, including proteolytic cleavage of the polypeptide, de novo synthesis of the fragment, or genetic engineering.

As used herein, the term "peptide binding pair" means a pair of proteins or polypeptides whose binding interaction results in activation of a signal transduction pathway in a cell or organism. The term "effector molecule" means a peptide or polypeptide that can be expressed as a fusion protein and, when so expressed, can activate an "indicator molecule," provided the effector molecule is translocated to the cell compartment containing the indicator molecule. As used herein, the term "indicator molecule" means a molecule acted upon by the effector molecule, either directly or indirectly through an intermediate molecule, such that activation of the indicator molecule produces a detectable signal. The term "activate" or "activation," when used in reference to an indicator molecule, means that the effector molecule has changed the indicator molecule such that the effector function can be detected as a signal generated by the changed indicator molecule or by a molecule subsequently acted upon by the changed indicator molecule. Various effector molecules and indicator molecules are known in the art, including, for example, the "effector proteins" and "reporter molecules," respectively, described in U.S. Pat. No. 5,776,689 (Karin et al.), which is incorporated by reference in its entirety herein.

The term "cell compartment localization domain" means a peptide or polypeptide sequence that directs translocation of a fusion protein containing the effector molecule to a particular cell compartment. Various cell compartment localization domains are known in the art, including, for example, plasma membrane localization sequences, nuclear localization signal sequences, mitochondrial membrane localization sequences, and the like. See, e.g., Karin et al., supra.

Provided by the present invention are methods and compositions for identifying compounds which affect the binding interaction between two key regulatory regions of voltage-gated ion channels, namely an intracellular receptor region of an .alpha.-subunit and an amino-terminal inactivation region of an ion channel protein. The present inventors have discovered that compounds that disrupt binding of these two key regulatory regions have significant modulatory effects on ion channel activity, and thus are expected to be clinically significant therapeutic agents for treating and/or preventing a wide variety of diseases and pathological conditions associated with ion channel dysfunction. Such compounds may also be useful as commercial or biological research reagents, for example, to further define interaction domains of ion channel proteins. Surprisingly, compounds identified using the methods of the invention have been found to selectively and dose-dependently eliminate "N-type" ion channel inactivation (discussed below) in modified host cells expressing these heterologous regulatory regions. Also surprisingly, these compounds exhibit potent anti-seizure activity both in vitro and in vivo. Thus, the present invention represents a significant advance in the pharmacological and pharmaceutical arts, by providing a reliable high-throughput screen which can identify potent and selective modulators of N-type inactivation of voltage-gated ion channels.

As discussed above, voltage-gated ion channels, and particularly potassium and sodium channels, are important determinants of membrane excitability. Each of these families of ion channels comprise several classes of proteins, including the pore-forming .alpha.-subunits and the auxiliary .beta.-subunits. The .alpha.-subunits comprise six transmembrane-spanning regions, usually referred to sequentially as S1 through S6. The sequences between segments S4 and S5, the "S4 S5 region," and sequences of segment S6 form part of the inner mouth and pore of ion channels, whereas part of the H5 region forms part of the outer mouth and outer half of the pore. See Heinemann, S., et al., J. Physiol. 88:173 180 (1994); Durrell, S. R. and R. Guy, Biophys. J. 62:238 250 (1992). Segment S4 contains several positively charged amino acids and is believed to be the voltage-sensing a helix. The amino-terminal domain of ion channels is involved in subunit assembly and channel inactivation. Li, M., et al., Science 257:1225 1230 (1992); Hoshi, T., et al., Science 250:533 538 (1990). Rapidly inactivating A-type ion channels have an amino-terminal inactivation domain which is able to close the open channel from the inside at depolarized membrane potentials, as will be discussed more fully below. This type of inactivation is often referred to as "N-type" inactivation. Hoshi, et al., supra.

