WO1999013895A1 - Polypeptide compositions that inhibit potassium channel activity and uses therefor - Google Patents

Abstract

Disclosed are novel polypeptide compositions that inhibit potassium channel activity, and in particular Kvl.3 channel activity. Various methods for making and using these synthetically-modified ShK-derived polypeptides and the nucleic acid sequences which encode them are disclosed, including the use of DNA segments as diagnostic probes and templates for protein production, and the use of peptides in a variety of therapeutic and diagnostic applications.

Description

DESCRIPTION

POLYPEPTIDE COMPOSITIONS THAT INHIBIT POTASSIUM

CHANNEL ACTIVITY AND USES THEREFOR

1.0 BACKGROUND OF THE INVENTION

The United States government has rights in the present invention pursuant to Grant R01-GM-54221 from the National Institutes of Health.

1.1 FIELD OF THE INVENTION The present invention relates generally to the fields of biological peptides and peptidomimetics. Disclosed are methods and compositions comprising modified ShK polypeptides, having improved potassium channel activity and decreased toxicity compared to native ShK polypeptides. Various methods for making and using these synthetically-modified ShK toxins, and polynucleotides encoding them are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of peptides in various immunological, diagnostic, and therapeutic applications.

1.2 DESCRIPTION OF RELATED ART

While many types of potassium channels are widely distributed in various tissues of the body, one of the delayed-rectifier channels, Kvl.3, is almost exclusively located in T lymphocytes (Cahalan et al, 1991; Lewis and Cahalan, 1995). This lymphocyte K channel has been shown to be homo-oligomeric, in contrast with many DR channels in the nervous and muscular systems, which can exist as hetero-oligomers containing more than one subunit. For instance, in the rat brain, most DR channels are of the Kvl.2 and Kvl .l types, and these two types of subunits also may be present in the same channel (Scott et al, 1994).

The mechanisms by which Kvl.3 channels affect lymphocyte proliferation are being investigated in several laboratories. The major evidence that they are involved is the ability of ChTx and margatoxin to inhibit lymphocyte proliferation and interleukin 2 production (Chandy et al, 1984; Price et al, 1989; Garcia-Calvo et al, 1993). Inhibition of Kvl.3 depolarizes the cell membrane sufficiently to decrease calcium influx, and this prevents elevation of free intracellular calcium concentration which is the stimulus for these two responses. The restricted tissue distribution of Kvl.3 and its immunosuppressive action upon T-cells has prompted several pharmaceutical companies to attempt development of specific Kvl.3 blockers for therapeutic use as immunosuppressants.

Until recently, K channel investigations were hampered by a paucity of selective neurotoxin probes, which have been so important for investigating sodium and calcium channels. But this has dramatically changed in the past few years. The sea anemone K channel toxins are the most recent addition to the K channel armamentarium. The dendrotoxins are relatively large peptides, and this has limited their utility in determining where they bind to the DR K channels, because of difficulties in preparing analogs or mutants of this toxin, of which the solid-phase synthesis has been recently accomplished. By exchanging functional domains of two DR channels, only one of which is sensitive to dendrotoxin, Stocker et al (1991) showed that the external loop between S5 and S6 contains at least part of the dendrotoxin receptor. Using K channel mutants, Hurst et al. (1991) reached a similar conclusion.

Chandy et al. (1995) have shown that the scorpion K channel toxins (charybdotoxin as prototype) are potent blockers of Kvl.l, Kvl.2, and 1.3 Shaker type DR channels. While charybdotoxin (ChTx) also blocks maxi-type K(Ca) channels, some newer ChTx homologs including margatoxin lack K(Ca) channel blocking activity. The scorpion K channel toxins are valuable tools for investigating these DR channels as well as the maxi-K(Ca) channels. Since they are also rather rigid molecules, they are also proving useful as "molecular calipers" for measuring distances between K channel amino acid residues in the outer vestibule of these channels (Stocker and Miller, 1994; Chandy, 1995).

Voltage-gated potassium (K⁺) channels regulate diverse biological processes (Chandy and Gutman, 1995). A short stretch of amino acids, the P-region, located Between the fifth and sixth transmembrane segments, contributes to the formation of the channel pore. Delineation of the spatial organization of the residues in the P-region would help define the structure of the ion conduction pathway and be valuable for understanding the mechanisms of ion permeation. Scorpion (ChTx, KTx) and sea anemone toxins (ShK) apparently interact strongly with residues in the P-region. It should be possible to deduce the spatial arrangement of the residues in the P-region by using these toxins as structural templates, provided the three- dimensional structures of the toxins is known. In preliminary studies using NMR and molecular modeling, The inventors have shown that four scorpion toxin-blockers of K⁺ channels, kaliotoxin (KTX), margatoxin (MgTX), noxiustoxin (NTX) and charybdotoxin (ChTX) have a similar tertiary fold.

Many times different molecules utilize the same functional groups to bind with their receptors. In the case of the Na-channel, the toxins tetrodotoxin and saxitoxin are heterocyclic organic compounds which utilize essential guanidinium functionalities to block Na channel function by binding to the Site I receptor (Catterall, 1980). Mu-conotoxins, short peptide toxins isolated from Conus venoms, also competitively bind to the same site I receptor. Interestingly, these toxins are able to discriminate between the tetrodotoxin/saxitoxin receptor on muscle and nerve sodium channels (Ohizumi et al, 1986). Structurally, these peptide toxins are highly constrained by three disulfide bonds which are utilized to correctly position a guanidinium functionality present on an invariant Arg residue (Arg 13 in μ-CgTX GUI A) for channel- blocking activity (Sato et al, 1991). Thus, in tetrodotoxin and saxitoxin, the essential binding features of μ-conotoxin have been naturally incorporated into a small organic type of scaffold. Peptides are characteristically highly flexible molecules whose structure is strongly influenced by their environment (Marshall et al, 1978). Nature introduces conformational constraints such as disulfide bonds to help lock a molecule into the biologically active structure. These types of constraints and other structures such as α-helix, β-sheet and reverse turns combine to form the architecture for a peptide/protein's three dimensional structure. The surface localization of turns in proteins, and the predominance of residues containing potentially pharmacophoric information has lead to the hypothesis that turns play a critical role in recognition events (Rose et al, 1985). The stability of α-helical conformations in peptides has also been found to be essential for biological activity in many different systems (Kaiser and Kezdy, 1983).

1.3 DEFICIENCIES IN THE PRIOR ART

What is lacking in the prior art is the identification of polypeptide and peptidomimetic compositions which selectively interact with Kv channels, and in particular, Kvl .3. Also lacking are compositions which decrease activation of T-cell lymphocytes, and which are useful in the treatment of autoimmune diseases and in immunosuppression regimens. 2.0 SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other limitations in the prior art by providing novel ShK-derived polypeptide compositions which selectively interact and reduce the activity of Kv channels, and in particular, Kvl .3 potassium ion channels.

2.1 SHK TOXIN-ENCODING DNA SEGMENTS

The present invention also concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the novel peptides disclosed herein. DNA segments encoding these peptide species may prove to encode proteins, polypeptides, subunits, functional domains, and the like of ShK toxin-related or other non- related gene products. In addition these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art.

As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a ShK toxin or peptide refers to a DNA segment that contains ShK-derived polypeptide coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained, which in the instant case is the genome of sea anemones of the genus Stichodactyla, and in particular, the species of Stichodactyla known as S. helianthus. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified ShK-derived polypeptide-encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein-, polypeptide- or peptide- encoding unit. As will be understood by those skilled in the art, this functional term includes both genomic sequences, operon sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding a ShK toxin, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode an ShK-derived polypeptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in any of SEQ ID NO:l to SEQ ID NO:3, or the amino acid sequences derived from any one of these sequences as described in Example 6.

The term "a sequence essentially as set forth in any of SEQ ID NO:l to SEQ ID NO: 3," means that the sequence substantially corresponds to a portion of the sequence of any of SEQ ID NO:l to SEQ ID NO:3, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences, or to any of the amino acid sequences derived from such sequences as described in Example 6.

The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of any of SEQ ID NO:l to SEQ ID NO:3 will be sequences that are "essentially as set forth in any of SEQ ID NO:l to SEQ ID NO:3."

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch encoding either of the peptide sequence disclosed in SEQ ID NO:l, or that are identical to or complementary to DNA sequences which encode any of the peptides disclosed in SEQ ID NO:l. For example, DNA sequences such as about 18 nucleotides, and that are up to about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 18, 19, 20, 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and including sequences of about 5200 nucleotides and the like.

It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of any of SEQ ID NO:l to SEQ ID NO:3. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences. The DNA segments of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon degeneracy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site- directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein.

2.2 DNA SEGMENTS ENCODING SHK POLYPEPTIDES AS HYBRIDIZATION

PROBES AND PRIMERS

In addition to their use in directing the expression of ShK toxins or peptides of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments encoding ShK or ShK analogs that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000 bp, etc. (including all intermediate lengths and up to and including the full-length sequence of 5200 basepairs will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to ShK toxin- encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 10 to about 14, or from about 15 to about 20, or about 30, or about 40, or about 50, or even of from about 100 to about 200 nucleotides or so, identical or complementary to a DNA sequence encoding ShK or ShK-derived polypeptides, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10 and 14 up to about 100 or about 200 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U. S. Patents 4,683,195 and 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating ShK toxin-encoding DNA segments. Detection of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U. S. Patents 4,965,188 and 5,176,995 (each incoφorated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al, 1990, 1994; Segal 1976; Prokop, 1991 ; and Kuby, 1994, are particularly relevant.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate ShK toxin-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross- hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

2.3 VECTORS EXPRESSING POTASSIUM CHANNEL INHIBITORY PEPTIDES

In other embodiments, it is contemplated that certain advantages will be gained by positioning a nucleic acid segment encoding one or more of the desired potassium-channel inhibitory polypeptides under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a ShK polypeptide or ShK-derived peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any animal, bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).

In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of ShK peptides or epitopic core regions, such as may be used to generate anti-ShK polypeptide antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful. Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequence from SEQ ID NO:l.

2.4 TRANSGENIC ANIMALS AND TRANSFORMED HOST CELLS EXPRESSING SHK AND SHK-DERIVED POLYPEPTIDES

In yet another aspect, the present invention provides methods for producing a transgenic cell which expresses a nucleic acid segment encoding the novel ShK and ShK- derived polypeptides of the present invention. The process of producing transformed cells is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operably linked to a coding region that encodes a

ShK or ShK-derived polypeptide, or a synthetically-prepared nucleic acid sequence which encodes such a polypeptide. Such a coding region is generally operably linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant ShK polypeptide expressed in a particular transformed cell, the invention also provides for the expression of nucleic acid segments encoding ShK antisense mRNAs. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic cells which express a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein. As used herein, the term "transgenic cell" is intended to refer to a cell that has incoφorated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed cell, such as genes which may normally be present in the non-transformed cell but which one desires to either genetically engineer or to have altered expression.

It is contemplated that in some instances the genome of a transgenic cell of the present invention will have been augmented through the stable introduction of one or more ShK transgenes, either native, synthetically modified, or mutated. In some instances, more than one transgene will be incoφorated into the genome of the transformed host cell. Such is the case when more than one ShK polypeptide-encoding DNA segment is incoφorated into the genome of such a cell. In certain situations, it may be desirable to have one, two, three, four, or even more S. helianthus ShK polypeptide-encoding nucleic acid segments (either native or recombinantly-engineered) incoφorated and stably expressed in the transformed transgenic cell.

A preferred gene which may be introduced includes, for example, a ShK polypeptide-encoding DNA sequence from sea anemone origin, and particularly one or more of those described herein which are obtained from Stichodactyla spp. Highly preferred nucleic acid sequences are those obtained from S. helianthus, or any of those sequences which have been genetically engineered to decrease or increase the activity of the ShK polypeptide in such a transformed host cell.

Means for transforming a host cell and the preparation of a transgenic cell line are well-known in the art, and are discussed herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed ShK polypeptides and peptide analogs. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified ShK polypeptide, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the transformed cell.

2.5 MUTAGENESIS OF POLYPEPTIDES AND POLYPEPTIDE ENCODING DNAs

In certain embodiments, it is desirable to prepare mutant polypeptides and/or polynucleotides that encode them. Once the structure of the desired peptide to be mutagenized has been analyzed, it may often be desirable to introduce one or more mutations into either the polypeptide sequencer, alternatively, into the DNA sequence encoding the ShK-derived polypeptide for the puφose of producing a mutated peptide with altered biological properties, and in particular, increased channel inhibitory activity, increased peptide stability, and or decreased toxicity.

To that end, the present invention encompasses both site-specific mutagenesis methods and random mutagenesis of a nucleic acid segment encoding a channel-inhibitory polypeptide of the present invention. Using the assay methods described herein, one may then identify mutants arising from these procedures which have improved channel inhibitory activity, increased peptide stability, and or decreased toxicity

The means for mutagenizing a DNA segment encoding a polypeptide are well- known to those of skill in the art. Modifications may be made by random, or site-specific mutagenesis procedures. The nucleic acid may be modified by altering its structure through the addition or deletion of one or more nucleotides from the sequence.

Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular polypeptide. In particular, site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incoφorating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site- specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. Alternatively, given the small size of the disclosed ShK polypeptides, de novo synthesis of either the peptide itself, or de novo synthesis of a polynucleotide that encodes it is contemplated to be particularly advantageous in designing improved peptides.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. A genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones incoφorating the mutagenic oligonucleotide. Alternatively, the use of PCR™ with commercially available thermostable enzymes such as Taq polymerase may be used to incoφorate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. The PCR™-mediated mutagenesis procedures of Tomic et al. (1990) and Upender et al. (1995) provide two examples of such protocols. A PCR™ employing a thermostable ligase in addition to a thermostable polymerase may also be used to incoφorate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol. The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent 4,237,224, specifically incoφorated herein by reference in its entirety

A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U. S. Patents 4,683,195, 4,683,202 and 4,800,159 (each of which is specifically incoφorated herein by reference in its entirety). Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase

(e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308, incoφorated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U. S. Patent 4,883,750, specifically incoφorated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase™, described in Intl. Pat. Appl. Publ. No. PCT US87/00880, incoφorated herein by reference in its entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[α-thio]triphosphates in one strand of a restriction site (Walker et al, 1992, incoφorated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA

Sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' end sequences of non-Cry-specific DNA and an internal sequence of a

Cry-specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe identified as distinctive products generating a signal which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a cry-specific expressed nucleic acid Still other amplification methods described in Great Britain Pat. Appl. No. 2 202 328, and in Intl. Pat. Appl. Publ. No. PCT US89/01025, each of which is incoφorated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR™ like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al, 1989; Intl. Pat. Appl. Publ. No. WO 88/10315, incoφorated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has crystal protein-specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second crystal protein-specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate crystal protein-specific sequences. Eur. Pat. Appl. Publ. No. 329,822, incoφorated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA

Intl. Pat. Appl. Publ. No. WO 89/06700, incoφorated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" (Frohman, 1990), and "one-sided PCR™" (Ohara, 1989) which are well- known to those of skill in the art.

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu and Dean, 1996, incoφorated herein by reference in its entirety), may also be used in the amplification of DNA sequences of the present invention.

2.6 PEPTIDE-SPECIFIC ANTIBODY COMPOSITIONS AND METHODS OF MAKING In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the ShK polypeptides and toxin analogs disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incoφorated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, rn-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine.

As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, incoφorated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified ShK or ShK derived polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen

7 8 from an immunized mouse contains approximately 5 x 10 to 2 x 10 lymphocytes. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,

LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3- NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line. Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (vol./vol.) PEG, (Gefter et al, 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^" to 1 x 10^" . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells. This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like. The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

2.7 ELISAs AND IMMUNOPRECIPITATION ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incoφorating ShK polypeptide antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween^®. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from about 2 to about 4 hours, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween , or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera- bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g. , incubation for 2 hours at room temperature in a PBS -containing solution such as PBS Tween ).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol puφle or 2,2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

The anti-ShK polypeptide antibodies of the present invention are particularly useful for the isolation of other ShK polypeptide antigens by immunoprecipitation.

Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. enzyme-substrate pairs. 2.8 WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-peptide antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immuno-precipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.

Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.

2.9 POLYPEPTIDE SCREENING AND DETECTION KITS

The present invention contemplates methods and kits for screening samples suspected of containing ShK polypeptides or ShK-derived polypeptides, or cells producing such polypeptides. A kit may contain one or more antibodies of the present invention, and may also contain reagent(s) for detecting an interaction between a sample and an antibody of the present invention. The provided reagent(s) can be radio-, fluorescently- or enzymatically-labeled. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.

The reagent(s) of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent(s) are provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent(s) provided are attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent(s) provided are a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided. In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the ShK or ShK-derived polypeptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect ShK or ShK-derived epitope-containing peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like. Generally, immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying puφoses, it is proposed that virtually any sample suspected of comprising either a ShK or ShK-derived polypeptides or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of ShK or ShK-derived polypeptides and/or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing ShK or ShK-derived polypeptides. Generally speaking, kits in accordance with the present invention will include a suitable ShK or ShK-derived polypeptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.

The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

2.10 EPITOPIC CORE SEQUENCES The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incoφorates an epitope that is immunologically cross-reactive with one or more anti- ShK polypeptide antibodies. In particular, the invention concerns epitopic core sequences derived from ShK or ShK-derived polypeptides. As used herein, the term "incoφorating an epitope(s) that is immunologically cross- reactive with one or more anti- ShK polypeptides antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a ShK or ShK-derived polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the ShK or ShK- derived polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

The identification of immunodominant epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U. S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al, 1988; U. S. Patent Number 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incoφorated into peptides, either through the application of either peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to about 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic ShK or ShK-derived polypeptides will provide advantages in certain circumstances, for example, in the preparation of immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution. It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to ShK or ShK-derived polypeptides. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the particular polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on the ShK or ShK-derived polypeptide-specific antibodies disclosed herein. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U. S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al, 1988). Computerized peptide sequence analysis programs (e.g., DNAStar software, DNAStar, Inc., Madison, WI) may also be useful in designing synthetic peptides in accordance with the present disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.

However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at about 4°C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use. 2.11 BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. In particular embodiments of the invention, mutated ShK or ShK- derived polypeptides are contemplated to be useful for the methods of the invention. The amino acid changes may be achieved by changing the codons of a nucleic acid segment encoding the polypeptide, or alternatively, by directly synthesizing the mutated polypeptide directly. The substituted amino acids may be either naturally-occuring amino acids, or alternatively, using non-natural amino acids such as ornithine, diaminopropionic acid (DAP), Norleucine (Nle), Homocitrulene, Bpa, Nph, Apa, and the like. For synthesis of polypeptides comprising naturally-occuring amino acids, a synthetic DNA segment may be constructed and translated using the codon table shown in Table 1.