N-type inactivation operates in a ball-and-chain type mechanism. Hoshi, et al., supra; Zagotta, W. N., et al., Science 250:568 570 (1990). The amino terminus of the .alpha.-subunit is the "ball" which swings into the open pore, binds to a receptor site (the intracellular receptor region) and thereby plugs the ion channel pore. This mechanism has been confirmed by several researchers. Mutations within the amino-terminal ball or a deletion of this ball abolishes rapid N-type inactivation. Also, mutations within the S4 S5 region disrupt N-type inactivation. Isacoff, E. Y., et al., Nature 353:86 90 (1991). The on-rate time constant for binding the inactivating domain to the receptor is voltage-dependent, such that depolarization of the membrane accelerates the binding. Conversely, the off-rate time constant is also voltage-dependent, but is significantly faster at negative than at more positive membrane potentials. S. Heinemann, supra; Ruppersberg, J. P., et al., Nature 353:657 660 (1991). Upon depolarization, the ball moves into the electric field of the membrane and obstructs the open channel pore. Upon repolarization of the membrane the off-rate is faster than the on-rate time constant. This causes the ball to swing away from the ion channel pore and to free the ion channel from inactivation. Thus, the ratio between on- and off-rate at negative membrane potentials may also be an important determinant for the refactory period which A-type ion channels require for recovery from inactivation. S. Heinemann, supra.

As described above for .alpha.-subunits, the amino terminus of .beta.-subunits also functions as a tethered inactivating ball which swings into the inner mouth of the ion channels and occludes the pore upon depolarization of the membrane. The amino terminus of .beta.-subunits has been shown to be structurally and functionally similar to the inactivating ball domain of .alpha.-subunits. S. Heinemann, supra. A hallmark of the inactivating domain of both .alpha. and .beta.-subunits is the presence of an amino terminal cysteine followed by a cluster of positively charged amino acids (lysines and arginines). Ruppersberg, et al., supra. The latter may be important for moving the inactivating ball into the electric field, the cysteine for interaction with the intracellular receptor region at or near the entrance of the ion channel pore. S. Heinemann, supra.

In one aspect, the invention provides methods for detecting a compound that inhibits binding of an intracellular receptor region of an .alpha.-subunit and an amino-terminal inactivation region of an ion channel protein, thereby keeping rapidly inactivating channels open longer. The methods comprise contacting the compound with the intracellular receptor region and the amino-terminal inactivation region, and determining the ability of the compound to interfere with the functional interaction or binding of these two regions. A decrease in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound inhibits binding interaction between these two regulatory regions. Although this method will work using any appropriately constructed in vitro or in vivo system which allows monitoring of these specific interactions, the invention is preferably practiced using a modified host cell which expresses these heterologous regulatory regions, such as the two-hybrid system described below. The method is generally applicable to voltage-gated ion channels which inactivate via an N-type inactivation mechanism, and particularly voltage-gated potassium and sodium ion channels.

In one embodiment, the method comprises adding a candidate compound to a modified host cell and comparing the exhibition of a selected phenotype in the presence and absence of the compound, wherein the modified host cell is adapted to exhibit a change in phenotype only in the presence of a molecule which inhibits the binding of the intracellular receptor region to the amino-terminal inactivation region. Preferably, the modified host cell comprises an inverse selection (also known as "counter" or "rescue") "two-hybrid" system, such as the modified yeast two-hybrid screen described herein.

In another aspect, the invention provides modified host cells which are useful for screening candidate compounds for ion channel modulatory activity. In a preferred embodiment, the modified host cell comprises a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage to either (i) an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel or (ii) an amino-terminal inactivation region of an ion channel protein, and a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to the intracellular receptor region if the DNA-binding domain is in polypeptide linkage to the amino-terminal inactivation region or to the amino-terminal inactivation region if the DNA-binding domain is in polypeptide linkage to the intracellular receptor region.

The intracellular receptor region of the .alpha.-subunit of a voltage-gated ion channel, for purposes of the present invention, are those regions of the .alpha.-subunit that bind to the amino-terminal inactivation region of an ion channel protein. By way of example, the amino acid sequences for the intracellular receptor regions of certain .alpha.-subunits are set forth herein in FIG. 1. These sequences can be easily identified in any .alpha.-subunit of a voltage-gated ion channel given the high degree of homology among these sequences. In the example of FIG. 1, the intracellular receptor regions ("loops") of the human Kv1.2 ("hKv1.2"), human Kv1.3 ("hKv1.3"), human Kv1.4 ("hKv1.4"), human Kv1.5 ("hKv1.5"), human Kv1.6 ("hKv1.6") and human Kv3.4 ("hKv3.4") are shown in alignment with human Kv1.1 ("hKv 1.1"). [As used herein and consistent with art-recognized usage, "Kv" refers to a voltage-gated potassium ion channel protein.] It is expected that intracellular receptor regions of currently unidentified .alpha.-subunits will contain a homology of at least 60%, preferably at least 75%, more preferably at least 85%, and most preferably at least 90 to 95%, based on the homologies present in the .alpha.-subunits of the hKv1 and hKv3 channel proteins. Due to the high degree of conservation of sequences among all known .alpha.-subunits of voltage-gated ion channels, additional members of the potassium channel family, as well as members of the sodium channel family, are expected to comprise intracellular receptor regions which are structurally and functionally equivalent to those of the hKv1.1 and hKv1.4 .alpha.-subunits exemplified in the Examples hereof. Thus, the general features contained and described herein will be applicable to newly discovered ion channel proteins.

The amino-terminal inactivation region of an ion channel protein, for purposes of the present invention, are those regions of the ion channel protein that bind to the intracellular receptor region of an .alpha.-subunit. By way of example, the amino acid sequences for the amino-terminal inactivation regions of certain ion channel proteins are set forth herein in FIG. 2. In the example of FIG. 2, the amino-terminal inactivation regions of human Kv.beta.1 b ("Kv.beta.1 b"; also known as "Kv.beta.1.2"), human Kv.beta.1c ("Kv.beta.1c"; also known as "Kv.beta.1.3"), Kv.beta.3 ("Kv.beta.3"), human Kv1.4 ("Kv1.4"), and human Kv3.4 ("Kv3.4") are shown in alignment with human Kv.beta.1 ("Kv.beta.1"). As can be seen in this figure, the amino-terminal inactivation regions in each of these subunits can be readily identified by the presence of an amino terminal cysteine residue connected to a string of positively charged amino acids (i.e., lysines and arginines). It is expected that amino-terminal inactivation regions of currently unidentified ion channel proteins will contain a homology of at least 60%, preferably of at least 75%, more preferably at least 85%, and most preferably at least 90 to 95%, based on the homologies present in the amino-terminal inactivation regions of the Kv.beta.1, Kv.beta.1.2, Kv.beta.1.3, Kv.beta.3, Kv1.4, and Kv3.4 channel proteins. Due to the characteristic chemical composition and structure of the globular domain on the amino-terminus of ion channel proteins, additional members of the potassium channel family, as well as members of the sodium channel family, are expected to comprise amino-terminal activation regions which are structurally and functionally equivalent to those of the hKv.beta.1 and hKv1.4 subunits exemplified in the Examples hereof. Thus, the general features contained and described herein will be applicable to newly discovered ion channel proteins.

In preferred embodiments, the voltage-gated ion channel is a potassium or sodium channel, the intracellular receptor region is an S4 S5 cytoplasmic receptor domain of an .alpha.-subunit or a biologically active fragment thereof, and the amino-terminal inactivation region is the amino-terminal domain of an .alpha.- or .beta.-subunit of a potassium or sodium channel protein, or a biologically active fragment thereof. Preferably, the intracellular receptor region comprises the S4 S5 cytoplasmic receptor domain of a potassium channel protein selected from the group consisting of Kv1.1, Kv1.4, and Kv3.4, and the amino-terminal inactivation region comprises the amino-terminal inactivation domain of a potassium channel protein selected from the group consisting of Kv.beta.1, Kv.beta.1.2, Kv.beta.1.3, Kv.beta.3, Kv1.4, and Kv3.4. In particularly preferred embodiments, the intracellular receptor region has an amino acid sequence as set forth in SEQUENCE (SEQ) ID NO:1 (GenBank Accession No. L02750) and SEQ ID NO:2 (GenBank Accession No. M55514), as well as DNA sequences encoding these sequences, such as the sequences shown in SEQ ID NO:3 (GenBank Accession No. L02750) and SEQ ID NO:4 (GenBank Accession No. M55514), and the amino-terminal inactivation region has an amino acid sequence as set forth in SEQ ID NO:5 (GenBank Accession No. X83127) and SEQ ID NO:6 (GenBank Accession No. L02751), as well as DNA sequences encoding these sequences, such as the sequences shown in SEQ ID NO:7 (GenBank Accession No. X83127) and SEQ ID NO:8 (GenBank Accession No. L02751). Also included are naturally occurring allelic sequences of SEQ ID NO:3, 4, 7 and 8, and equivalent degenerative codon sequences of the above.