TABLE 1

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC uuu

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG ecu

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Tφ w UGG

Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incoφorate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (—1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (— 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within +0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101, incoφorated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within +0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A. Electrophysiological analysis of ShK block of mKvl.3 and mKvl.l. Shown are typical Kvl.3 currents expressed in RBL cells or in a L929 cell line stably expressing these channels, studied in the whole-cell configuration and blocked with ShK added to the external bathing solution.

FIG. IB. Electrophysiological analysis of ShK block of mKvl.3 and mKvl.l. Shown is a Hill plot of data in FIG. 2A.

FIG. IC. Electrophysiological analysis of ShK block of mKvl.3 and mKvl.l. Shown are typical Kvl.l currents in L929 cells studied in the whole-cell configuration and blocked with external ShK.

FIG. ID. Electrophysiological analysis of ShK block of mKvl.3 and mKvl.l. Shown is a Hill plot of data in FIG. 2C.

FIG. 2. Peptide binding studies. Points reveal the mean +SEM percent displacement (n=4) by each peptide concentration of specifically-bound I-ChTX in membranes transfected with hKvl.3, as described in Materials and Methods. Lines were iteratively fitted by Origin 4.1 software (Microcal Coφ.) to the following expression: 100 / [1+ (IC₅₀/x)ⁿ ], where x is toxin concentration, IC₅₀ is the concentration producing one-half maximal block, and nH is the Hill factor. Fitted parameters obtained from the individual studies were: ShK: IC₅₀ 118 ± 20 pM; nH =1.60 + 0.18, n = 5; ShK-DAP²²: IC₅₀ = 102 + 17 pM; nH =1.53 ± 0.15, n = 8; ShK-Nle²²: IC₅₀ = 663 + 172 pM, nH = 1.62 + 0.27, n = 6; MgTX: IC₅₀=79 ± 10 pM, nH = 1.71 ± 0.3, n = 6.

FIG. 3A. Identifying Kvl.3:peptide interactions by mutant cycle analysis. Mutant cycles for the pairs Kvl.3 [His⁴⁰⁴ - Val⁴⁰⁴]-ShK[DAP²²^Nle²²] and Kvl.3 [His⁴⁰⁴^ Val⁴⁰⁴]-ShK[Lys²²^Nle²²]. FIG. 3B. Identifying Kvl.3:peptide interactions by mutant cycle analysis.

Three dimensional bar graph showing coupling energies DDG (kcal.mol^" ) for various peptide: channel interactions. The horizontal line represents the 0.8 kcal.mol^" cutoff. Data for the mutant cycle Kvl.3[Asp³⁸⁶ - Lys³⁸⁶]-ShK[Arg²⁹ - Ala²⁹] are as follows: Asp³⁸⁶:Arg²⁹ K_d = 21 pM; Lys³⁸⁶:Arg²⁹ K_d = 660 pM; Asp³⁸⁶:Ala²⁹ K_d = 13 pM; Lys³⁸⁶:Ala²⁹ K_d = 1920 pM; W = 4.7.

FIG. 4A. Typical Kvl.3 currents expressed in RBL cells or in a L929 cell line stably expressing these channels, studied in the whole-cell configuration and blocked with external ShK-DAP²².

FIG. 4B. Hill plot of data in FIG. 5 A. FIG. 4C. External ShK-DAP²² is significantly less potent on Kvl .1.

FIG. 4D. Hill plot of data in FIG. 5C.

FIG. 5. MgTX, ShK, ShK-DAP²² and ShK-Nle²² suppress anti-CD3 induced H-thymidine incorporation by human peripheral blood T-cells. Points show the mean percent (+SEM; n=4-10) inhibition produced by each peptide, as determined by the assay protocol described. Lines were iteratively fitted by Origin using to the following expression:

A-i / [1+ (IC₅₀/x)ⁿ ], where x is toxin concentration, Aj is the maximal block achieved, IC₅₀ is the concentration producing one-half maximal block, and nH is the Hill factor for the fitted line. Fitted parameters obtained were: ShK: A,=53%; IC₅₀=50 pM; nH = 0.77; ShK-DAP²²: A,=50%; IC₅₍ =170 pM; nH = 0.64; MgTX: A,=59%; IC₅₀=390 pM; nH = 0.69. FIG. 6A. Parameters characterizing the final 20 structures of ShK-DAP , plotted as a function of residue number. Values for ShK (Tudor et al. , 1996) are shown on the left-hand side for comparison. Upper-bound restraints used in final round of structural refinement shown as long-range (black), medium-range (cross-hatched), sequential (diagonal shading) and intra-residue (unshaded).

FIG. 6B. Parameters characterizing the final 20 structures of ShK-DAP²², plotted as a function of residue number. Values for ShK (Tudor et al , 1996) are shown on the left-hand side for comparison. RMS differences from mean structure for N, C^α and C atoms following supeφosition over the whole molecule. FIG. 6C. Parameters characterizing the final 20 structures of ShK-DAP²², plotted as a function of residue number. Values for ShK (Tudor et al, 1996) are shown on the left-hand side for comparison. Angular order parameters (S) for the backbone dihedral angle φ.

FIG. 6D. Parameters characterizing the final 20 structures of ShK-DAP²², plotted as a function of residue number. Values for ShK (Tudor et al, 1996) are shown on the left-hand side for comparison. Angular order parameters (S) for the backbone dihedral angle ψ.

FIG. 7A. ShK wild type toxin. Plot showing the IC50 = 2.41 nM, Slope = 1.2.

FIG. 7B. ShK variant, A21-DAP22. Plot showing the IC50 = 507 nM, Slope = 0.66. The ratio of the IC50 A21-DAP22/ IC50 ShK wild type = 210. Kvl .3 = 5 nM.

FIG. 7C. ShK variant, Nle21-DaP22. Plot showing the IC50 = 1,940 nM, Slope = 0.74. The ratio of the IC50 Nle21-DAP22/ IC50 ShK wild type = 805. Kvl.3 = 50-100 pM.

FIG. 8. Sequence comparison between ShK toxin (Karlsson et al, 1992) BgK toxin (Aneiros et al, 1993). Boxed-in residues are either conservative substitutions or identical.

FIG. 9. Schematic representation of ShK disulfide pairings.

FIG. 10. Sequence of wild-type ShK toxin (Karlsson et al, 1992), BgK toxin (Aneiros et al, 1993; revised, Karlsson et al, 1992), AsK (Schweitz et α/. , 1995) and ChTX (Sugg et α/., 1990). FIG. 11. Amino acid sequence and disulfide pairings of ShK toxin peptides. 4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 DEFINITIONS

The following words and phrases have the meanings set forth below.

Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene. Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incoφorated into a chromosome or is capable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.

Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

4.2 PROBES AND PRIMERS

In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected ShK polypeptide gene sequence, e.g., a sequence such as that shown in SEQ ID NO:l. The ability of such DNAs and nucleic acid probes to specifically hybridize to a ShK polypeptide- encoding gene sequence lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample. In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of a ShK polypeptide-encoding gene from S. helianthus using PCR™ technology. Segments of related ShK polypeptide genes from other species may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of a ShK polypeptide-encoding sequence, such as that shown in SEQ ID NO:l. A size of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 14 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR™ technology of U. S. Patents 4,683,195, and 4,683,202, herein incoφorated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.

4.3 EXPRESSION VECTORS

The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression vector is an isolated and purified DNA molecule comprising a promoter operatively linked to a coding region that encodes a polypeptide of the present invention, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.

As used herein, the term "operatively linked" means that a promoter is connected to a coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art. In a preferred embodiment, the recombinant expression of DNAs encoding the ShK polypeptides of the present invention is preferable in a Stichodactyla host cell, such as S. helianthus. Promoters that function in bacteria are well-known in the art. An exemplary and preferred promoter for the Stichodactyla ShK polypeptides include any of the known ShK polypeptide gene promoters, including the ShK gene promoters. Alternatively, mutagenized or recombinant ShK polypeptide-encoding gene promoters may be engineered by the hand of man and used to promote expression of the novel gene segments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encoding the ShK polypeptides of the present invention is performed using a transformed Gram-negative bacterium such as an E. coli or Pseudomonas spp. host cell. Promoters which function in high- level expression of target polypeptides in E. coli and other Gram-negative host cells are also well-known in the art.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed.

These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in U. S. Patent Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are incoφorated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

4.4 IMMUNOSUPPRESSANTS Immunosuppressants such as cyclosporin and FK506 exhibit severe side effects which limit their therapeutic use. Cyclosporin's side effects are due to the interaction of this drug with the protein cyclophilin which is present in many different tissues. Similarly, FK-506 causes toxicity because its target protein, FK-binding protein, is found in many different tissues. There has therefore been a major effort to identify novel immunosuppressants without serious side-effects, the goal being to identify novel targets expressed principally in T- lymphocytes.

The Kvl.3 potassium channel in T- lymphocytes plays an important role in regulating T-cell activation. Expression of this gene is highly restricted to T-cells, although Kvl.3 mRNAs are also detected faintly in brain, β-lymphocytes, microglia, macrophages, osteoclasts and platelets; only in T-cells does this channel dominate the membrane potential, and therefore, only in T-cells does Kv 1.3 -blockade have functional consequences. Due to its distinct mechanism and restricted tissue distribution, a Kvl .3 blocker would not likely display the toxic side-effects of cyclosporin and FK-506, and therefore may prove useful for treatment of chronic autoimmune diseases as well as transplantation therapy. Recent studies by scientists at Merck Shaφe and Dohme have shown that the potent

Kvl.3 peptide-antagonist, margatoxin (MgTX), is effective in suppressing the immune response in animal models (pig) and has minimal side-effects. This peptide is however, not specific for Kvl.3, and blocks the closely related Kvl.2 channel with similar potency. Since the Kvl.2 channel is expressed in the heart and brain, its blockade might have serious deleterious effects. The inventors have therefore searched for other novel peptides that might be truly selective for

Kvl.3.

The sea-anemone toxin, ShK, is known to potently block the Kvl.3 channel. The inventors assessed the selectivity of this toxin on a panel of cloned Kv channels and found that ShK blocked Kvl.l with similar potency as Kvl.3; other related channels were >100-fold less sensitive to the ShK polypeptide. Although the native toxin is not specific for the lymphocyte channel, the inventors screened ShK mutants using a panel of cloned channels to identify a Kv 1.3 -selective antagonist. These results are described in Example 13.

4.5 ION CHANNEL TOXINS: 3D STRUCTURES AND CHANNEL-BINDING SURFACES Polypeptide ion channel toxins are proving to be valuable therapeutic leads in the treatment of a range of conditions. Among their advantages are high potency, good target specificity, high solubility and rapid onset of action. As they are often small proteins crosslinked by several disulfides, they generally also have quite stable structures in solution, which are readily determined using H NMR spectroscopy. Once the solution structure has been solved it is possible to map onto that structure the likely channel binding surface, identified initially by alanine scanning, then characterized further by additional residue substitutions. If a model of the ion channel is also available then possible docking interactions of the toxin can be tested by complementary mutagenesis. This information provides the basis for the design of smaller peptidic analogs of the toxin, and eventually of peptidomimetic analogs. ShK polypeptide is a potent blocker of Kvl.3 potassium channels in

T-lymphocytes. The solution structure of ShK polypeptide consists of two helices and a series of turns, making it quite different from scoφion toxins that interact with the same channel (Tudor et al, 1996). Key residues for channel binding have been defined using synthetic analogs. For both toxins the structural effects of disulfide bond removal and truncation have been investigated as a first step towards development of a peptidic analog.

4.6 SHK METHODS OF SYNTHESIS

Synthesis of a peptide via solid-phase methods includes the use of a solid-phase resin such as but not limited to polystyrene, polyacrylamide, cotton or other stable polymer. Derivatization of the solid-phase resin with a suitable handle such as chlorotrityl chloride, 2- chlorotrityl chloride, hydroxymethylphenyl, Sasrin as a means to produce the C-terminal acid functionality. A C-terminal amide may also be prepared as a means of proteolytic stabilization via a resin linker such as but not limited to 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)- phenoxymethyl group. Chain assembly shall include any of the protecting group strategies where the a- amino protecting group is either t-butyloxycarbonyl (Boc) or 9-fluorenyl-methyloxycarbonyl. Side chain protecting groups shall include any combination of either no protecting groups or t- butyl, benzyl, trityl, methyltrityl, benzyl-methylbenzyl, tosyl, benzyloxymethyl, t- butyloxycarbonyl, 2-chlorobenzyl, 2-bromobenzyl, methoxybenzyl, formyl, acetamidomethyl, pentamethylchroman sulfonyl, pentamethyldihydrobenzofuran-sulfonyl, nitro for sidechain amines, guandines, phenols, alcohols, acids, imidazoles, thiols, and indoles. This includes side chain protecting groups which could be invented which accomplish the same goal of eliminating side chain reactions during primary chain assembly.

Synthesis of the amide bond may be accomplished by using any of the acid activation methods including but not limited to symmetrical anhydrides (carbodiimide), HOBT esters, acyl fluorides, uronium activators such as but not limited to TBTU, HATU or HBTU, phosphonium activators such as but not limited to BOP, PyBOP, PyBrOP. These are all methods of activation of the carboxyl group which those practicing the art of peptide synthesis would be expected to know.

Synthesis of analog structures which include substitution of unnatural amino acids into the sequence of ShK may also be useful for certain embodiments of the invention.

Synthesis of ShK via convergent methods whereby fragments of the peptide are assembled in a fashion whereby the ultimate product is ShK or a related toxin analog. Final cleavage and deprotection and folding of the toxin may be but not limited to either HF or TFA depending on the strategy employed for synthesis. Disulfide bond formation includes any orthagonal approach where differential Cys protection could be used to position the disulfide bonds in the correct C3-C35, C12-C28 and C17-C32 linkage. Also included would be any oxidative folding procedure wherein the same disulfide array is realized via air oxidation or glutathione mediated shuffling reaction.

In order to produce a peptide with a higher half life in vivo, analog structures of ShK whereby key proteolytic digestion sites may be substituted to reduce protease susceptibility. This may include replacement or substitution of nonessential residues with conservative isosteric replacements (e.g., Lys to Lys(acetyl) or Gin) and or neutral replacements (Ala). Also, acetylation of the N-terminus or amidation of the C-terminus may provide stability from exopeptidases. Also, endopeptidase sites may have an Na-methylated substitution to reduce proteolytic degradation. Internal or external truncations may also be prepared from any of the disclosed peptides. These may include removing one or more residues from either the C-terminus or N- terminus or removal of one or more internal non-essential residues or sequence.

4.7 Low MOLECULAR WEIGHT ANALOGS OF SHK POLYPEPTIDE AND SHK-DERIVED

POLYPEPTIDE ANALOGS

The three-dimensional structure of ShK polypeptide in aqueous solution (Tudor et al, 1996) consists of two short α-helices (residues 14-19 and 21-24) and a number of reverse turns. A number of the residues essential for ShK binding to the T-lymphocyte (Kvl.3) and rat brain K⁺ channels have been identified using analogs made by peptide synthesis (Pennington et al, 1996a, Pennington et al, 1996b; Pennington et al, 1997). It appears that Lys22 and Tyr23, which are part of the second helix, are important for binding to both types of K channel, while Argi l is one of the key residues responsible for preferential binding to Kvl.3. These residues are on the same face of ShK polypeptide, making it practical to design and synthesize mimetics that present these residues in the bioactive conformation.

Two methods may be used to development of low molecular weight analogs of ShK polypeptide. The first is polypeptide minimization, where the size of a polypeptide is reduced in such a way that the amino acid residues important for activity are maintained in character and conformation even though much of the molecule may be deleted. This has the advantage that it can provide useful new analogs directly, possibly with improved pharmocokinetics and bioavailability. Moreover, it simplifies the task of identifying non-peptidic scaffolds for the development of peptidomimetics.

Minimization is achieved by compensating for the deleted intramolecular interactions of the native molecule (including disulfide bonds) by stabilizing the remaining structure. This may be done by stabilizing the local conformations of the two helices in ShK polypeptide (residues 14-19 and 21-24) then incoφorating covalent links between them to maintain the bioactive spatial orientation found in the native toxin.

There are several proven ways of stabilizing helices. One is to incoφorate lactam bridges between carboxyl-bearing residues (aspartate and glutamate) and lysine residues separated by three intervening residues in the amino acid sequence (Houston et al, 1995). This has been achieved for analogs of human growth hormone releasing factor (Felix et al, 1988) and the C-terminal helix of neuropeptide Y (Rist et al, 1996; Kirby et al, 1997). Other examples are found in Kemp et al. (1991), Chorev et al. (1993) and Kanmera et al. (1995). Further helix stabilization is afforded by incoφoration of overlapping /^' to i+4 lactam bridges in the helix (Bracken et al, 1994), although it may be more difficult to incoφorate two such bridges in ShK analogs without affecting the K-channel binding surface.

Another means of stabilizing helices involves positioning stereoisomers of Cys to enable formation of an i to i+4 disulfide bridge between the L-Cys and D-Cys residues (Krstenansky et al, 1988).

Having stabilized the two short helices of ShK polypeptide, the next step is to lock them into a conformation similar to that found in the native toxin structure. Several methods are possible to achieve this, including non-native disulfide bridges, linkage via 4-(aminomethyl)phenylacetic acid (AMP A) (Yu and Taylor, 1996) between amino- and carboxyl-bearing residues, or linkage via an alkanediyl chain between the side-chain nitrogen atoms of glutamine residues (Phelan et al, 1997). The remaining requirement is to initiate the first helix while at the same time making provision for inclusion of a functional group equivalent to Argi l of the native toxin. Helical initiators derived from aspartic acid and glutamic acid are known (Meara et al, 1995). Another way to achieve helix initiation is to retain the reverse turn involving residues 9-12 in the native toxin or to incoφorate a mimetic for this turn (Zhang et al, 1996; Kieber-Emmons et al, 1997). The turn mimetic could then be suitably functionalized to include a side-chain guanidino group to mimic Arg 11.

A bioactive, minimized peptidic analog of ShK polypeptide may be ftirther modified by inclusion of selected D-amino acids or by synthesis of a retro-inverso analog, where all residues are D-handed and the amino acid sequence is reversed (Jameson et al, 1994; Juvvadi et al, 1996). Such modifications are expected to further increase its stability in vivo.