The invention further provides methods for detecting a compound that inhibits binding of an intracellular receptor region of an .alpha.-subunit and an amino-terminal inactivation region of an ion channel protein utilizing an improved two-hybrid system, such as the yeast two-hybrid screen exemplified herein. The yeast two-hybrid screen is generally known in the art. See, e.g. Fields, et al., Nature 340:245 246 (1989), and as modified by Young, K. H. and B. A. Ozenberger in PCT WO 95/34646 (Dec. 21, 1995), the whole of which is incorporated by reference herein. The present invention provides an improved two-hybrid system by utilizing two vectors which have not heretofore been utilized in such a system. In particular, the present invention provides an improved two-hybrid system, wherein the improvement comprises a first vector containing nucleic acid sequences encoding a fusion protein of a DNA binding domain of a transcriptional activator and either (i) an intracellular receptor region of an .alpha.-subunit of a voltage-gated ion channel or (ii) an amino-terminal inactivation region of an ion channel protein, and a second vector containing nucleic acid sequences encoding a fusion protein of an activation domain of a transcriptional activator and the intracellular receptor region if the first vector encodes a fusion protein comprising the amino-terminal inactivation region or to the amino-terminal inactivation region if the first vector encodes a fusion protein comprising the intracellular receptor region. As will be appreciated by those skilled in this art, the expression of the DNA binding fusion protein and the activation fusion protein can be interchanged, such that the intracellular receptor region is expressed as a fusion with either the transcription DNA binding domain or the activation domain of the transcriptional activator.

Briefly, using a two-hybrid system, a candidate compound is introduced into the system (a host cell), and a change in a reporter or marker protein product is assayed. Any compound which alters the level of expression of the reporter or marker, as monitored by a suitable assay, is a potential drug candidate and may be suitable for further, in-depth studies of therapeutic applications. The candidate compound may be of any form suitable for entry into the cytoplasm and/or nucleus of the modified host cell. Under appropriate conditions, the candidate compound may be allowed to freely diffuse into the cell, or the delivery of the compound may be facilitated by techniques and substances which enhance cell permeability, a wide variety of which are known in the art. Methods for increasing cell permeability include, without limitation, the use of organic solvents such as dimethylsulfoxide, hydrolytic enzymes (which degrade cell walls), yeast cell mutants (e.g., erg-), liposomes, application of electrical current, and physical means such as compound-coated teflon pellets.

The host organism ("modified host cell") may be any eukaryotic or prokaryotic cell, or multicellular organism. Many strains of yeast cells known to those skilled in the art may be available as host cells for practicing the present invention. Suitable host cells may also be mammalian cells, such as Chinese hamster ovary cells (CHO), the monkey COS-1 cell line, and the mammalian cell CV-1, or amphibian cells, such as a Xenopus egg cell. Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277 298 (Plenum Press 1986) and references cited therein. In preferred embodiments, the modified host cell is a yeast or mammalian cell. More preferably, the modified host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. In a particularly preferred embodiment, the modified host cell is a yeast cell derived from a Saccharomyces organism having the genotype MATa, gal80, gal 4, his3, ade2-101, leu2-3, 112 trp1-901, ura3-52 cyh.sup.r LYS2:GAL.sub.UAS-HIS3.