The second major approach to the development of low molecular weight analogs of ShK polypeptide is to generate non-peptidic (peptidomimetic) analogs. In these studies, analogs of ShK polypeptide are designed and synthesized based on non-peptidic scaffolds which contain key functional groups from the potassium channel binding surface of the parent polypeptide. Conformationally-directed database searches are undertaken to identify potential lead compounds from existing chemical libraries. Potential candidates are identified, including some which present the key functional groups in the appropriate conformation and some of which require synthetic modification. Using these methods, one may prepare novel ShK polypeptide analogs that are largely or entirely non-peptidic in nature and which display selectivity for particular target tissues. There are many examples where naturally occurring low molecular weight, non- peptidic compounds have been shown to mimic or antagonize polypeptide or protein ligands. Similarly, peptidomimetic compounds have been designed and synthesized for a number of therapeutically relevant polypeptides. For example, a loop present on the CD4 receptor which binds to HIV gpl20 protein (Chen et al, 1992). This compound effectively blocked gpl20 binding to CD4 receptor at low micromolar concentrations and effectively reduces syncytium formation 50% at 250 μ/ml. Another example is FTI-276, a mimetic of the C-terminal region of the Ras protein that is a potent blocker of oncogenic Ras signaling (Lemer et al, 1995).

Compounds showing a degree of similarity to the ShK pharmacophore are tested for K-channel binding, and those having binding affinity constitute valuable new leads, which may be further modified with the aim of improving binding affinity and channel sub-type specificity.

4.8 HIGH-LEVEL EXPRESSION OF Kvl.3 CHANNELS FOR SHK BINDING STUDIES

Integral membrane proteins including receptors, transporters, and ion channels are critical for the transfer of both signals and substrates between the external and internal environments of cells. Although the genes for many of these mammalian proteins have been isolated, and site-specific mutagenesis studies have mapped functional domains, not a single mammalian integral membrane protein has had its 3 -dimensional structure solved. Such information would clearly define how protein structure relates to function, and could guide pharmaceutical efforts to develop novel therapeutic agents. The primary impediment to structural analysis is the lack of a method for large scale expression and purification of intact protein. The disadvantages of existing heterologous non-mammalian expression systems for purifying mammalian proteins includes inappropriate posttranslational modification and protein accumulation in inclusion bodies. A vaccinia virus based heterologous expression system has been developed for over-expression and rapid purification of appropriately folded and modified Kvl .3 at adequate amounts for direct structural analyses. The gene for Kvl.3 was cloned into a vaccinia transfer vector (pTMl) in-frame with an 111 bp sequence encoding a polyhistidine repeat, a segment from gene 10 of bacteriophage T7, and an enterokinase cleavage site. Expression of this fusion protein in African Green monkey kidney cells, CV-1, produced 1-5 x 105 functional K+ channels which are biophysically identical to native Kvl.3. The heterologously expressed channel was glycosylated like its native counteφart in lymphocytes. These Kvl.3 expressing cells have been used for radiolabeled ChTX binding studies, and are adaptable to ShK binding.

Gel filtration and sucrose density gradient sedimentation analyses suggested that purified Kvl.3 protein retained its native tetrameric structure. As few as 10 cells yielded 50 mg of homogeneous Kvl.3 protein, making it easily possible to upscale this method to produce adequate quantities for structural studies such as 2-dimensional crystallography, electron microscopy and spin-label topology mapping. The purified protein when reconstituted into lipid bilayers produces functional channels which are blocked by ShK and by MgTX. The protein also binds radiolabeled MgTX. The method described here has wide implications for the purification and direct structural analyses of any integral membrane protein.

4.9 ANIMAL MODELS OF AUTOIMMUNE DISEASES AND TRANSPLANT REJECTION

Over the years, several animal models of autoimmune diseases have been developed. It is important that animal models mimic as closely as possible the human disease and that they respond to treatment in similar ways as the human disease. Small animal models, such as rodents, are preferred because they are inexpensive, can be used in relatively high numbers, and have well characterized genetics. Although small animal models are often adequate models, large animals, particularly primates, are more suitable for some types of diseases. Small animals are less related to humans, but in many cases will react to treatment in the same way as humans do. While primates may be better models for some human diseases, they tend to be expensive, and handling can be difficult.

4.9.1 PSORIASIFORM SKIN DISEASE

CD-I 8 deficient mice backcrossed onto a PL/J strain background have been used as an animal model for psoriasiform skin disease. Homozygotes for a null mutation in CD 18 within the 129/Sv background are characterized by a mild leukocytosis, an impaired response to chemically-induced peritonitis, and delays in transplantation rejection (Wilson et al, 1993). Bullard et al. (1996) report that when the CD 18 homozygote null mice are crossed to the PL/J strain of mice, the backcrossed mice develop an inflammatory skin disorder. The skin disease shows several histological and clinical similarities to human hypeφroliferative inflammatory skin disorders, such as psoriasis (Camisa et al, 1994). These include epidermal hypeφlasia, hyperkeratosis, parakeratosis, subcorneal microabscesses, lymphocyte exocytosis, and dilation of dermal capillaries.

Adult CD 18 homozygous mice developed a progressive dermatitis characterized by erythema, alopecia, and scale and crust formation (Bullard et al, 1996). Visible signs of the disease first appeared as red, scaly skin on the ears, paws, tail, and facial area. Similar to the human disease, the CD 18 null mice responded to administration of corticosteroids. Response to corticosteroids was assessed by daily subcutaneous injections of 20 μg of dexamethasone. Improvement was seen in all affected mice; dramatic improvement with disappearance of scales, crust, and erythema occurred after 2 weeks and was accompanied by regrowth of hair (Bullard et al, 1996). Acute withdrawal of the dexamethasone dose or reduction of the dose to

10 μg/day resulted in a severe exacerbation of the dermatitis (Bullard et al, 1996).

The gross moφhology, anatomical distribution, disease course, and response to anti-inflammatory drug, such as dexamethasone, treatment are all features with similarity to human psoriasis and other inflammatory skin disorders. The inflammatory skin disorder of the CD 18 null PL/J mice has generally been accepted as a model of dermatitis because of its similarities to human psoriasis and autoimmune skin disease.

4.9.2 INFLAMMATORY BOWEL DISEASE

An animal model for inflammatory bowel disease is described by Leach et A/. (1996). In this study, chronic inflammation develops spontaneously in the large intestine of

C.B-17 scid mice restored with the CD45RB ^lg subset of CD4+ T cells obtained from normal BALB/c mice. The changes in the large intestine of these mice are similar to those seen in patients with idiopathic inflammatory bowel disease (Crohn's disease and ulcerative colitis). This murine model appears to be useful for studying mucosal immunoregulation as it relates to the pathogenesis and treatment of chronic inflammatory bowel diseases in the large intestine of human patients (Leach et al, 1996). CB-17 scid mice injected with CD45RB^high CD4+ T cells from BALB/c mice consistently develop chronic inflammatory and epithelial lesions that extended profusely from the cecum to the rectum (Leach et al, 1996). Moφhological features in the large intestine of these mice are similar to those seen in the colon of human patients with Crohn's Disease or ulcerative colitis (Leach et al, 1996). Similarly, these mice seem to have immunopathological findings similar to those found in patients with CD or UC. Therefore, this model provides an excellent system to test the efficacy of anti-immune or anti-inflammatory compositions, such as the polypeptides of the present invention, for treating Crohn's disease or ulcerative colitis.

4.9.3 EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (MULTIPLE SCLEROSIS)

Experimental autoimmune encephalomyelitis (EAE) describes a group of inflammatory diseases of the central nervous system (CNS) that are induced in susceptible animals by immunization with myelin antigens or by adoptive transfer of sensitized T-cells to syngeneic recipients (Alvord et al, 1984; Pettinelli et al, 1985). In inbred rodents, chronic and relapsing remitting forms of EAE that have been described resemble human multiple sclerosis

(MS) (Zamvil et al, 1985; McFarlin et al, 1974; Raine et al, 1984). EAE has served in the testing of scores of therapies for MS, yet applicacy has often not been a predictor of benefit in humans. Autogenetic differences between inbred rodents and outbred humans, have limited the usefulness of EAE as an MS model. EAE has been described in macaques, yet acute CNS lesions in these species are hyperacute, hemorrhagic and destructive, unlike those in MS

(Alvord et al, 1979). Additionally, the outbred nature of non-human primates has limited their value as disease models, since adoptive transfer of genetically compatible T-cells between animals is valuable for elucidating the role of specific T-cell populations in EAE.

Massacesi et al. (1995, incoφorated herein by reference) describe the induction and the characteristics of EAE in the common marmoset Callithrix jachus, a new world monkey.

Actively induced EAE in C. jachus is characterized clinically by mild neurological signs and a relapsing-remitting course, and pathologically by mononuclear cell infiltration primary, primary demyelination, and reactive gliosis. A further advantage of the use of the marmosets as the model for EAE is that they are born as naturally occurring bone marrow chimeras (Picus et al, 1985). While individual animals from multiple births arrive from separate ova that are fertilized independently, the placenta of the developing animals fuse, resulting in a cross- circulation of bone marrow-derived elements between the developing fetuses. Thus, while animals are genetically distinct, they share and are tolerant of each other's bone marrow-derived cell populations.

In this model, one is capable of adoptively transferring EAE by T-cell transfer between members of a chimeric set of twins. Acute and chronic EAE, created in a species whose immune and nervous system genes are phylogenetically close to those of humans, represents a unique disease model and may be useful in elucidating immune mechanisms of CNS demyelination. Furthermore, it provides an excellent system for testing the efficacy of compositions, such as the polypeptides of the present invention, at treating such disorders.

4.9.4 TRANSPLANTATION REJECTION

Transplantation of organs into a new host causes an immune response against the new organ, similar to autoimmune diseases. Thus, transplantation model systems in animals also are very useful in testing the efficacy of anti-inflammatory or autoimmune compounds, such as the polypeptides of the present invention. Animal transplantation models include a lung transplantation model in swine (Schmidt et al, 1997), a kidney transplantation model in swine (Granger et al, 1995), a kidney transplantation model in canines (Tanabe et al, 1994), and an intrasplenic hepatocyte transplantation model in canines (Benedetti et al, 1997).

4.10 IMMUNOASSAYS

As noted, it is proposed that native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that the utility of ShK-derived proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures. In preferred ELISA assays, proteins or peptides incoφorating ShK, rShK, or ShK- derived peptide antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti- human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS-Tween®). After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol puφle or 2,2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer. ELISAs may be used in conjunction with the invention. In one such ELISA assay, proteins or peptides incoφorating antigenic sequences of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

4.11 PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incoφorated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incoφorated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incoφorated into sustained-release preparation and formulations. The active compounds may also be administered parenterally or intraperitoneally.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incoφorating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incoφorated into the compositions.

For oral prophylaxis the polypeptide may be incoφorated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incoφorating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incoφorated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

4.12 LIPOSOMES AND NANOCAPSULES

In certain embodiments, the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).

Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1977; 1988). Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. In addition to the teachings of Couvreur et al. (1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs. Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsoφtion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.

4.13 AFFINITY CHROMATOGRAPHY Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody. The column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix. The column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted. The requirements for successful affinity chromatography are:

1) that the matrix must specifically-adsorb the molecules of interest;

2) that other contaminants remain unadsorbed;

3) that the ligand must be coupled without altering its binding activity;

4) that the ligand must bind sufficiently tight to the matrix; and 5) that it must be possible to elute the molecules of interest without destroying them.

4.14 THERAPEUTIC KITS COMPRISING SHK COMPOSITIONS

A therapeutic kit comprising, in suitable container means, one or more ShK composition(s) of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention. The ShK composition(s) may comprise:

1) one or more ShK polypeptide;

2) one or more truncated ShK polypeptides;

3) one or more site-specifically or randomly mutated ShK polypeptides; 4) one or more ShK-encoded peptide epitopes, domains or motifs;

5) one or more antibodies which bind to native, truncated, site-specifically or randomly mutated ShKs, or ShK-encoded peptide epitopes, domains or motifs;

6) one or more nucleic acid segments encoding all or a portion of one or more ShK genes, These nucleic acid segments may encode native ShKs, truncated ShKs, site-specifically or randomly mutated ShKs, or ShK-derived peptide epitopes, domains or motifs, and may be either native, recombinant, or mutagenized DNA or RNA segments; or, alternatively, 7) a combination of one or more of the compositions 1) through 6). The kit may comprise a single container means that contains the ShK composition(s). The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the ShK composition(s) and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous composition (e.g., a collagenous composition), a powder, solution, matrix, lyophilized reagent, or any other such suitable means. In certain cases, the container means may itself be a syringe, pipette, or other such like apparatus, from which the ShK composition(s) may be applied to a tissue site, skin lesion, or wound area. However, the single container means may contain a dry, or lyophilized, mixture of one or more ShK composition(s), which may or may not require pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one or more containers would contain each of the ShK composition(s), either as sterile solutions, powders, lyophilized forms, etc., and the other container(s) would include a matrix, solution, or other suitable delivery device for applying the ShK composition to the body, bloodstream, or to a tissue site, skin lesion, wound area, or other sites. Such delivery device may or may not itself contain a sterile solution, diluent, gelatinous matrix, carrier or other pharmaceutically-acceptable components.

The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the ShK component into a more suitable form for application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition. The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The matrix and gene components may also be aliquoted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow- molded plastic containers into which the desired vials or syringes are retained. Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate matrix-gene composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

4.15 METHODS FOR GENERATING AN IMMUNE RESPONSE

Also disclosed in a method of generating an immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a peptide composition disclosed herein. Preferred peptide compositions include the ShK polypeptides disclosed in any of SEQ ID NO:l to SEQ ID NO:3.

The invention also encompasses ShK and ShK-derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, as may be employed in the formulation of particular therapeutics. The identification or design of suitable ShK epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp, as enabled in U. S. Patent 4,554,101, incoφorated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences. For example, Chou and Fasman (1974a,b; 1978a,b; 1979); Jameson and Wolf (1988); Wolf et al. (1988); and Kyte and Doolittle (1982) all address this subject. The amino acid sequence of these "epitopic core sequences" may then be readily incoφorated into peptides, either through the application of peptide synthesis or recombinant technology. It is proposed that the use of shorter antigenic peptides, e.g., about 15 to about 35, or even about 20 to 25 amino acids in length, that incoφorate epitopes of one or more ShKs will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a ShK-reactive antibody, such as a biological sample from a patient, and contacting the sample with a first ShK or peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.

Contacting the chosen sample with the ShK or peptide under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the protein or peptide composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, i.e., to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non- specifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected. The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, e.g., Nakamura et al. (1987), incoφorated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.

Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.

In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of ShK-specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associated with a ShK or peptide, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first ShK or peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is a ShK peptide that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. 4.16 POLYPEPTIDE COMPOSITIONS

An ShK composition of the present invention is understood to comprise one or more polypeptides that are capable of eliciting antibodies that are immunologically reactive with one or more ShK polypeptides as described in any of SEQ ID NO: 1 to SEQ ID NO:3. A ShK composition of the present invention is also understood to comprise one or more polypeptides that elicit an immune response in an animal. Likewise, an ShK composition is also understood to comprise the polypeptide of SEQ ID NO:l substituted in one or more amino acids with one or more distinct natural or non-natural amino acids. The inventors contemplate any such modified ShK polypeptides to be useful in the practice of the disclosed methods so long as the polypeptide has Kv ion channel inhibiting activity, and in particular, selective Kvl.3 channel inhibiting activity.

As used herein, an active fragment of a ShK includes a whole or a portion of a ShK which is modified by conventional techniques, e.g., mutagenesis, or by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure and function as a native ShK as described herein.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1 ~ METHODS EMPLOYED IN THE PRESENT INVENTION 5.1.1 REAGENTS

Natural ShK and Dendroaspis polylepsi I (DTX) were provided by Dr. E. Karlsson

(Biomedical Center, University of Uppsala, Uppsala, Sweden). Sequencing-grade and HPLC-grade solvents and reagents were obtained from Applied Biosystems (Foster City, CA),

Achromobacter lysyl endoproteinase from Wako Bioproducts (Richmond, VA) and thermolysin TLCK-α-chymotrypsin and TPCK-trypsin from Boehringer-Mannheim (Indianapolis, IN).A11 other reagents were the finest grade commercially available.

5.1.2 SYNTHESIS OF SHK PEPTIDE Fmoc-amino acids (Bachem Feinchemikalien, CH-4416 Bubendorf, Switzerland) included Arg(Pmc), Asp(OtBu), Cys(Trt), Gln(Trt), His(Trt), Lys(Boc), Ser(tBu) and Thr(tBu). Stepwise assembly was carried out on an Applied Biosystems 431 A peptide synthesizer at the 0.25 mmol scale starting with Fmoc-Cys(Trt)-R. Residues 34-22 were single-coupled. At this point, half of the resin was removed to effect better mixing. The remainder of the peptide sequence was double coupled to the remaining resin aliquot. All couplings were mediated by dicyclohexyl-carbodiimide in the presence of 2 equiv. of 1-hydroxy-benzotriazole. Following final removal of the Fmoc-group, the peptide resin (2.42 g) was cleaved from the resin and simultaneously deprotected using reagent K (King et al, 1990) for 2 h at room temperature. Following cleavage, the peptide was filtered to remove the spent resin beads and precipitated with ice-cold diethyl ether. The peptide was collected on a fine filter funnel, washed with ice-cold ether and finally extracted with 20% AcOH in H₂O. The peptide extract was subsequently diluted into 2 L of H₂O, the pH adjusted to 8.0 with NH₄OH, and allowed to oxidize in air at room temperature for 36 h. Following oxidation of the disulfide bonds, the peptide solution was acidified to pH 2.5 and pumped onto a Rainin Dynamax C₁₈ column (5.0 x 30 cm). The sample was eluted with a linear gradient from 5 to 30% acetonitrile into H₂O containing 0.1% TFA. The resulting fractions were analyzed using two analytical RP-HPLC systems, TFA and TEAP (Rivier and McClintock, 1983). Pure fractions were pooled and lyophilized. Upon lyophilization, 120 mg of ShK polypeptide was obtained, representing a yield of 24% of theory (from the starting resin).

5.1.3 AMINO ACID ANALYSIS

Synthetic peptide samples were hydrolyzed in 6 N HCl at 110°C for 22 h in vacuo. Amino-acid analysis was performed on a Beckman 126AA System Gold amino-acid analyzer. The masses of the natural and synthetic ShK polypeptide samples used in the [ I]DTX binding comparison were determined by Dr. Jan Pohl (Emory Microchemical Facility, Atlanta, GA). 5.1.4 FAB-MS ANALYSIS

FAB-MS analysis was performed by M-Scan (West Chester, PA) on a ZAB 2-SE high-field mass spectrometer.

5.1.5 LIGAND BINDING ASSAY WITH DTX

Iodination of DTX was performed by the chloramine T method (Hunter and

125

Greenwood, 1962). After removal of the unreacted I by gel chromatography, the specific

195 radioactivity of [ I]DTX was 34 Ci/mmol. Male Sprague-Dawley rats (Harlan, 175-250 g) were decapitated, and whole brains were removed and homogenized at 0°C in 10 vol. of saline (0.15 M NaCl, 0.03 M Tris HCl, pH 7.0) (wt./vol.) using a Teflon pestle. The homogenate was centrifuged (10 min, 17000 x g, 4°C), and the resulting pellet was resuspended in the saline and again homogenized. Centrifugation and homogenization were repeated once more before the membranes were used for the DTX binding displacement assay. Binding of ShK polypeptide to rat brain membranes was indirectly investigated by competition with 1 nM [ IJDTX.

125 Membranes (0.5 mg protein) were incubated with test compounds and [ IjDTX in a

Tris-buffered saline (0.15 M NaCl, 0.03 M Tris HCl, BSA 2 mg/ml, pH 7.0) at room temperature in a final volume of 0.25 ml. After 1 h incubation, membrane suspensions were diluted with two 0.7 ml portions of saline (0.15 M NaCl, 0.03 M Tris HCl, pH 7.0), and membranes with bound radioligand were separated by filtration under vacuum through glass filters (Whatman GF/C) at room temperature and washed twice with 3.5 ml of the same buffer.

Filters were presoaked for 10 min in 0.5% (vol./vol.) polyethylenimine before filtration. Nonspecific binding was measured in the presence of 0.5 μM cold DTX. Membrane protein concentration was determined by the Coomassie Blue method (Bradford, 1976).

5.1.6 [¹²⁵I]CHTX BINDING TO Kvl.3 T-LYMPHOCYTE POTASSIUM CHANNELS

Jurkat T lymphocytes (ATCC) were suspended in a saline solution (NaCl 5 mM, KC1 5 mM, sucrose 320 mM, HEPES 10 mM, glucose 6 mM, pH adjusted to 8.4 with Tris base). Cells (2 x 10 /tube) were incubated in polypropylene 1 ml deep wells in the presence of

195

30 pM [ I]ChTX ± test agents for 20 min at 22°C. Nonspecific binding was determined in the presence of 10 nM ChTX. Binding reactions were terminated by filtration through GF/C glass filters that had been presoaked in 0.6% polyethylenimine. Samples were washed with twice with 1 ml of ice-cold wash saline (NaCl 200 mM, HEPES 20 mM, adjusted to pH 8.0 with Tris base). Radioactivity bound to filters was measured in a Betaplate liquid scintillation to counter (Wallace, Gaithersburg, MD).

5.1.7 MEASUREMENT OF POTASSIUM CURRENT IN JURKAT T LYMPHOCYTES

All recordings were made on cells bathed with saline of the following composition (in mM): NaCl 160, KC1 4.5, MgCl₂ 2, CaCl₂ 1, HEPES 10, pH 7.4. Patch pipettes (see below) were filled with (in mM): KF 154, EGTA 11, CaCl₂ 1.1, MgCl₂ 2, HEPES 10, pH 7.3 with KOH. For electrophysiological and binding studies, cells were allowed to settle to the bottom of the chamber and adhere for approximately 5 min prior to flow being initiated. Studies were performed at room temperature (21-24°C) with constant flow perfusion rates of 4- 5 ml/min. Voltage-clamp recording utilized an Axopatch IC or 200A amplifier (Axon Instruments, Foster City, CA), and data were digitized with a LabMaster 125 kHz DMA board and a Compaq Deskpro 386 computer or ALR 486 computer. All data acquisition analyses were performed with the pCLAMP software package (Axon Instruments). Current records were digitized at 2 kHz and filtered at 0.5 kHz. Series resistance compensation was employed in all studies. The membrane potential was held at -80 mV and the Kvl .3 channel current was measured by giving 150 ms voltage steps to +30 mV once every minute. Pharmacological inhibition was assessed by obtaining outward current values during the voltage step described above before and after a 6 min exposure to ShK polypeptide (with no applied pulses) and plotting percentage current block versus toxin concentration. A single drug concentration was tested on each cell.

5.1.8 PROTEOLYTIC CLEAVAGES

(1) ShK polypeptide (60 μg) was dissolved in 0.1 M Tris-HCl, pH 8.5, containing 2

M urea (60 μl) and was digested with lysyl endoproteinase (E:S = 1 :50 wt./wt, 30°C).

Aliquots (1.5 μl) of the reaction mixture were withdrawn at time intervals from 20 min to 29 h, acidified in 0.1% aqueous TFA (50 μl) and the ShK fragments were purified by RP-HPLC and characterized as described below. (2) ShK polypeptide (15 μg) was dissolved in 0.05 M HEPES, pH 6.5, containing 10 mM CaCl₂ (30 μl) and was digested with thermolysin (E:S = 1:20, wt./wt., 25°C, 3.5 h), or with a mixture of trypsin and chymotrypsin (E:S = 1 :1 :50, wt./wt., 30°C, 6 h). The digestion was terminated by acidification with 10% aqueous TFA (3 μl), and the solution was centrifuged (13000 g, 5 min). The supernatant was directly fractionated by RP-HPLC. Selected

HPLC-purified tryptic-chymotryptic peptides of ShK polypeptide were reconstituted in 0.05 M HEPES, pH 6.5, and 10 mM CaCl₂ and were subdigested with thermolysin (E:S = 1 :150, 25°C, 2 h) before fractionation by RP-HPLC.

5.1.9 RP-HPLC

The peptides were fractionated using a microbore RP-HPLC system consisting of Applied Biosystems 140A pumps and a 1000S diode-array detector (2.3 μl flow cell, 0.0025 inch i.d. tubing). Fractionation of the thermolytic and tryptic-chymotryptic peptides was performed on a Zorbax-SB c₁₈ column (1 x 150 mm, d_p ~ 5 μm, Microtech Scientific, Saratoga, CA) equilibrated in 0.1% aqueous TFA, and eluted at a flow rate of 80 μl/min using a linear gradient of acetonitrile/water/TFA (80:20:0.1). The column effluent was monitored at 215 nm. The thermolytic peptides, generated by subdigestion of the tryptic-chymotryptic peptides, were reconstituted in 0.1% heptafluorobutyric acid (HFBA) and further purified on the same column, equilibrated of acetonitrile/water/HFBA (80:20:0.1). The column eluent was manually collected and stored at -20°C until analysis.

5.1.10 SEQUENCE ANALYSIS

Automated Edman degradation of the peptides was performed on Applied Biosystems pulsed-liquid 477A/120A, gas phase 470A/120A, and 491A/140S Procise sequencing systems, as described previously (Pohl, 1994). Reagent 3 (TFA) and reagent 4

(25% aqueous TFA) contained 0.002% dithiothreitol. HPLC separation of the PTH amino acids was performed on-line. The solvent system for PTH separation (Pohl, 1994) was modified by replacing sodium acetate, pH ~3.95 in solvent A (3.5%, vol./vol., aqueous tetrahydrofuran) with the Premix buffer (13.5 ml/1). On all three sequencer systems, diPTH-Cys was recovered and identified as the peak coeluting with PTH-Tyr (Haniu et al, 1994; Crankshaw and Grant, 1993). PTH-Tyr coelution with diPTH-Cys was not problematic, since none of the disulfide-linked peptides contained tyrosine. In addition to diPTH-Cys, PTH-Ser (PTH-dehydroalanine adduct with DTT) and PTH-Ser (formed by rehydration of PTH-dehydroalanine) were also present as side products in the cycles containing cystine (see Pohl, 1994).

5.1.11 MALDI-TOF MASS SPECTROMETRY

The peptides were analyzed by matrix-assisted laser desoφtion/ionization mass spectrometry (MALDI) using a Kratos KOMPACT MALDI III mass spectrometer (Manchester). Each fraction (0.3 μl) was spotted on a target site of a 20-sample slide, followed by addition of 0.3 μl matrix (saturated α-cyano-4-hydroxy cinnamic acid; Aldrich, Milwaukee, WI) dissolved in 1 : 1 ethanol/water. The sample matrix was allowed to dry at room temperature for 5 min. Each sample was desorbed with 50 laser shots, each giving a spectrum. The shots were averaged to give the final spectrum. The instrument was calibrated using external standard peptides.

5.1.12 FAB MASS SPECTROMETRY ANALYSIS

FAB-MS analysis of synthetic ShK polypeptide was performed by M-can (West

Chester, PA) on a ZAB 2-SE high-field mass spectrometer. The sample was dissolved in 5% AcOH and a matrix of m-nitrobenzyl alcohol was used. A cesium ion gun was used to generate ions for the spectra, which were recorded using a PDP 11-250J data system. Mass calibration was performed using Csl.

5.1.13 SYNTHESIS OF SHK POLYPEPTIDE ANALOGS Fmoc-amino acids (Bachem Feinchemikalien, Bubendorf, Switzerland) included:

Ala, Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Gly, His(Trt), Homocitrulline, He, Leu, Lys(Boc), Met, Nle, Orn(Boc), Phe, p-aminoPhe(Boc), /?-nitroPhe, Pro, Ser(tBu), Thr(tBu), and Tφ. Stepwise assembly was carried out starting with 10 g of Fmoc-Cys(Trt)-resin (0.65 mmol/g) on a Labortec SP640 peptide synthesizer through 10 synthetic cycles (residues 34 through 25). At this point, resin aliquots were removed and placed on an Applied Biosystems 431 A peptide synthesizer at the 0.25 mmol scale and the remaining amino acid sequence incoφorating the substitution was assembled as described above. K30A analog was synthesized entirely on an ABI 431 A according to the procedure used to prepare wild-type ShK polypeptide described above except for the substitution of Ala for Lys30. Following final removal of the Fmoc-group, each of the peptides was cleaved from the resin and simultaneously deprotected using reagent K (King et al, 1990) for 2 h at room temperature. After cleavage, the peptide was filtered to remove the spent resin beads and precipitated with ice cold diethyl ether. The peptide was collected on a fine filter funnel, washed with ice cold ether and finally extracted with 20% AcOH in H₂O. Oxidative folding of the disulfide bonds and subsequent purification were as previously described in Example 3. Pure fractions were pooled and lyophilized. Structures and the purity of all the analogs were confirmed by HPLC, circular dichroism spectroscopy, amino acid and FAB-MS analysis.

5.1.14 BINDING ASSAYS AND ELECTROPHYSIOLOGICAL CHARACTERIZATION

The procedures for measuring displacement of ¹²⁵I-DTX to rat brain membranes and for blockade of voltage-activated Kvl.3 channels in Jurkat T lymphocytes were described previously (Pennington et al, 1995).

5.2 EXAMPLE 2 - A RECOMBINANT VACCINIA VIRUS EXPRESSING Kvl.3 FOR

HIGH- AFFINITY BINDING STUDIES Studies by Moss and collaborators have demonstrated the utility of the vaccinia virus system for heterologous expression of proteins in mammalian cells. A vaccinia transfer vector, pTMl was developed for this puφose. the key features of this construct are as follows: i) It contains an upstream non-coding region from EMC virus and an initiator methionine codon (AUG) which provides a very efficient translation initiation site. ii) The transcription initiation and termination sites for the T7 polymerase facilitate efficient transcription of full-length mRNA by this polymerase, which is provided by a separate vector. iii) The two halves of the thymidine kinase gene can be used to transfer the cloned sequence into an infectious vaccinia virus construct by homologous recombination. iv) Two origins of replication allow for production of either double-stranded or single-stranded DNA in E. coli, and an ampicillin-resistance gene permits drug selection.

This vector was modified for protein purification (pTHl), as follows. The initiator methionine codon (AUG) in pTMl was fused in-frame to six tandem histidines. This histidine motif allows for binding and elution from a Ni2⁺-bearing column as the first step in protein purification. This is followed by a gene- 10 sequence derived from bacteriophage T7 that can be targeted by a commercially available monoclonal antibody, permitting the detection of protein by Western blotting and its isolation by immunoprecipitation. Third, an enterokinase site and a multiple cloning site links the epitope sequence to the inserted gene; it- can be used, if desired, to remove the superfluous sequence.

The pTMl and pTHl constructs can be used for transient expression in mammalian cells in either of two ways: by transfection/infection, or by double infection. In the first case, transfection of target cells with a construct containing the sequence of interest (VV:Kvl.3), is followed by infection with T7 polymerase-encoding vaccinia virus (VV:T7), resulting in T7- dependent transcription and subsequent translation of protein. Alternatively, VV:Kvl.3 can be recombined into a virus and used to infect target cells simultaneously with the VV:T7, with similar results. The advantage of the latter method is that infection is more efficient than transfection in introducing DNA into target cells; on the other hand, while the plasmid required for transfection can be made easily, the process of producing recombinant viruses for the dual infection takes considerably more time.

To find an appropriate cell type for VV-mediated expression of channels, the inventors have tested Kvl.3 expression in several cell lines (CV-1, HeLa, Jurkat T cells, Rat Basophilic Leukemic cells, U937, NIH-3T3-fibroblasts); CV-1 cells provide the highest yield of the protein. CV-1 cells do not express endogenous voltage-gated or inwardly rectifying K⁺ channels, and therefore provide an electrically silent background for electrophysiological analysis of K⁺ channels. Another advantage is that these cells express biophysically "normal" Kvl.3 channels even after block of glycosylation by tunicamycin, suggesting that immature forms of protein are functional. Additionally, CV-1 cells can be adapted to spinner cultures. Each of these cell lines will be examined for their sustain high-level expression of functional

Kvl.3 channels. 5.3 EXAMPLE 3 ~ PHOTOAFFINITY ANALOGS OF SHK POLYPEPTIDE

Although the primary sequence of the K-channel receptor has been deduced from corresponding cDNA channels, the extracellular residues contributing directly to the formation of the ShK polypeptide binding pocket are entirely unknown. On the other hand, residues on

ShK which contact the receptor may be identified during the course of these analog studies. At this time, Lys9, Argi l, Lys22 and Tyr23 appear to interact with receptors on the Kvl.2 and Kvl .3 channels. To map site-site interactions between ShK polypeptide and the K-channel receptor, the inventors will prepare photoactivatable ShK polypeptide analogs. Photoactivatable amino acid derivatives are easily incoφorated into standard solid-phase peptide synthesizers. The receptor (primarily Kvl.3, but also rat brain K channels) radioligand binding characteristics of toxin analogs containing a photoreactive group may be studied prior to performing photolabeling experiments. The photoreactive amino acid derivatives include p- azido-phenylalanine and >-benzoyl-phenylalanine (Bpa). /?-azido-phenylalanine has been successfully employed to affinity label the human thrombin receptor with a synthetic analog which incoφorated a />-azido-Phe residue in place of Leu (Bischoff et al, 1994). This amino acid residue is stable to synthesis conditions. Furthermore, it is a suitable replacement for aliphatic residues such as He, Leu and Val as well as the aromatic residues Phe, Tyr and Tφ.

An alternative photolabel is ?-benzoyl-Phe (Bpa). This amino acid derivative is also very stable to the solid-phase synthesis conditions and has a high efficiency for forming cross-links (Kauer et al, 1986). Furthermore, Bpa has the added feature of incoφorating a benzophenone moiety which undergoes a n_π* transition to give a triplet biradical that has a high reactivity for C-H bonds which likely line the surface of the ShK polypeptide receptor. This amino acid derivative has been incoφorated successfully into a calmodulin-binding peptide (Kauer et al, 1986) substance P (Boyd et al, 1991) and into several semisynthetic insulin analogs (Shoelson et al, 1992). Positioning of this derivative within the ligand chain has been shown to affect the ability to form covalent cross-links with the receptor (Shoelson et al, 1992). This data has been inteφreted as suggesting that these residues are probably not buried within the receptor-ligand complex. This derivative is more sterically encumbering and may be useful in cases where poor low-efficiency cross-linking with the p-azido-Phe derivative is encountered. In order to identify where the ShK-toxin affinity probe has inserted into the K- channel receptor, radiolabeling or biotinylating of ShK affinity label is required, radiolabeling may be accomplished either by radioiodination or derivatization with label containing [ H] or [ C] such as N-terminal capping with acetic anhydride in the last synthetic step. The N- terminal Argl has been replaced with Ser without any change of biological activity. This suggests that the N-terminus is not essential for binding and acetylation of the N-terminal a- amino group should be tolerated. If iodination is utilized, incoφoration of iodine at either the His or Tyr residue will be limited to His if the photolabel Bpa or .-azido-Phe for Tyr23 is used. If radiolabeling is a problem or not desired, the N-terminal amino group may be selectively biotinylated (Lobl et al, 1989; Pennington, 1994) allowing the covalently attached ShK polypeptide-K-channel receptor to be identified using a biotin-avidin type interaction.

Lastly, amino groups may be conveniently modified on the solid-phase with ?-benzoyl-benzoic acid (BBA) provided that selective orthogonal protection amino groups are utilized (Gorka et al, 1989; Pennington, 1994). By employing a Lys or Orn derivative with an Alloc or Methyltrityl protecting group, selected positioning of a Lys(BBA) or Orn(BBA) may be achieved. This will allow the inventors to not only look at those residues present in hydrophobic "patches" within the ShK sequence but in the more hydrophilic regions as well. Provided that the substitution is not extremely disruptive for binding, an additional set of contact points on the receptor may be identified. Additionally, this BBA derivative is commercially available in a tritiated form to facilitate analysis of the proteolytically derived receptor peptides containing the BBA insertion.

Irradiation at 366 nm causes the BBA and Bpa containing ShK analogs to photchemically insert into the receptor surface (Kauer et al, 1986; Shoelson et al, 1992). Irradiation at 300 and 350 nm causes the j_>-azido-Phe containing ShK analogs to photochemically insert as well (Bischoff et al, 1994). The photochemically derivatized toxin- receptor complex may now be digested with proteases such as: thermolysin, chymotrypsin, Glu-C and Asp-C (Pohl et al, 1995). The receptor derived covalently modified peptide or peptides may be purified by microbore RP-HPLC if the radiolabel approach is utilized or by biotin-avidin chromatography of the biotinylation approach is followed. These approaches have been utilized successfully to define the scoφion toxin receptor site and the brevetoxin receptor site on voltage-sensitive Na channels (Tejedor and Catterall, 1988; Trainer et al, 1994). Purification of particular segments of Kvl.3 can be facilitated by immunoprecipitation with an antibody against the gene 10 tag at the N-terminus.