The transcriptional activation protein ("transcriptional activator") may vary widely as long as the DNA binding domains and the activation domains are known or can be deduced by available scientific methods. The transcriptional activator may be any protein having two components, a DNA binding component and an activation component, wherein the transcriptional activator contains an acidic a-helix for the activation of transcription. Preferably, the transcriptional activator is selected from the group consisting of Gal4, Gcn4, Hap1, Adr1, Swi5, Stel2, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qal F, VP16, LexA, non-mammalian nuclear receptors (e.g., ecdysone) or mammalian nuclear receptors (e.g., estrogen, androgens, glucocorticoids, mineralocorticoids, retinoic acid and progesterone). See Picard, D., et al., Gene 86:257 261 (1990). Preferably, the transcriptional activator is a yeast protein, and more preferably, the transcriptional yeast protein is Gal4, Gcn4 or Adr1. In general, any DNA binding protein which functions with an activation domain may be used. A DNA binding protein may be substituted for the DNA binding domain of a transcriptional activation protein if the recognition sequences operatively associated with the reporter gene are correspondingly engineered. Illustrative of non-yeast DNA binding proteins are mammalian steroid receptors and bacterial LexA. See Wilson, T. E., et al., Science 252:1296 1300 (1990).

The modified host cell may comprise a reporter gene whose transcription is dependent upon binding between the first and second hybrid proteins, thereby reconstituting a transcriptional activator. The reporter gene is generally selected in order that the binding of the domains of the transcriptional activation protein can be monitored by well-known and straightforward techniques. Preferably, the reporter gene is selected based on its cost, ease of measuring its activity, and low background (i.e., the activity can be determined at relatively low levels of expression of the reporter gene because of a high signal to background ratio and/or minimal or no uninduced activity). Suitable reporter genes include, for example, genes which confer a selectable phenotype to cells in which the reporter gene is efficiently expressed, and/or encode a gene product (e.g., enzyme) which is conveniently detected such as by in situ assay, or the like. Illustrative of reporter genes which may be used in the present invention are reporter genes selected from the following: genes which confer sensitivity to a chemical, such as CYH2 (cyclohexamide sensitivity) and CAN1 (canavine); genes which confer resistance to a chemical (e.g., an antibiotic), such as neo.sup.r and KAN; genes which complement auxotrophic mutations in a host organism, such as HIS3, URA3, LEU2, ARG, MET, ADE, LYS, and TRP, and the like; genes which encode toxic gene products, such as ricin; and LACZ, LAC1, firefly luciferase, bacterial luciferase, green fluorescent protein, CAT (chloramphenicol acetyl transferase), alkaline phosphatase, horseradish peroxidase, and the like.

In one embodiment, the present invention may be practiced using a conventional two-hybrid system which relies upon a positive association between two Gal4 fusion proteins, thereby reconstituting a functional Gal4 transcriptional activator which then induces transcription of a reporter gene operably linked to a Gal4 binding site. Transcription of the reporter gene generally produces a positive readout, typically manifested either as an enzyme activity (e.g., .beta.-galactosidase) that can be identified by a calorimetric enzyme assay, or as enhanced cell growth on a defined medium (e.g., HIS3). Using conventional two-hybrid systems, a compound which is capable of inhibiting N-type inactivation of a voltage-gated ion channel is identified by its inhibitory affect on reporter gene expression (e.g., reduced enzyme activity or cell growth).

In a preferred embodiment, the methods of the present invention are practiced using an "inverse" (also known as "counter selection" or "reverse") two-hybrid system. Using an inverse two-hybrid system, compounds which are capable of affecting N-type inactivation of a voltage-gated ion channel will generate a selectable and/or detectable readout (e.g., complementation of an auxotrophic phenotype, expression of a detectable reporter molecule, and the like). Typically, an inverse two-hybrid system produces a positive readout under conditions wherein an agent blocks or otherwise inhibits the intermolecular binding of the interacting polypeptides (i.e., an intracellular receptor region of an .alpha.-subunit and an amino-terminal inactivation region of an ion channel protein). A positive readout condition is generally identified as one or more of the following detectable conditions: (1) an increased transcription rate of a reporter gene, (2) an increased concentration or abundance of a polypeptide product encoded by a reporter gene, typically such as an enzyme which can be readily assayed in vivo, and/or (3) a selectable or otherwise identifiable phenotypic change in the organism harboring the inverse two-hybrid system. Generally, a selectable or otherwise identifiable phenotypic change that characterizes