5.4 EXAMPLE 4 - PEPTIDOMIMETIC ANALOGS OF SHK POLYPEPTIDE The present example relates to the synthesis of peptidomimetic prosthetic units to replace the three basic building blocks of ShK polypeptide: (α -helix, β-sheets and reverse turns). Literature reports have focused around the importance of reverse turns in biological recognition events (Ball and Alewood, 1990). Reverse turns are capable of participating in biological recognition events in either an active role, where the precise spatial orientation of pharmacophoric information is critical (Smith and Pease, 1980), or in a more passive manner of properly positioning two peptide chains as they enter and exit the reverse turn (Niwa et al, 1993). The strategy focuses on turn mimetics which will address both of these turn situations simultaneously. Analysis of the tertiary structure of ShK in combination with the contact point refinement studies permits the definition of the pharmacophore surface of ShK polypeptide. β-Turns constitute tetrapeptide units which cause a reversal of in direction of the peptide chain. Turns are described as the distance from the C_a of the first residue to the C_a of the fourth residue. If this distance is less than 7A and the tetrapeptide sequence is not in an α- helical region it is considered a β-turn. Additionally, three residue reverse turns (γ-turns) are also possible but less common. These procedures are adaptable to either solid-phase or solution phase methods. Synthesis of the reverse turn mimetic involves the coupling of the first modular component piece (1), to the amino terminus of a growing peptide chain (2). Coupling of the second modular component (3), removal of the protecting group P' and subsequent coupling of the third modular component (4) provides the nascent β-turns (5). The critical step in this sequence involves the use of an azetidinone as an activated ester to effect the macrocyclization reaction (Wasserman, 1987). Upon nucleophilic opening of the azetidinone by the X-moiety, a new amino terminus is generated for continuation of synthesis. An important feature of this scheme is the ability to alter the X-group linker, both in regard to length and degree of rigidity/flexibility. The synthesis allows commercially available building blocks of either L or D stereochemistry to be utilized. Furthermore, deleting the second modular component (3) provides access to γ-turn mimetics. By utilizing these types of turn mimetics, none of the pharmacophoric information is lost and a stable reversal of chain direction is achieved. A peptidomimetic compound was recently prepared in six steps utilizing Scheme 1 which effectively mimics a loop present on the CD4 receptor which binds to HIV gpl20 protein (Chen et al, 1992). This compound effectively blocked gpl20 binding to CD4 receptor at low micromolar concentrations and effectively reduces syncytium formation 50% at 250 μg/ml. Relatively few attempts have been made to initiate or stabilize α-helices with small synthetic molecules (Kemp et al, 1991). Most efforts in this area have relied upon positioning Lys and Asp residues on the same face of amphiphilic helical surfaces spaced i+4 residues apart and form an isopeptide bond between the sidechains to stabilize the helix (Chorev et al, 1993; Kanmera et al, 1995). Other attempts at positioning stereoisomers of Cys with the same distance forming an i+4 disulfide bridge between the L-Cys and D-Cys residues has also been reported (Krstenansky et al, 1988). The problem with this approach is that any sidechain interaction at these two positions is effectively lost. Thus, mimetics which enhance the initiation of an α-helix in peptides may offer a better alternative.

Helical initiator compounds 1 and 2 are cyclic compounds derived from aspartic acid and glutamic acid, respectively (Meara et al, 1995). Each of these cyclic helical initiator compounds may be incoφorated at the N-terminus of the helical segment of ShK, and the biological activity of this short helical mimetic assessed. Additionally, this helical initiator modular component may be conveniently incoφorated into a solid-phase assembly permitting the synthesis of full-length analogs incoφorating this compound. The pharmacophore surface assessment and the analog-based contact point refinement data forms the basis for the design of peptidomimetic compounds. The contact point side-chain refinement data allows the optimization of potential K-channel interaction points by having a better understanding of geometry, distances, charge and hydrophobicity of the reciprocal K-channel interaction site. The analogs studied in modifying the K-channel selectivity of ShK polypeptide may be utilized in designing peptidomimetic compounds which are specific for a particular K-channel subtype.

Compounds which show a degree of similarity to the ShK pharmacophore will be tested for K-channel binding. Any compounds found to have binding affinity will constitute valuable new leads, which could then be modified with the aim of improving binding affinity and channel sub-type specificity. Even if matching compounds do not display binding initially, they may still serve as useful leads in the development of mimetic compounds in that they could represent useful structural scaffolds for further synthetic manipulation. In either case a close interaction between modelling and conformational analysis by NMR will be maintained in order to guide the synthetic program.

5.5 EXAMPLE 5 -- SHK-K22DAP: A POTENT KV1.3-SPECIFIC IMMUNOSUPPRESSIVE

PEPTIDE

This example describes the ShK mutant, ShK-K22DAP, which potently and selectively blocks the T-lymphocyte potassium channel, Kvl.3. The half-blocking dose for Kvl.3 is 28 pM. ShK-K22DAP is 100-fold less potent as a blocker of closely related channels: Kvl.l (Kd = 3.4 nM), Kvl .2 (5 nM); Kvl.5 (Kd > 100 nM), Kv3.1 (Kd > 100 nM), small conductance calcium-activated potassium channel in T-cells (Kd = 10 nM). The mutant is more selective than the native peptide, ShK, which blocks Kvl.l and Kvl.3 with similar potency (Kd = ~10 pM).

5.5.1 A POTENT KV1.3-SPECIFIC IMMUNOSUPPRESSIVE POLYPEPTIDE

The voltage-gated potassium channel in T lymphocytes, Kvl.3, is an important molecular target for immunosuppressive agents. A structurally-defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus, inhibited Kvl.3 potently, and also blocked Kvl.l, Kvl .4 and Kvl.6 at sub-nanomolar concentrations. Using thermodynamic mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kvl.3, and utilizing the structure of ShK, a likely docking configuration was determined for this peptide in the channel.

99

Based upon this topological information, the critical critical Lys in ShK was replaced with the

99 positively-charged, non-natural amino acid, diaminoproprionic acid (ShK-Dap ), and

99 generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap , at sub-nanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte H- thymidine incoφoration in vitro. Toxicity with this mutant peptide was low in a rodent model with median lethal dose of -300 mg/kg body weight following intravenous administration. The

99 overall structure of ShK-Dap in solution, as determined from NMR data, is similar to that of native ShK polypeptide, but there are some differences in the region of residues involved in potassium channel binding. Based on these results ShK-Dap 22 or a structural analog may be used as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.

Many potent polypeptide inhibitors of the Kvl.3 channel have been isolated from scoφion venom. These polypeptides adopt well-defined conformations constrained by 3 or 4 disulfide bonds, and bind with extremely high affinity to a shallow vestibule at the external entrance to the Kvl.3 pore (Aiyar et al, 1995; Aiyar et al, 1996). The most selective of these, margatoxin (MgTX), suppresses T-lymphocyte activation in vitro and is immunosuppressive in vivo (Kath et al, 1997), suggesting the possibility of using MgTX as an injectable immunosuppressant. However, MgTX potently blocks the closely-related Kvl.2 channel (Grissmer et al, 1994), which is expressed in the brain, peripheral nerves, and heart (Chandy and Gutman, 1995), raising concerns about potential cardiac and neuronal toxic side-effects.

Extensive efforts are therefore ongoing to identify other more selective and potent peptide inhibitors of Kvl.3.

Despite differences in the scaffolds, the sea anemone and scoφion toxins share a conserved diad of residues that is essential for block of potassium channels (Pennington et al,

11

1996a;b; Dauplais et al, 1997). This diad consists of a critical lysine (Lys in the scoφion toxins and Lys²²/Lys²⁵ in ShK and BgK) and a neighbouring aromatic residue (Tyr in ChTX, Tyr²³ in ShK, Tyr²⁶ in BgK) separated by ~ 7 A. In the scoφion toxins, Lys interacts with a tyrosine (Tyr⁴⁰⁰ in Kvl.3, Tyr⁴⁴⁵ in Shaker) in the potassium channel selectivity filter (Aiyar et al, 1995; Aiyar et al, 1996; Ranganathan et al, 1996). A better understanding of the interactions between ShK and the Kvl .3 channel may guide the design of specific ShK mutants that have the potential to be used clinically as injectable immunosuppressants. This issue has been addressed using three approaches. Complementary mutagenesis of the ShK peptide and Kvl.3 coupled with thermodynamic mutant cycle analysis and molecular modeling, allowed us to determine a likely docking configuration of the ShK polypeptide in the channel. This information was used to guide the identification of a Kvl.3 -specific ShK mutant that inhibits T- cell activation in vitro and is minimally toxic in vivo. The structure of the mutant peptide was solved and compared with native ShK to discern the reasons for its specificity for Kvl.3. 5.5.1.1 PEPTIDE SYNTHESIS

Fmoc-amino acid derivatives were obtained from Bachem A.G. (CH-4416

Bubendorf, Switzerland). Solid-phase assembly was initiated with Fmoc-Cys(Trt)-2- chlorotrityl resin in order to minimize potential racemization of the C-terminal Cys residue (Fujiwara et al, 1994). Automated stepwise assembly was carried out entirely on an ABI-431A peptide synthesizer (Applied Biosystems, Foster City, CA). Fmoc-Dap(Boc) was substituted in

99 place of Lys in the assembly of the polypeptide. Following removal of the final Fmoc

99 protecting group, the Dap substituted polypeptide was cleaved and deprotected with reagent K (King et al, 1990) containing 5% triisopropylsilane. The crude product was precipitated into diethyl ether and subsequently dissolved in 20% AcOH. Oxidative folding of the polypeptide was initiated by dilution of the solubilized product into water (Dauplais et al. , 1997) and adjustment of the pH to 8.0 with NH₄OH. After folding for 2 hr, oxidized and reduced glutathione were added to a final concentration of 1 mM, and folding allowed to continue

99 overnight. The ShK-Dap analog was purified using RP-HPLC as described previously (Pennington et al, 1996a;b). HPLC-pure fractions were pooled and lyophilized. The structure and purity were confirmed by RP-HPLC, amino acid analysis and ESI-MS analysis. All other ShK analogs were synthesized, purified and characterized as reported previously (Pennington et al, 1995; Pennington et al, 1996a;b; Pennington et al, 1996a;b; Pennington et al, 1997). Samples were weighed and adjusted to account for peptide content prior to bioassay.

5.5.1.2 REAGENTS

Cell lines stably expressing mKvl.l, rKvl.2, mKvl.3, hKvl.5 and mKv3.1 (Grissmer et al, 1994) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and G418 (1 mg/ml). Human IK^., channels were studied in activated human T cells as described previously (Grissmer et al, 1994). All the mKvl.3 mutants and mKvl .4 used in this study have been described previously (Aiyar et al, 1995; Aiyar et al, 1996; Grissmer et al, 1994). Rat Kvl .6 and rKv3.4 were gifts from Dr. Olaf Pongs (ZMNH Hamburg, Germany). ¹²⁵I-ChTX was purchased from NEN Life Science Products (Boston, MA). Fetal calf serum and L-glutamine, penicillin, and streptomycin were obtained from GIBCO BRL, Grand Island, NY. Anti-CD3 monoclonal antibody was acquired from Biomeda

Ine (Foster City, CA). 5.5.1.3 ¹²⁵I-CHTX BINDING ASSAY

Membranes were prepared from a cell line stably transfected with the hKvl.3 channel. The membranes were suspended at 50 mg/ml in incubation buffer (5 mM NaCl, 5 mM KC1, 10 mM HEPES, 6 mM glucose, pH 8.4) in Falcon 96-well polystyrene plates. Peptides

99 were added in triplicate to wells at various concentrations. Cold ShK, ShK-Dap or MgTX were added to the membranes for 30 min, then I-ChTX (25 pM; 2200 Ci/mmol) was added, and the reaction allowed to proceed at 22°C for a further 20 min. The reaction was stopped by harvesting the membranes onto Packard GF/C Unifilter 96-well filter plates and by washing twice rapidly with ice-cold wash buffer (200 mM NaCl, 20 mM HEPES, pH 8.0). The filter plates were dried overnight, scintillation cocktail (Packard Microscint-20; Packard Bioscience, Meriden, CT) was added, and the plates counted in a scintillation counter (Packard Top Count). Specific binding was determined by subtracting non-specific binding (defined by 100 nM unlabeled ChTX) from total binding. This binding assay was protein dependent, saturable (B_max = 916 + 37 fmol/mg protein) and of high affinity (K_d = 23 pM).

5.5.1.4 MOUSE ACUTE-TOXICITY DETERMINATIONS

99

Several doses of ShK or ShK-Dap were administered by i.v. tail vein injection into 15-20 g Swiss- Webster male mice. Because of the relatively low toxicities and high expense of both synthetic peptides, only small groups (n=5) of mice were used. Mortality was assessed 24 hr after injection.

5.5.1.5 ACTIVATION OF HUMAN T-CELLS BY ANTI-CD3 ANTIBODY

Polymoφhonuclear cells (PMNs) were isolated over a Ficoll-Hypaque density gradient (Histopaque-1077 from SIGMA, St. Louis, MO). The isolated PMNs were incubated

(37°C, 5% CO₂) for < 2 days in RPMI-1640 supplemented with 10% fetal calf serum, 1 mM L- glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. The assay was conducted in a 96- well plate by first adding monoclonal anti-CD3 and various polypeptide concentrations to wells in triplicate. Anti-CD3 was titrated to dilutions that produced a 4-25 fold increase in H- thymidine incoφoration. PMNs were resuspended in fresh media and then added to wells at a final concentration of 0.3 x 10 cells/well (final volume 200 ml). For determination of background uptake, anti-CD3 was not added to six wells in each plate, and the averaged H- thymidine uptake from these wells subtracted from wells containing anti-CD3. Plates were incubated for 48 hr, and ³H-thymidine added during the last 6 hr. The contents of the wells were harvested onto glass fiber filters (Packard GF/C unifilters) using a multi-well harvester and cells were lysed with water. Filters were air-dried overnight. Scintillation cocktail (Packard Microscint-20) was added and H-thymidine incoφoration measured by counting in a scintillation counter.

5.5.1.6 ELECTROPHYSIOLOGICAL ANALYSIS 5.5.1.6.1 MAMMALIAN CELLS

Each construct was linearized with EcoRI and transcribed in vitro with the SP6 Cap-Scribe System (Boehringer Mannheim, Germany). The resulting cRNA was phenol/chloroform extracted and stored at -75 C. cRNA was diluted with a fluorescent FITC-dye (0.5 % FITC-Dextran in 100 mM KC1; Fluorescein-dextran MW 10,000 was obtained from Molecular Probes, Eugene, OR, U.S.A. and from Sigma, Deisenhofen, Germany) to a final concentration of 1 mg/ml. The cRNA/FITC-solution was filled into injection capillaries (Femtotips, Eppendorf, Germany) and rat basophilic leukemic (RBL) cells, chosen because they lack endogenous Kv channels (McCloskey and Cahalan, 1990), were injected using an Eppendorf microinjection system (Micromanipulator 5171 and Transjector 5246), as described previously (Nguyen et al, 1996; Ikeda et al, 1992). Fluorescent cells were visualized 2-6 hr later and electrical currents measured using the patch-clamp method. Studies were performed at room temperature (21°-25° C) as described before (Nguyen et al, 1996; Rauer and Grissmer, 1996). Cells measured in the whole-cell configuration were normally bathed in mammalian Ringer solution containing (in mM): 160 NaCl, 4.5 KC1, 2 CaCl₂, 1 MgCl₂, 10 HEPES adjusted to pH 7.4 with NaOH, with an osmolarity of 290-320 mOsm. A simple syringe-driven perfusion system was used to exchange the bath solutions in the recording chamber. The internal pipette solution for Kv channel recordings contained (in mM): 134 KF, 1 CaCl₂, 2 MgCl₂, 10 HEPES, 10 EGTA, adjusted to pH 7.2 (with KOH) and an osmolarity of 290-320 mOsm. The internal pipette solution for the K^ channel recordings contained (in mM): 135 K-aspartate, 2 MgCl₂ 10 HEPES, 10 EGTA, 8.7 CaCl₂ adjusted to pH 7.2 (with KOH), and an osmolarity of 290-320 mOsm ([Ca²⁺]_free of 10^"6 M). Series resistance compensation (80%) was used if the current exceed 2 nA. Capacitative and leak currents were subtracted using the P/8 or P/10 procedure. The holding potential in all studies was -80 mV.

5.5.1.6.2 OOCYTES cRNA was transcribed in vitro and injected into oocytes (Xenopus laevis purchased from NASCO, Fort Atkinson, WI) as described previously (Aiyar et al, 1995; Aiyar et al, 1996). Potassium currents were measured at room temperature using the two-voltage clamp technique (Aiyar et al, 1995; Aiyar et al, 1996) and data were analyzed using pClamp software (version 5.5.1, Axon instruments, Burlingame, CA). Whole oocytes were held at -100 mV and depolarized to +40 mV over 500 ms; time between pulses was 30 s. Capacitative and leak currents were subtracted prior to analysis using the P/4 procedure. The dissociation constant was calculated assuming a 1 :1 binding of toxin to Kvl.3 as described (Aiyar et al, 1995; Aiyar et al, 1996).

5.5.1.7 THERMODYNAMIC MUTANT CYCLE ANALYSIS

This semi-quantitative method provides a simple and powerful way to evaluate the strength of interaction between any two pairs of residues in proteins (Schreiber and Fersht, 1995; Hidalgo and MacKinnon, 1995). For each mutant cycle the potency (K_d values) of ShK and each of its mutants was measured on Kvl .3 and each of the channel mutants.

0 1 1 29

Three positively-charged ShK residues, Lys , Arg and Lys , were chosen for mutagenesis studies. Each of these positively-charged residues was replaced individually by the neutral residues alanine (Ala) or norleucine (Nle). In the mutant cycle studies, the wild-type polypeptide was compared against the corresponding neutral polypeptide. In addition, Lys was replaced with the non-natural positively-charged lysine analogs, diaminopropionic acid

(Dap) or ornithine (Orn). These residues at ShK position 22 vary in their side-chain lengths (Dap = 2.5 A; Orn = 5.0 A; Nle = 5.0 A, Lys = 6.3 A). The positively-charged position-22

99 mutants (along with Lys ) were treated as "wild-type" in the mutant cycle analysis and

22 compared against the mutant polypeptide containing the neutral residue, Nle . The choice of channel residues to mutate was guided by earlier molecular model of the Kvl.3 external vestibule based on mapping studies with kaliotoxin and ChTX (Aiyar et al, 1995). Four residues of the Kvl.3 channel were selected for mutagenesis, His , Asp^{4 2}, Tyr⁴⁰⁰ and Asp . His was replaced with the hydrophobic residue Val and Asp with Lys. Since substitutions at positions 400 and 402 result in non-functional channels, dimeric constructs were generated containing one wild-type subunit and one mutant subunit. The resulting tetramers would be composed of [Asn ₂,Asp⁴⁰² ₂] and [Val ₂,Tyr⁴⁰⁰ ₂]). All of these channel mutants have been used previously in mapping studies with kaliotoxin (Aiyar et al, 1995; Aiyar et al, 1996). The mutant cycles for Kvl .3 -His and various ShK residues are shown below.

. Kvl.3[His⁴⁰⁴ - Val⁴⁰⁴]-ShK[Argⁿ - Ala^u]

• Kvl .3[His⁴⁰⁴^ Val⁴⁰⁴]-ShK[Lys²² - Nle²²]

• Kvl .3 [His⁴⁰⁴^ Val⁴⁰⁴]-ShK[Dap ²² - Nle²²]

. Kvl.3[His⁴⁰⁴^ Val⁴⁰⁴]-ShK[Orn²² - Nle²²]

Similar cycles were constructed to measure coupling interactions between Asp

(A_Λ„sp³⁸⁶_ ->_Lτys386N ), A_A sp402 ( ,A_Asp402₄_- A_Asn402₂,A_Asp402_2Λ),_TT-yr400 (.-.Tr.yr400₄^χ_ΛV₇-aιl400₂,_τTyr400₂^) and, polypeptide positions 9, 11 and 22.

The change in coupling energy, DDG, for a given pair of Shk-Kvl.3 residues and their mutants was calculated using the formula DDG = kT/«Ω, where Ω is a dimensionless value given by the formula, Ω = K_d (Wt ShK-Wt Kvl.3) x K_d (mut ShK-mut Kvl.3)/ K_d (mut ShK-Wt Kvl.3) x K_d (Wt ShK-mut Kvl.3). Schreiber and Fersht (Schreiber and Fersht, 1995) demonstrated a strong correlation between DDG values obtained by mutant cycle analysis and inter-residue distance derived from crystal structures of Barnase and Barstar; DDG values of > 0.5 kcal.mol^"1 (2σ error) corresponded to an inter-residue distance of < 5 A, and higher DDG values matched shorter inter-residue distances. To be conservative, a DDG value of >0.8 kcal.mol^" was used as an indicator of a close interaction between a pair of peptide and channel residues. Note that, although high DDG values indicate tight interactions, residues that are physically close may be energetically "silent" and not be detected by this method (Clackson and Wells, 1995).

5.5.1.8 STRUCTURE DETERMINATION Two-dimensional H NMR spectra were recorded at 600 MHz on a ca. 2 mM solution of synthetic ShK-Dap²² in 90% H₂O/ 10% ²H₂O (v/v) or 100% ²H₂O at pH 4.9 and 293 K, essentially as described previously (Tudor et al, 1996; Pallaghy et al, 1997) but with water suppression by means of pulsed field gradients using the WATERGATE scheme and a 3-9-19 selective pulse (Sklenar et al, 1993). Spectra were also recorded at 278 K in an attempt to

9 91 99 9^ shaφen some of the backbone amide resonances, eg. from Ser , Cys , Met , Dap and Tyr .

Chemical shifts for the resonances of Dap in the synthetic polypeptide GlyGlyDapGlyGly-OH were measured from both ID and TOCSY spectra at 293 and 298 K in 90% H₂O/10% ²H₂O at pH 5.0, using 2,2-dimethyl-2-silapentane-5-sulfonate as internal standard.

A summary of the sequential assignments, slowly exchanging amides, backbone

99 coupling constants and medium-range NOEs for ShK-Dap was prepared in combination with a Table of 1H chemical shifts (Table 2). Methods for obtaining distance and angle restraints, generating structures in D YANA (Gϋntert et al. , 1997) and refining the structures by restrained simulated annealing and restrained energy minimization in X-PLOR (Brunger, 1992), were as described previously (Tudor et al, 1996; Pallaghy et al, 1997). The final NMR restraint list (from which values redundant with the covalent geometry had been eliminated by DYANA) consisted of 82 intra-residue, 82 sequential, 105 medium-range (|i-j|<5) and 74 long-range (|i- j|>5) upper bound restraints, no lower bound restraints, and 30 backbone and 6 side-chain dihedral angle restraints. Of the 50 CHARMM-minimized structures, the best 25 were chosen on the basis of their stereochemical energies (i.e. excluding the electrostatic term). Of these, the best 20 were chosen on the basis of their Ramachandran plots and the consistency of their secondary structures with the NMR restraints. These structures and the NMR restraints on which they were based have been submitted to the Protein Data Bank (Bernstein et al. , 1977) (PDB ID code lbei). Structures were analyzed using Insight II (Molecular Simulations Ine, San Diego) and MOLMOL (Koradi et al, 1996). Hydrogen bonds were identified in MOLMOL using a maximum C-N distance of 2.4 A and a maximum angular deviation of 35° from linearity. TABLE 2 PROTON CHEMICAL SHIFTS OF SHK-DAP²² AT 20°C AND PH 4.9^A

Residue: NH αH βH γH δH b

Argl 4.10 1.95 1.69 3.23 N^εH 7.30

Ser2 8.92 4.48 3.80

Cys3 9.03 4.85 2.98

Ile4 7.78 4.66 1.95 C^γH₂0.99, 1.19; C^γH₃0.86; C^δH₃ 0.81

Asp5 8.63 5.31 3.26. 2.70

Thr6 9.49 4.48 4.57 1.25

Ile7 7.27 4.79 1.90 C^γH₂ 1.36, 0.85; C^γH₃ 0.49; C^δH₃ 0.53

Pro8 - 4.27 2.42, 1.76 1.95, 3.40, 2.07 3.82

Lys9 8.36 3.89 2.03, 1.86 1.52 1.75 C^εH₂ 3.07

Ser 10 8.46 4.10 3.91

Argi l 8.15 4.44 1.94, 2.29 1.76 3.28, N^εH 7.47 3.14

Cysl2 7.99 5.04 3.28, 2.91

Thr 13 7.28 4.38 4.78 1.31 O^γH

5.84

Alal4 8.88 3.98 1.47

Phel5 8.53 4.15 3.24, 2.88 C(2,6) 7.09; C(3,5) 7.06 C(4) 6.86

Glnl6 7.80 4.18 1.95, 1.49 2.28, N^εH₂ 6.49,

2.35 6.47

Cys 17 8.52 4.22 3.20, 2.94

Lysl8 7.52 4.01 1.57, 1.41 1.14 0.94 C^εH₂2.85

His 19 7.79 4.46 3.07. 2.35 C(2)H 8.34; C(4)H 6.50

Ser20 8.35 5.05 4.10, 3.90

Met21 9.40 4.13 2.17, 2.67 2.56 εH 2.06

Dap22 - 4.19 3.09, 2.75 N^γH₃ ⁺ 7.15 Tyr23 8.10 3.97 3.38, 2.61 C(2,6) 7.50; C(3,5) 6.95

Arg24 8.06 3.94 2.25, 1.77 1.70, 3.36, N^εH 7.37 N^ηH₂ 6.51, 1.52 3.22 6.83

Leu25 8.20 4.43 1.77. 1.49 1.68 0.89, 0.85

Ser26 7.21 4.74 3.55, 3.38

Phe27 7.48 5.30 3.27, 2.50 C(2,6) 6.20; C(3,5) 7.22; C(4) 7.15

Cys28 8.60 5.83 3.28. 3.13

Arg29 8.43 3.91 1.63, 1.83 1.46 3.27, N^εH 7.15 N^ηH₂ 7.21, 3.40 6.64

Lys30 7.21 4.17 1.84 1.32 1.63 C^εH₂3.09

Thr31 10.87 3.87 4.08 1.32

Cys32 9.16 4.78 3.34, 2.91

Gly33 7.87 4.08, 4.08

Thr34 8.73 4.16 4.42 1.14

Cys35 7.79 4.33 3.34, 2.94

Chemical shifts are in ppm and referenced to an impurity peak at 0.15ppm. For methyl groups, two chemical shifts are listed where both protons could be assigned. Resonances assigned stereospecifically are underlined with the first entry having the lower branch number. This resonance could not be assigned due to fast exchange with water.

5.5.1.9 MODEL OF Kvl.3 AND SHK DOCKING

The configuration of ShK bound to the Kvl.3 channel was investigated by docking the closest-to-average ShK structure (from the family of NMR-derived solution structures, ref. 19) into a model of the pore and vestibule of Kvl.3 (residues 380-410). To create the Kvl.3 model, residues Phe⁴²⁵, Lys⁴²⁷, Thr⁴⁴⁹, Gly⁴⁵², Phe⁴⁵³ and Tφ from a recent model of the Shaker potassium channel (Durell et al, 1998) were changed to the corresponding Kvl.3 residues, Gly³⁸⁰, Asn³⁸², His⁴⁰⁴, Thr⁴⁰⁷, He⁴⁰⁸ and Gly⁴⁰⁹, respectively, using Insight II. Modifications to the backbone and side-chain dihedral angles were then made so that the local and global structure of the channel model better resembled the corresponding region of the recently published structure of KcsA, a potassium-channel from Streptomyces lividans (Doyle et al, 1998). Following conjugate gradient minimization of the model using Discover (MSI), ShK was juxtaposed with the channel so as to preclude steric contact between the two. The backbone atoms (N, C^a and C) of Kvl.3 were fixed in Cartesian space during the simulation, while the backbone fold of ShK was maintained by 16 medium- and 3 long-range distance constraints. Inter-molecular distance constraints were added to the peptide-channel complex in conjunction with a 50 kcal mol^"1 force constant in Discover so as to reflect data from thermodynamic mutant cycle analyses (see Results), with Lys N^z (ShK) being kept within 5 A of Tyr⁴⁰⁰ C^g (to mimic an interaction with the phenolic ring) from each of the four Kvl.3 subunits, and Arg C^z being kept within 5 A of a single His N . A lower limit of 6 A was maintained between Arg C^z and Asp C⁸ to restrict any interaction between these two residues, which show no coupling. The complex was energy minimized using 10,000 steps of conjugate-gradient minimization, then a 250 ps molecular dynamics simulation was performed in vacuo at 300 K with a 1 fs time-step, a distance-dependent dielectric and a 15 A non-bonded interaction cut-off. After equilibration of the complex, the conformation with the lowest van der

Waals repulsive energy was chosen for further energy minimization, carried out as above.

5.5.1.10 ABBREVIATIONS

The abbreviations used are: AcOH, acetic acid; Boc, t-butyloxy carbonyl; Dap, diaminopropionic acid; ChTX, charybdotoxin; MgTX, margatoxin; Kv: Voltage-gated K⁺ channel; Fmoc, fluorenylmethyloxy carbonyl; ESI-MS, electrospray ionization-mass spectroscopy; NOE, Nuclear Overhauser Enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; PMN, polymoφhonuclear cells; TOCSY, total correlation spectroscopy; RBL, rat basophilic leukemic; RMS, root mean square; RP-HPLC, reverse phase HPLC. 5.5.2 RESULTS

5.5.2.1 SHK, A POTENT BLOCKER OF THE Kvl.3 CHANNEL IN T LYMPHOCYTES

The 35-residue polypeptide toxin ShK blocks mKvl.3 currents with a K_d of 11 + 1.4 pM (n=4; mean ± SEM; FIG. 1A, Table 3) and with 1 :1 stoichiometry (FIG. IB). Similar results were obtained for block of Kvl .3 channels in human peripheral blood T cells.

TABLE 3 SELECTIVITY OF SHK AND SHK-DAP²² ON KV CHANNELS

CHANNEL ShK ShK-Dap^z

(pM)^fl

mKvl.l 16 ± 3° (3) 1,800 ± 577 (4) rKvl.2 9,000 ± 300 (2) 39,000 + 3200 (3) mKvl.3 11 ± 1.4* (4) 23 ± 3* (4) mKvl.4 312 ± 51⁶ (2) 37,000 + 11,000 (2) hKvl.5 > 100,000 (3) > 100,000 (3) hKvl.6 165 ± 3^b (2) 10,500 + 900 (2) mKvl.7 11,500 ± 2340 (3) >100,000 (3) mKv3.1 >100,000 (3) >100,000 (3) rKv3.4 > 100,000 (3) >100,000 (3) hKCa4 28,000 ± 3,300 (3) >100,000 (3)

IC50 in pM + SEM; number of determinations shown in parentheses. The native intermediate conductance Kc_a channel in T lymphocyte is blocked with potency similar to the cloned hKCa4 channel.

125

The ShK polypeptide inhibited I-ChTX binding to its receptor in the external vestibule of hKvl.3. FIG. 2 shows the concentration-dependent displacement of specifically

195 bound I-ChTX by ShK. Fitting the concentration-response curve to a Hill equation reveals an IC₅₀ value for ShK of 118 + 20 pM (n = 5; mean + SEM) and 1 :1 peptidexhannel stoichiometry. Two ShK mutants, ShK-Dap²² and ShK-Nle²², also displaced ¹²⁵I-ChTX binding to hKvl.3 with 1 :1 stoichiometry and IC₅₀ values of 102 + 17 pM (n=8) and 663 + 172 pM (n = 6), respectively. MgTX had an IC₅₀ value of 78 + 10 pM (n = 6) in the same binding assay. Collectively, the electrophysiology and binding data indicate that ShK and the ShK-

22

Dap are potent blockers of the Kvl.3 channel, and these sea anemone polypeptides interact with a receptor in the external vestibule of the Kvl.3 channel that is identical or overlapping the receptor surface for the scoφion toxins.

To evaluate the selectivity of this polypeptide for Kvl.3, it was tested against a panel of eight K channel targets (Table 3). All the channels tested, with three exceptions, are

>100-fold less sensitive to block by ShK compared to Kvl .3 (Table 3). ShK, however, blocks mKvl.l, a cardiac and neuronal channel, with roughly the same potency as it does mKvl.3 (FIG. IC, FIG. ID), and two other channels, mKvl.4 and rKvl.6, are also blocked in the picomolar range (Table 3). Thus, ShK is not selective for Kvl.3, necessitating a search for a ShK mutant that might be more specific.

5.5.2.2 IDENTIFYING POLYPEPTIDE.CHANNEL INTERACTIONS

Determination of the docking configuration of ShK in the Kvl.3 channel external vestibule might help identify ShK mutants that exhibit Kvl.3 specificity. Guided by the solution structure of the ShK peptide (Tudor , 1996) and by a molecular model of the channel

(FIG. 3A), complementary mutants of these toxins and Kvl.3 were generated. Utilizing thermodynamic double mutant-cycle analysis (Aiyar et al, 1995; Aiyar et al, 1996; Schreiber and Fersht, 1995; Hidalgo and MacKinnon, 1995), specific pairs of peptidexhannel interactions were identified. Three residues in ShK were chosen for mutagenesis: Arg and Lys on the surface thought to interact with Kvl.3, and Lys on the opposite surface (Pennington et al, 1995; Pennington et al, 1996a;b; Pennington et al, 1996a;b; Pennington et al, 1997; Tudor et al, 1998). Four channel residues, His⁴⁰⁴, Asp⁴⁰², Tyr⁴⁰⁰ and Asp³⁸⁶ (FIG. 3 A) were targeted that have been shown previously to be important for scoφion toxin binding (Aiyar et al, 1995; Aiyar et al, 1996). His⁴⁰⁴ lies in the base of the Kvl .3 vestibule at the outer entrance to the ion conduction pathway (FIG. 3A). The ring of four His residues is unique to Kvl.3, and compounds that target this ring might therefore be selective for the lymphocyte channel (Kath et al, 1997). The highly conserved tyrosine (Tyr⁴⁰⁰) and aspartate (Asp ) in the critical signature sequence (GYGD) are thought to form part of the ion selectivity filter (Aiyar et al. , 1996; Chandy and Gutman, 1995; Ranganathan et al, 1996; Heginbotham et al, 1994). Asp⁴⁰² lies near His , while Tyr interacts with the essential Lys of scoφion toxins in a K⁺ ion- dependent manner (Aiyar et al, 1996; Ranganathan et al, 1996). Asp lies -10-14 A from the center of the pore and about 7-10 A from His⁴⁰⁴ and interacts with the positively charged Arg in kaliotoxin and Arg in charybdotoxin (Aiyar et al, 1995; Aiyar et al, 1996).

A fundamental assumption that guides the inteφretation of the mutant cycles is that the ShK peptide and its mutants sit in the channel with a similar geometry; this allows us to study the coupling energy (ΔΔG) for each pair of channel-peptide residues. Based on the criterion of Schreiber and Fersht (1995), it was assumed that a coupling energy of > 0.8 kcal mol^" corresponds to an inter-residue distance of < 5 A for a given pair of ShK-Kvl.3 residues. All the peptide-mapping studies were performed on channels expressed in Xenopus oocytes, whereas the studies described in FIG. 1A-FIG. ID were performed on channels expressed in mammalian cells. In general, there was very good correspondence between the K_d values

99 measured on channels expressed in mammalian cells and oocytes, although ShK-Dap was about 6-fold more potent in the oocyte system (K_d = 3.3 + 1.9 pM, n = 12) compared to mammalian cells (see FIG. 1 A-FIG. ID). Examples of two mutant cycles are presented in FIG. 4A. Replacing His in

Kvl.3 with the hydrophobic valine (Val⁴⁰⁴) significantly altered the interaction of Dap²² with the channel (DDG =2.0 kcal.mol^"1), but not that of the longer Lys²² analog (DDG =0.19 kcal.mol^" ). Using a similar approach, the coupling energies for several other Kvl.3-peptide pairs (FIG. 4B) were determined. His⁴⁰⁴ coupled tightly to Arg and with intermediate strength

99 Q to Orn . In contrast, Lys , the residue on the opposite surface of ShK, did not show any energetic coupling. These results suggest that Kvl .3-His lies close to ShK- Arg¹¹ and ShK-

Similar mutant cycles showed modest interactions between Asp in the channel,

99 99 and Dap and Orn in the peptide; there was no evidence for energetic coupling between Asp⁴⁰² and Lys⁹, Lys²² or Arg¹¹ (FIG. 4B). Strong energetic interactions were detected between

Tyr⁴⁰⁰-Orn²² and Tyr⁴⁰⁰-Lys²² (FIG. 4B), and a weaker interaction between Tyr⁴⁰⁰-Dap²² (ΔΔG = 0.7 kcal.mol^" which is below the threshold of 0.8 kcal.mol^" ); Arg and Lys did not show any coupling with Tyr (FIG. 4B). Additional mutant cycle studies showed a strong interaction between

(FIG. 4B).

5.5.2.3 ESTIMATED DIMENSIONS OF THE Kvl.3 VESTIBULE

Seven pairs of peptide: channel interactions have been identified involving four channel positions (ShK:Kvl.3 - Arg^u:His⁴⁰⁴, Dap²²:His⁴⁰⁴, Dap²²: Asp³⁸⁶, Dap²²: Asp⁴⁰²;

Orn²²:Asp⁴⁰², Orn²²:Tyr , Lys ²:Tyr⁴⁰⁰). Knowing the spatial relationships between these interacting residues (Tudor et al, 1998), th topological map of the Kvl .3 vestibule has been refined (FIG. 3B).

22 22

Mutant cycle data suggest that Lys and Orn protrude into the pore with their terminal ammonium groups positioned within 5 A of Tyr . However, the actual distance may be greater than 5 A since the observed coupling may be an overestimate due to interactions with more than one of the four Tyr „400 residues. These pairs of peptidexhannel residues might

99 99 interact via cation pi interactions. Based on the side-chain lengths of ShK-Lys and ShK-Orn (6.3 A and 5.0 A, respectively), Tyr⁴⁰⁰ must lie within 10-11 A of the peptide backbone of the toxin when it sits docked in the vestibule. Asp⁴⁰² interacts strongly with both Dap and Orn²² but not Lys ², suggesting that it must be located within 7.5 A of the external vestibule (i.e. length of side-chain of Dap + 5 A). Since His forms a tight energetic contact with Dap but not with the longer analogs, it must be positioned closer to the external surface than Asp⁴⁰².

Thus, one can imagine a cylinder lined by His⁴⁰⁴ at the external end, and the GYGD motif positioned vertically below this ring, with Asp⁴⁰² closer to the outside than Tyr . These estimated dimensions are similar to the dimensions of the external vestibule of the two transmembrane bacterial K⁺ channel, KcsA, determined by x-ray crystallography (Doyle et al. , 1998).

5.5.2.4 DOCKING SHK IN THE EXTERNAL VESTIBULE OF Kvl.3

1 1 99

The placement of Arg and Lys relative to the channel imposes constraints on possible docking configurations of the toxin in the external vestibule. For heuristic puφoses, ShK has been docked in the Kvl.3 vestibule, using restrained molecular dynamics simulations, such that Lys²² is kept in close proximity to the circle of Tyr residues in the channel pore and Arg in close proximity to His⁴⁰⁴ from one channel subunit. As shown in FIG. 3B this configuration places the three remaining His⁴⁰⁴ residues in the tetramer in close proximity to

91 90 90 ^Rή peptide residues Met and Arg . In addition, the model places Arg near Asp in the channel subunit diagonally opposite that which interacts with Arg . Two lines of evidence support this placement. First, introduction of the positively charged lysine at channel position 386 (D386K) causes a significant reduction in peptide potency (Kvl.3: 11.8 + 4.8 pM; n = 11 ; Kvl.3 D386K: K_d = 563 + 340 pM, n = 10), possibly via electrostatic repulsion of Arg²⁹ on ShK. Second, energetic coupling between Asp and Arg (DDG =0.88 kcal.mol^" ) was detected in mutant cycle studies (FIG. 4A, legend).

In summary, the Kvl.3-ShK docking model is supported by considerable experimental data. This docking configuration could guide the identification of ShK mutants

99 that exhibit Kvl.3 specificity. For example, the ShK-Dap mutant that couples strongly with the ring of four His residues unique to Kvl .3 and makes novel contacts with Asp might be sseelleeccttiivvee ffoorr tthhee llyymmphocyte channel. To test this idea, the ShK-Dap 22 mutant was evaluated in a selectivity screen.

5.5.2.5 SHK-D AP²² IS A POTENT AND SELECTIVE BLOCKER OF Kvl .3

The ShK-Dap²² mutant blocked mKvl.3 currents with a K_d of 23 + 3 pM, (n = 4; mean + SEM; FIG. 5A) and a Hill coefficient close to unity (FIG. 5B). Human Kvl.3 channels

99 1 5 are blocked with a similar potency. ShK-Dap displaced I-ChTX binding to hKvl .3 with an IC₅₀ of 102 + 17 pM (n=8; FIG. 2) and with 1:1 stoichiometry, indicating that the peptide binds in the external vestibule in a site overlapping the ChTX receptor. These results corroborate the mutant cycle data presented above. In a selectivity screen, ShK- Dap²² was found to be a highly selective inhibitor of

Kvl.3. ShK- Dap²² blocked mKvl.l, mKvl.4, rKvl.6, and other potassium channel targets with significantly less potency than Kvl.3 (Table 3). 5.5.2.6 SHK, SHK-DAP²² AND MGTX INHIBIT HUMAN T-CELL ACTIVATION WITH SIMILAR POTENCY

The ability of ShK, ShK-Dap²² and MgTX to suppress anti-CD3 -stimulated ³H- thymidine incoφoration by human peripheral blood T cells was analyzed. All three toxins inhibited lymphocycte activation to a maximum level of -50-60% of total stimulated H- thymidine incoφoration. However, the midpoint of inhibition (IC₅₀) for each toxin was below 500 picomolar, in keeping with their affinity for the Kvl.3 channel. Consistent with these results, an earlier study reported that peripheral blood T cells isolated from mini-pigs during intravenous MgTX infusion never showed more than a -60% inhibition of mitogen-stimulated H-thymidine incoφoration in an ex vivo proliferation assay (Koo et al, 1997). As MgTX is an effective immunosuppressant in vivo, despite its inability to completely inhibit mitogen-

• T 99 stimulated H-thymidine incoφoration in vitro, ShK-Dap , which is a more selective and potent inhibitor of Kvl.3, might be equally efficient as an immunosuppressant, but not exhibit the side-effects of MgTX.

5.5.2.7 SHK-DAP²² DOES NOT EXHIBIT ACUTE TOXICITY FOLLOWING INTRAVENOUS INJECTION INTO RODENTS

22

As an initial evaluation of the toxicity of ShK and ShK-Dap , mice were injected intravenously with each polypeptide. ShK polypeptide displayed a remarkably low toxicity when injected into mice, the median lethal dose being approximately 0.5 mg per 20 g mouse, or

99

25 mg/kg body weight. ShK-Dap was even less toxic; a 5.0 mg dose failed to cause any symptoms (hyperactivity or seizures) or mortality, and the median lethal dose was -300 mg/kg body weight.

5.5.2.8 SOLUTION STRUCTURE OF SHK-DAP²² AND COMPARISON WITH THE STRUCTURE OF NATIVE SHK

22

As a basis for future docking studies of ShK-Dap , and to determine if the

99 replacement of Lys by Dap had caused significant local conformational changes in the region of the potassium channel binding surface of the toxin, the solution structure of ShK-Dap²² has been determined using NMR data. 5.5.2.9 STRUCTURE OF SHK-DAP²²

99

The structural statistics for ShK-Dap (Table 4) show that the structures are in good agreement with the experimental restraints and have good stereochemistry. Moreover, 91% of the residues have φ-ψ values in the generously allowed regions of a Ramachandran plot,

33 Gly being the only residue with a positive f angle. The angular order parameters (S) (Hyberts et al, 1992; Pallaghy et al, 1993) of the final 20 structures indicate that residues 2-21 and 23- 35 are well defined locally, with S > 0.8 for both f and y angles. The backbone RMSD from the mean structure also shows that the structure is well defined over most of the molecule. For the puφoses of global superimposition residues 2-21 and 23-35 were considered as being well defined. Mean pairwise RMS differences calculated over the backbone heavy atoms (N, C^a, C) and all heavy atoms, respectively, of the whole molecule were 0.63 ± 0.15 and 1.41 ± 0.23 A, and for the well-defined region 0.51 + 0.13 and 1.04 + 0.14 A.

TABLE 4

STRUCTURAL STATISTICS FOR THE 20 ENERGY-MINIMIZED STRUCTURES OF SHK-DAP²² FROM X-PLOR⁴

RMS deviations from experimental distance restraints (A) (343)° 0.028 ± 0.001

RMS deviations from experimental dihedral restraints (deg) (36) 0.47 + 0.15

RMS deviations from idealized geometry: bonds (A) 0.0107 + 0.0006 angles (deg) 2.66 ± 0.05

Impropers (deg) 0.37 + 0.02

Energies (kcal mol )

ENOE 14.1 + 1.1

E_Cdih 0.53 ± 0.28

EL-J -126 + 7

^elec -513 + 29

Mean pairwise RMSD (A): Backbone heavy atoms All heavy atoms

Residues 1-35 0.63+0.15 1.41 + 0.23 Residues 2-21, 23-35 0.51+0.13 1.04 + 0.14

The best 20 structures after energy minimization in the distance geometry force field of X-PLOR were subsequently energy minimized in the CHARMM force field, using a distance-dependent dielectric. Values represent mean ± SD. b The numbers of restraints are shown in parentheses. None of the structures had distance violations > 0.3 A or dihedral angle violations > 5°.

22

The main secondary structure elements of ShK-Dap are two short a-helices encompassing residues 14-19 and 21-24. The first of these is stabilized by a capping box involving Thr¹³ and Gin¹⁶ as well as the flanking half-cysteine residues 12 and 17. The N- terminus adopts an extended conformation up to residue 8, where a pair of interlocking turns commences; in 25% of the structures this pair of turns satisfies the criteria for a 3₁₀-helix centered on residues 9-10 (with an 1 1-→8 hydrogen bond found in all 20 structures). There is also a short stretch of helix between residues 29-32 (with a 32→28 hydrogen bond in all 20 structures) that is a mixture of α- and p-helix. Backbone hydrogen bonds associated with these secondary structure elements account for many of the slowly exchanging backbone amide protons observed by NMR following dissolution in in H₂O. Several other backbone amide protons found to be slowly exchanging were shielded from solvent. Of the three disulfides, only 3-35 adopts a well-defined (right-handed) conformation, while the 12-28 and 17-32 disulfides exist as mixtures of left- and right-handed conformations.

5.5.2.10 COMPARISON WITH SHK

99

The overall structures of ShK and ShK-Dap are quite similar. Pairwise RMS differences over the backbone heavy atoms N, C^a and C between the closest-to-average structures for ShK and the analog are 1.82 A over residues 1-35, 1. A over residues 2-21 and 23-35 (the well-defined region of the analog), and 1.38 A over the well-defined region of ShK

(residues 3-33). The structure of the analog was calculated with NOEs and dihedral angle constraints similar in number to those for native ShK but with a different distribution.

The main secondary' structure elements of the two molecules are the same, but

22

ShK-Dap also has a recognizable helix near the C-terminus, involving residues 29-32. In ShK, this region has a similar structure but does not satisfy the criteria for a helix. The only appreciable differences between the backbone dihedral angles of the two structures occur at

Pro (ψ), Thr (φ) and the three C-terminal residues (φ).

5.5.2.11 POTASSIUM CHANNEL BINDING RESIDUES IN SHK-DAP²² The structures of ShK-Dap²² and ShK were aligned over N, C^a, C and C^b of residues 11-23, which includes the most important residues for potassium channel binding (Pennington et al, 1996a;b; Pennington et al, 1996a;b). In this view, the side chains of Arg

93 and Tyr have similar orientations, although they have moved closer together. The distances from Tyr²³ C^γ to Arg¹¹ C^γ are 3.9 ± 0.2 and 7.4 ± 0.7 A, respectively, in ShK-Dap²² and ShK.

93 The functionally more important distances from the centroid and phenolic oxygen of Tyr to

Arg¹¹ C^z are, respectively, 4.9 ± 0.2 and 3.3 ± 0.2 A in ShK-Dap²² and 6.7 ± 1.1 and 4.7 ± 1.4 A

92 in ShK. In ShK, the Lys side chain is not as well defined as other side chains in this region.

99 22

The shorter Dap side chain of ShK-Dap is better defined and in most structures is oriented

93 towards the Tyr ring (there may be a weak hydrogen bonding interaction between the positively charged NH₃ ⁺ group and the aromatic ring). Distances from the centroid and phenolic oxygen of Tyr to N^s or C⁸ of residue 22 are, respectively, 4.5 + 1.1 and 4.9 ± 1.0 A in ShK-Dap²² and 6.6 ± 0.8 and 8.1 ± 0.8 A in ShK. Corresponding distances from C^z of Arg¹¹ to N⁸ or C⁸ of residue 22 are, respectively, 8.0 ± 1.1 in ShK-Dap²² and 11.7 ± 1.5 A in ShK. Thus, it appears that these three functionally important residues, 11, 22 and 23, have moved closer together in ShK-Dap .

7 97

Associated with this, there has been a shift in the positions of the He and Phe side-chains; the centroid of the aromatic ring of Tyr is 6.3 ± 0.2 A from the centroid of the phenyl ring of Phe²⁷ in ShK-Dap²², compared with 4.5 ± 0.4 A in ShK, and 6.8 ± 0.2 A from C of He⁷, compared with 7.9 ± 0.7 A in ShK, although distances from N⁸ or C^g of residue 22 to the centroid of the Phe ring are unchanged at about 6.2 A. The shorter side chain of Dap

22

(compared with that of Lys in ShK) might be expected to increase the solvent accessibility of

99 1 Q nearby residues. The largest increase in ShK-Dap (1.4-fold) was for His , with the flanking residues showing little deviation.

Using the ShK peptide as a structural template and applying thermodynamic mutant cycle analysis to determine the spatial proximity of particular ShK and Kvl.3 residues, the geometry of the external vestibule and the outer pore of the lymphocyte channel was defined. These data, along with those obtained from earlier mapping studies with scoφion toxins (Aiyar et al, 1995; Aiyar et al, 1996), allowed refinement of the molecular model of the Kvl.3 vestibule and to dock the ShK peptide into the channel. Second, the docking model was used to identify the Kvl.3 specific ShK mutant, ShK-DAP²². ShK-DAP²² inhibited mitogen-stimulated human T-cell activation in vitro with sub-nanomolar potency and exhibited minimal toxicity in

11 vivo in a rodent model. Third, the structure of ShK-DAP was solved by NMR. By comparing the structures of the native and mutant peptides an understanding for the basis for the Kvl .3

99 specificity of ShK-DAP was obtained. These three complementary studies suggest that ShK-

22

DAP might be a clinically useful immunosuppressant provided additional improvements are made with regards to peptide stability, plasma half-life, and oral availability.

Peptide toxins as candidate immunosuppressive agents ~ The Kvl.3 channel is widely regarded as a novel therapeutic target for T-cell immunosuppression (e.g., 2, 9). Due to its restricted tissue distribution and unique role in regulating T-cell function, selective and potent blockers of this channel might not have the toxic side-effects of currently used drugs such as cyclosporin, FK-506 and rapamycin (Cahalan and Chandy, 1997; Kath et al, 1997).

Kvl.3-specific antagonists may therefore be useful in the prevention of graft rejection and treatment of diverse autoimmune diseases.

Several scoφion toxins potently and reversibly block this channel with IC₅₀ values in the low picomolar to nanomolar range, and with 1 :1 stoichiometry (Aiyar et al, 1995; Aiyar et al, 1996). By blocking Kvl.3, these polypeptides attenuate the calcium signaling response and inhibit mitogen-activation of T cells in vitro (Price et al, 1989; Leonard et al, 1992; Lin et al, 1993; Nguyen et al, 1996; Kath et al, 1997; Koo et al, 1997). The most potent and selective of these, MgTX, has also been shown to effectively suppress delayed-type hypersensitivity and alloimmune responses in vivo in micro- and mini-pigs (Koo et al, 1997). However, MgTX also potently blocks the closely related channels, Kvl.l and Kvl.2 (Grissmer et al, 1994; Kocj et al, 1997; Koschak et al, 1998), which are expressed in the brain and peripheral neurons, and is therefore potentially toxic. An equally potent but more selective peptide blocker of Kvl.3 might not exhibit these side effects.

99

This example describe a structurally-defined peptidic inhibitor, ShK-Dap , that exhibits the requisite potency and specificity for the Kvl.3 channel target. This polypeptide shows significant inhibitory activity in an in vitro human T cell proliferation assay, and does not produce in vivo toxic effects in mice following intravenous injection at a dose (25 mg/kg) many times that required for immunosuppressant activity.

99

Comparison of structures of ShK and ShK-Dap ~ The overall structure of ShK-

22

Dap determined by NMR is similar to that of the native ShK peptide toxin (Tudor et al. , 1998; Figs. 7 and 8), but there are some differences in the region of side chains involved in potassium channel binding. Are these differences significant, or do they reflect differences between the number and distribution of NMR-based restraints in key regions in the structure. Such differences could arise from differential peak overlap in the two NOESY spectra. It is therefore appropriate to consider how the differences between the calculated structures compare with differences in measured parameters such as chemical shifts, coupling constants and NOEs. The

H chemical shifts of the two molecules are very similar, the only differences > 0.1 ppm being

91 99 3 for Met NH (Dd 0.25 ppm), Dap and residues 26-28 (Table 2). The J_HNCBH coupling constants, which are dependent on backbone φ angles, also differed by > 1 Hz for residues 26, 27 and 29 (other residues in this category were 9, 10, 16 and 35). The backbone amide

99 91 23 99 resonance of Dap was not observed at all, and those of Met and Tyr in ShK-Dap were broader than in ShK. As a result, there were fewer NOEs to these protons and this region of the

99 99 structure is not as well defined in ShK-Dap . Part of the reason for the broader Dap NH resonance is that the intrinsic linewidth is greater, as found in the penta-peptide GlyGlyDapGlyGly (spectra not shown); this presumably reflects the proximity of the side-chain ammonium group of Dap to the backbone. However, this is unlikely to be the explanation for the flanking residues, suggesting that this region has greater conformational flexibility in ShK-

22 99

Dap . In order to confirm the difference between ShK-Dap and ShK, a NOESY spectrum was recorded on a mixture of the two at pH 4.7 and 293 K. Resonance overlap prevented any

93 21 99 comparison for Tyr , but it was quite clear that the cross-peaks from Met of ShK-Dap were

91 broader and weaker than those of ShK. Bearing in mind that the chemical shift of Met NH was also perturbed, it appears that there are some genuine differences in the local structure and dynamics of ShK-Dap²² around the substituted residue. The backbone amides of ShK-Dap²² also show slightly faster exchange than those of ShK (although respective rate constants are within a factor of 2 of one another), suggesting that the overall structure of ShK-Dap²² may be slightly more flexible than that of ShK. In other regions, particularly the less well-defined regions at the N- and C-termini, the apparent structural differences partly reflect differences in the number and distribution of

NOEs. For example, there are differences in the backbone dihedral angles of the three C- terminal residues (φ and to a lesser extent ψ), but their chemical shifts differ by < 0.03 ppm in the two molecules, and the J_HNCaH coupling constants for Thr and Cys are similar (although

32 22 the value for Cys is 1 Hz higher in ShK-Dap , at 6.7 Hz). These data imply that the structures are very similar, making it likely that the apparent differences stem partly from the

99 presence of a few NOEs unique to one of the restraint sets. The fact that the ShK-Dap structures are better defined than those of ShK at both termini may imply that they are more representative of the 'real' structure in solution for both polypeptides. However, it is important to note that there is some flexibility in these regions of both structures, and that both may change when bound to potassium channels.

5 30

In ShK there is evidence for an electrostatic interaction between Asp and Lys (Tudor et al, 1998), which may also be important in folding, at least in vitro (Pennington et al,

99 1996a or b?). In ShK-Dap , both the side-chain functional groups and the a-carbons are about

1 A further apart than in ShK, implying that this interaction may be slightly weaker.

22

Finally, the similarity between the structures of ShK-Dap and ShK confirms that the structure of this sea anemone-derived potassium channel blocker is different from that of the homologous BgK toxin (Dauplais et al, 1997), with which it shares 13 residues. BgK contains two longer helices, involving residues 9-16 and 24-31, although its overall topology is similar to that of ShK.

5.6 EXAMPLE 6 — ADDITIONAL SHK MUTANTS

The inventors have also constructed the following mutant forms of ShK: ShK 106 K-14-Asp- Lactam Bridge; ShK 108 Ile-7Cys, C12 Abu; ShKl lO Abu 121-Abu28, Ala21;

ShKl l l Abul7-Abu32, Ala21 ; ShK112 Abu3-Abu35, Ala21.

5.6.1 SHK-117 DES(ARG1-SER2)

ShK-117 des(Argl-Ser2) - 3-mercaptopriopionyl- Lysl4-Aspl8 (lactam bridge between side chains) Nle21, DAP22 Cys35-amide. This peptide contains disulfide bonds between the 3-mercaptopriopionyl and Cys35, Cysl2-Cys28 and Cysl7-Cys32. This peptide lacks the N-terminal Arg-Ser and begins with des-amino Cys (i.e. 3-mercaptopriopionyl). This maintains the disulfide pairing integrity of the peptide while eliminating protease susceptibility at the N-terminus. The C-terminus is amidate as a means of stabilizing the C-term to carboxypeptidase type of proteases. The DAP22 substitution has been retained to maintain the selectivity. The Nle21 is replacement for Met to prevent oxidation. Lastly, the lactam bridge between Lys 14 and Asp 18 stabilizes the helix at this position as well as minimizing protease susceptibility at these positions. Any combination and/or all of these substitutions should be protected as they may have different effects in different combinations.

5.6.2 SHK ARGII

This analog substitutes Argi l to Alal l to better assign the docking configuration into the Kvl.3 model.

5.6.3 SHK NLE21, DAP22 This double mutant contains selectivity determinant DAP22 and the Nle for Met at position 21 to stabilize versus oxidation. This peptide has a Ki on rat brain of 2 μM but maintains a Ki of 10 pM for Kvl.3. The peptide has an IC₅₀ of 1,940 nM. (see FIG 7C). The control wildtype ShK data is shown in FIG. 7A.

5.6.4 SHK ALA21, DAP22

Double mutant contains selectivity determinant DAP22 and the Ala for Met substitution at positon 21 stabilizes the molecule. This peptide has an IC₅₀ of 507nM (see FIG. 7B).

5.6.5 SHK- HYP20

This substitution replaces Ser20 with an amino acid which maintains the hydroxy group while stabilizes the kink between the two helices. 5.6.6 POLYPEPTIDES MAY BE TRIMETHYLATED OR TRIETHYLATED AT LYS22 AND OTHER LYS/ARG POSITIONS TO REDUCE PROTEASE DEGRADATION

This substitution may help prevent protease degradation at the basic amino acid residues by increasing the steric bulk at these positions while retaining the cationic charge. These may be substituted for Lys22 or any other basic amino acid positions. Likewise, we could also produce the trimethylated or triethyl DAP to maintain specificity and eliminate degradation.

5.6.7 SUMMARY OF IMPORTANT COMBINATORIAL SHK MUTANTS Given these potential substitutions, the inventors show the following exemplary mutants having various substitutions in the primary ShK sequence, and contemplate their use in the formulation of compositions having potassium channel-inhibitory activity. Based on the peptide sequences set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3, the following substitutions are made at the residues indicated: 1. Nle21 , trimethylated-Lys22

2. Nle21 , triethy lated-Ly s22

3. Nle21, DAP22

4. Nle21 , trimethylated-D AP22

5. Nle21 , triethylated-D AP22 6. trimethylated-Lys22

7. triethylated-Lys22

8. DAP22

9. trimethylated-DAP22

10. triethy lated-DAP22 11. Hyp20, Nle21, trimethylated-Lys22

12. Hyp20, Nle21, triethylated-Lys22

13. Hyp20, Nle21, DAP22

14. Hyp20, Nle21 , trimethylated-D AP22

15. Hyp20, Nle21 , triethylated-D AP22 16. Hyp20, trimethylated-Lys22

17. Hyp20, triethylated-Lys22 18. Hyp20, DAP22

19. Hyp20, trimethylated-D AP22

20. Hyp20, triethylated-DAP22

21. Alal 1 , Nle21 , trimethylated-Lys22 22. Alal l, Nle21, triethylated-Lys22

23. Alal l, Nle21, DAP22

24. Alal 1 , Nle21 , trimethylated-D AP22

25. Alal 1, Nle21, triethylated-DAP22

26. Alal 1 , trimethylated-Lys22 27. Alal l, triethylated-Lys22

28. Alal l, DAP22

29. Alal 1 , trimethylated-D AP22

30. Alal 1 , triethylated-D AP22

31. Alal 1 , Hyp20, Nle21 , trimethylated-Lys22 32. Alal l, Hyp20, Nle21, triethylated-Lys22

33. Alal l, Hyp20, Nle21, DAP22

34. Alal 1 , Hyp20, Nle21 , trimethylated-D AP22

35. Alal 1 , Hyp20, Nle21 , triethylated-D AP22

36. Alal 1 , Hyp20, trimethylated-Lys22 37. Alal l, Hyp20, triethylated-Lys22

38. Alal l, Hyp20, DAP22

39. Alal 1 , Hyp20, trimethylated-D AP22

40. Alal 1 , Hyp20, triethylated-D AP22

Further stabilization of the peptides occurs, as stated above, by creating disulfide bonds between the 3-mercaptopropionyl and Cys35. Alternatively, disulfide bonds may be formed between Cysl7 and Cys22. As a reference, FIG. 8 shows the primary sequence of native ShK toxin (Karlsson et al, 1992) and compares it to BgK toxin (Aneiros et al, 1993). In FIG. 9, a schematic representation of ShK disulfide pairings is shown, and the sequence of wild-type ShK toxin (Karlsson et al, 1992), BgK toxin (Aneiros et al, 1993; revised, Karlsson et al, 1992), AsK (Schweitz et al. , 1995) and ChTX (Sugg et α/., 1990) are shown in FIG. 10.

5.7 EXAMPLE 7 - IMMUNOSUPPRESSIVE ACTIVITY OF SHK AND SHK-KSSDAP Peripheral blood human lymphocytes were activated by anti-CD3 antibody by routine methods. Briefly, cells were isolated by Ficoll-Hypaque density sedimentation, and placed in media (RPMI-1640 supplemented with 10% fetal calf serum, 1 -glutamine and penicillin/streptomycin). The cells were incubated alone, or with anti-CD3 antibody, or with anti-CD3 antibody plus various concentrations of ShK or ShK-KSSDAP. Following 48 hours of incubation at 37°C in 5% CO₂ atmosphere, cells were pulsed with H-thymidine for 6-16 hours, the cells then harvested, and the thymidine uptake determined. ShK and ShK K22DAP suppressed T-cell activation with an IC50 of about 1 nM.

5.8 EXAMPLE 8 ~ TREATMENT OF AUTOIMMUNE DISEASES The CD18-null PL/J mice provide an excellent system for the study of the efficacy of ShK and ShK derived mutants for the treatment of hypeφroliferative skin disorders. A recognized animal model for the assessment of the therapeutic activity of a composition for the treatment of inflammatory skin disease is described by Bullard et al. (1996). The inventors contemplate that this model may be utilized to identify ShK polypeptide compositions useful in the treatment of inflammatory skin diseases. The ShK polypeptide may be administered to

CD18-null PL/J mice in a manner similar to the administration of dexamethasone in the Bullard study (Bullard et al, 1996). In preferred embodiments, the ShK polypeptide is administered inteφeritoneally, intraveneously, of subcutaneously.

CD18-deficient 129/Sv are backcrossed onto the PL/J strain for several generations (N₄, N₇, and N₈). Homozygous mutants are used for analysis. Ten CD 18 homozygous mice displaying severe dermatitis and ten non-mutant littermate controls are given daily subcutaneous injections of an effective amount of a compound of the present invention for at least six weeks. A variety of concentrations of the compound may be given to determine the dose effect. The compound then is withdrawn completely or the concentration is lowered over a period of several weeks. Improvement and exacerbation of the dermatitis is clinically assessed on a daily basis. Histological and immunological analyses may be performed as described in Bullard et al (1996). Improvement during the period of administration of the compound followed by exacerbation upon the withdrawal or reduction of the concentration of administered compound is indicative of an effective compound for the treatment of inflammatory skin disease. Similar to autoimmune diseases, transplantation of organs into a new host causes an immune response against the new organ. Immunosuppressive compounds are routinely given to patients following organ transplantation to decrease the probability of rejection of the newly transplanted organ. Therefore, transplantation model systems in animals also may be employed to test the efficacy of anti-inflammatory or autoimmune compounds, such as the polypeptides of the present invention.

The inventors contemplate that the polypeptides of the present invention may be used as an in immunosuppressant in transplantation procedures. For example, Granger et al. (1995) describe a the determination of the efficacy of rapamycin monotherapy for immunosuppression following kidney transplantation in swine. The procedures of Granger et al. may be repeated using a polypeptide of the present invention in place of rapamycin.

RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 1 )

CIDTIPKSRCTAFQCKHSNDYRLSFCRKTCGTC (SEQ ID NO:2)

RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO:3)

6.0 REFERENCES

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(1) GENERAL INFORMATION:

(i) APPLICANT:

(A NAME: The University of Florida (B STREET: 223 Grinter Hall (C CITY: Gainesville (D STATE : Florida (E COUNTRY: USA (F POSTAL CODE (ZIP) : 32S11

(A NAME: The Regents of the University of California (B STREET: 300 Lakeside Drive (C CITY: Oakland (D STATE: California

(E : COUNTRY: USA

(F POSTAL CODE (ZIP) : 94612-3550

(A NAME: Bachem Bioscience, Inc. (B STREET: 3700 Horizon Drive (C CITY: King of Prussia (D STATE : Pennsylvania (E COUNTRY: USA (F POSTAL CODE (ZIP) : 19406

(A NAME: Biomolecular Research Institute (B STREET: 343 Royal Parade (C CITY: Parksville (E COUNTRY: AUSTRALIA (F POSTAL CODE (ZIP) : 3052

(ii) TITLE OF INVENTION: POLYPEPTIDE COMPOSITIONS THAT INHIBIT POTASSIUM CHANNEL ACTIVITY AND USES THEREFOR

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US 97/22096

(B) FILING DATE: 26-NOV-1997

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/059,126

(B) FILING DATE: 17-SEPT-1997

(2) INFORMATION FOR SEQ ID NO : 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1:

Arg Ser Cys lie Asp Thr lie Pro Lys Ser Arg Cys Thr Ala Phe Gin 1 5 10 15

Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30

Gly Thr Cys 35

(2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2:

Cys lie Asp Thr lie Pro Lys Ser Arg Cys Thr Ala Phe Gin Cys Lys 1 5 10 15

His Ser Asn Asp Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 20 25 30

Cys

(2) INFORMATION FOR SEQ ID NO : 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Arg Ser Cys lie Asp Thr lie Pro Lys Ser Ala Cys Thr Ala Phe Gin 1 5 10 15

Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30

Gly Thr Cys 35 All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS:

I . A method of inhibiting potassium channel activity in a lymphocyte cell of an animal, comprising providing to said cell an amount of an amino acid substituted ShK polypeptide composition comprising the amino acid sequence of any of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3, wherein the amino acid substitution comprises substituting Met21 and Lys22 with modified amino acids, the composition effective to inhibit Kvl.3 channel activity in said cell.

2. The method of claim 1, wherein said animal is a human.

3. The method of claim 2, wherein said human is diagnosed with autoimmune disease, psoriasis, lupus, Sjogren's syndrome, rheumatoid arthritis, ulcerative colitis, Crohn's disease, sympathetic ophthalmia, or has received or will receive a transplanted organ or tissue.

4. The method of claim 1, wherein said polypeptide comprises the amino acid sequence of

SEQ ID NO:l, or a polypeptide comprising the amino acid sequence of SEQ ID NO:l, wherein the amino acid substitution comprises substituting Met21 with Nle and Lys22 with trimethylated-Lys, triethylated-Lys, DAP, trimethylated-DAP, or triethy lated-DAP.

5. The method of claim 4, further comprising any of the polypeptide having a lactam bridge between Lysl4 and Aspl╬┤, a disulfide bond between Cysl2-Cys28, a disulfide bond between Cysl7-Cys32, and an amide group at Cys35.

6. The method according to claim 5, comprising the polypeptide having a lactam bridge between Lys 14 and Asp 18.

The method of claim 5, comprising the polypeptide having one or both a disulfide bond between Cysl2-Cys28 and a disulfide bond between Cysl7-Cys32.

8. The method of claim 5, comprising the polypeptide having an amide group at Cys35.

9. The method of claim 1, wherein said polypeptide comprises the amino acid sequence of

SEQ ID NO:2, or a polypeptide comprising the amino acid sequence of SEQ ID NO:l, wherein the amino acid substitution comprises substituting Met21 with Nle and Lys22 with trimethylated-Lys, triethylated-Lys, DAP, trimethylated-DAP, or triethylated DAP.

10. The method of claim 9, further comprising the polypeptide having any of a 3-mercaptopriopionyl group at the amino terminal end of the polypeptide, a lactam bridge between Lysl4 and Aspl8, a disulfide bond between Cysl2-Cys28, a disulfide bond between Cysl7-Cys32, and an amide group at Cys35.

11. The method according to claim 10, comprising the polypeptide having a lactam bridge between Lys 14 and Asp 18.

12. The method of claim 10, comprising the polypeptide having disulfide bonds between Cysl2-Cys28 and Cysl7-Cys32.

13. The method of claim 10, comprising the polypeptide having a disulfide bond between 3-mercaptopriopionyl group and Cys35.

14. The method of claim 10, wherein the polypeptide comprises a 3-mercaptopriopionyl group at the amino terminal end of the polypeptide, a lactam bridge between Lys 14 and Aspl8, a disulfide bond between Cysl2-Cys28, a disulfide bond between Cysl7-Cys32, a disulfide bond between 3-mercaptopriopionyl group and Cys35, and an amide group at Cys35.

15. The method of claim 1, wherein said polypeptide comprises the amino acid sequence of

SEQ ID NO:3, or a polypeptide comprising the amino acid sequence of SEQ ID NO:l, wherein the amino acid substitution comprises substituting Met21 with Nle and Lys22 with trimethylated Lys, triethylated-Lys, DAP, trimethylated-DAP, or triethy lated-DAP.

16. The method of claim 15, further comprising the polypeptide having any of a lactam bridge between Lysl4 and Aspl8, a disulfide bond between Cysl2-Cys28, a disulfide bond between Cysl7-Cys32, and an amide group at Cys35.

17. The method according to claim 16, comprising the polypeptide having a lactam bridge between Lys 14 and Asp 18.

18. The method of claim 16, comprising the polypeptide having one or both a disulfide bond between Cysl2-Cys28 and a disulfide bond between Cysl7-Cys32.

19. The method of claim 16, comprising the polypeptide having an amide group at Cys35.

20. The method of claim 16, comprising the polypeptide having a lactam bridge between Lys 14 and Asp 18, a disulfide bond between Cysl2-Cys28, a disulfide bond between Cysl7-Cys32, and an amide group at Cys35.

21. The method of claim 1, wherein said polypeptide is administered to said animal with at least a first immunosuppressive agent.

22. The method of claim 21, wherein said immunosuppressive agent is selected from the group consisting of cyclosporin, rapamycin, azathioprine, prednisone, deoxyspergualin, and a salt or analog thereof.

23. A composition comprising an amino acid substituted-peptide having the amino acid sequence of any of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3, wherein the amino acid substitution comprises substituting Met21 and Lys22 with modified amino acids, the composition effective to inhibit Kvl .3 channel activity in said cell.

24. The composition of claim 23, wherein said immunosuppressive agent is a peptidomimetic.

25. The composition of claim 23, wherein said immunosuppressive agent is selected from the group consisting of cyclosporin, rapamycin, azathioprine, prednisone, deoxyspergualin, and a salt or analog thereof.

26. A kit comprising, in suitable container means, a therapeutically-effective amount of a polypeptide composition in accordance with claim 23 and a pharmaceutically acceptable excipient.

27. The kit of claim 26, comprised within a single container means.

28. The kit of claim 26, wherein said polypeptide and said excipient are comprised within distinct container means.

29. The kit of claim 26, wherein said polypeptide is suitable for parenteral, intramuscular, or intravenous administration.

30. The kit of claim 26, wherein said polypeptide is suitable for oral or topical administration.

31. The kit of claim 26, further comprising at least a first immunosuppressive agent.

32. The kit of claim 31, wherein said immunosuppressive agent comprises a polypeptide or a peptidomimetic.

33. A method of suppressing an immune response in an animal, comprising administering to said animal an effective amount of a polypeptide composition in accordance with claim 23.

34. A method of treating an autoimmune disease in an animal, said method comprising the steps of:

(a) identifying an animal suspected of having an autoimmune disease; and

(b) administering to said animal an amount of an ShK polypeptide composition comprising the amino acid sequence of any of amino acid substituted SEQ ID NO:l to SEQ ID NO:3 sufficient to treat said autoimmune disease in said animal.

35. The method of claim 34, further comprising administering to said animal at least a first immunosuppressive agent.

36. The method of claim 34, wherein said autoimmune disease is selected from the group consisting of psoriasis, lupus, Sjogren's syndrome, rheumatoid arthritis, ulcerative colitis, Crohn's disease, and sympathetic ophthalmia.

37. The method of claim 34, wherein said polypeptide inhibits potassium channel activity in a lymphocyte T-cell of said animal.

38. The polypeptide composition of claim 34, further comprising a pharmaceutically acceptable excipient.

39. A method of selectively decreasing potassium channel activity in a lymphocyte T-cells cell, comprising contacting said cell with an amount of a polypeptide composition in accordance with claim 23 effective to selectively decrease said channel activity.

40. A method of suppressing T-cell activation in the immune system of an animal, comprising contacting a population of T-cells with an amount of a polypeptide composition in accordance with claim 23, effective to suppress T-cell activation in said animal.

41. The method of claim 40, wherein said T-cell is a mammalian T-cell.

42. The method of claim 41 , wherein said mammalian T-cell is a human T-cell.

43. The method of claim 40, wherein said T-cell activation is caused by an immune response in said animal.

44. The method of claim 43, wherein said immune response is the result of heterologous organ rejection or an autoimmune disease.

45. The method of claim 40, wherein said T-cell activation is the result of psoriasis.

46. The method of claim 40, wherein said T-cells are contacted with said polypeptide by injection or ingestion of said polypeptide into said animal.

47. The method of claim 44, wherein said organ is a heart, a heart-lung, a liver, a kidney or a pancreas.

48. A composition which selectively inhibits T-cell lymphocyte channel activity, said composition characterized as a modified ShK polypeptide that interacts with amino acid residue His404, Asp402 or Tyr400 in said T-cell lymphocyte channel.

49. The composition of claim 48, wherein said polypeptide comprises an Argi l to Alal l amino acid substitution.

50. The composition of claim 49, wherein Lys22 in SEQ ID NO:l is substituted by a distinct amino acid.

51. The composition of claim 50, wherein said distinct amino acid is diaminopropionic acid.

52. The composition of claim 50, wherein said distinct amino acid is trimethylated- diaminopropionic acid.

53. The composition of claim 50, wherein said distinct amino acid is triethylated- diaminopropionic acid.

54. The composition of claim 49, wherein Met21 in SEQ ID NO:l is substituted by a distinct amino acid.

55. The composition of claim 54, wherein said distinct amino acid is Nle or Ala.