WO2013113060A1

WO2013113060A1 - Peptide

Info

Publication number: WO2013113060A1
Application number: PCT/AU2013/000075
Authority: WO
Inventors: Livia HOOL
Original assignee: The University Of Western Australia
Priority date: 2012-01-31
Filing date: 2013-01-30
Publication date: 2013-08-08

Abstract

Peptides are described which interact with the beta subunit of the L-type Ca²⁺ channel in cardiac cells of a subject and can be used to modulate the uptake of calcium in the cardiac cells making these peptides of use in attenuating reperfusion injury following a period of ischemia.

Description

Peptide

TECHNICAL FIELD

The present invention relates to peptides which bind the L-type Ca²⁺ channel and use thereof to reduce cardiac damage following reperfusion.

BACKGROUND ART

The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

Pathological cardiac hypertrophy may arise in a human or another animal as a response to stress; disease such as hypertension; heart muscle injury including myocardial infarction; neurohormones; or pollution causing hypoxia due to atmospheric carbon monoxide.

Patients that present with cardiac hypertrophy as a result of complete or partial occlusion of a coronary artery are commonly treated with reperfusion therapy, for example using thrombolytic therapy, percutaneous coronary intervention (PCI), or bypass surgery. However, following reperfusion therapy, when blood supply returns to cardiac tissue after a period of ischemia, reperfusion injury can result. The absence of nutrients and oxygen from blood during the period of ischemic causes a condition in which restoration of circulation results in inflammation and oxidative damage. This results through the induction of oxidative stress rather than restoration of normal function.

The long- or L-type Ca ⁺ channel is the main route for calcium influx into cardiac myocytes producing the invaluable muscle contraction for the pumping heart. Increases in intracellular calcium and oxidative stress are involved in the pathophysiology of cardiac hypertrophy with increased influx through the L-type Ca²⁺ channel or over-expression of the alpha subunit of the channel inducing the hypertrophy.

It is understood that the primary structure of the pore-forming L-type Ca²⁺ channel alpha- 1 (α-ι) subunit is composed of 4 homologous repeating motifs (I— IV), each of which consists of 6 putative transmembrane segments (S1-S6) (Figure 1). Cytoplasmic loops between the transmembrane segments are named according to the motifs they link. There are also ct_2, δ and Y subunits which contain transmembrane domains, and the L-type Ca²⁺ channel beta-2 (β₂) subunit which is entirely intracellular. The structure of the β₂ subunit comprises four beta subunit isoforms (βι-β₄). All isoforms are hydrophilic, nonglycosylated, and intracellular with no membrane-spanning region. The β₂ isoform is tightly bound to a highly conserved motif in the cytoplasmic linker between repeats I and II of all cloned high voltage-activated a_\ subunit isoforms, called the alpha-interaction domain (AID).

The alpha subunit of the channel has been the target of a number of therapies which aim to protect the cardiac muscle during reperfusion and calcium overload. Examples .of these therapies include monoclonal antibodies to the alpha subunit; Ca²⁺ channel antagonists such as the Dihydropyridines, the Benzodiazepines and the Phenylalkylamines. Although Ca²⁺ channel blockers bind specifically to regions of the cite subunit of the L-type Ca²⁺ channel, these drugs have been found to have limited success with ischemia-associated heart failure. Thus, there is a need for new therapies which address reperfusion injury arising during treatment of cardiac hypertrophy.

It is against this background that the present invention has been developed. SUMMARY OF INVENTION

In a first aspect, the invention provides a peptide comprising the amino acid sequence:

Gin Gin Xi Glu X₂ X₃ Leu X4 Gly Tyr X₅ X₆ Trp lie X₇ X₈ X₉ Glu wherein X₁.9 are naturally occurring amino acids (SEQ ID No: 1). Preferably: is an amino acid selected from the group comprising: Leu, lie, Met, Val, Phe, Ala, Cys, Trp, Tyr, Pro, Thr;

X₂ is an amino acid selected from the group comprising: Glu, Asp, Gin, Lys, Gly, His, Arg, Asn, Ser, Ala, Pro, Thr, Met, Val;

X₃ is an amino acid selected from the group comprising: Asp, Glu, Asn, His, Lys, Gly, Gin, Arg, Pro, Ser, Thr, Ala;

X4 is an amino acid selected from the group comprising: Lys, Arg, Glu, Gin, Asn, His, Asp, Trp, Tyr, Ser, Gly, Met, Pro, Ala, Thr, lie, Val;

X₅ is an amino acid selected from the group comprising: Leu, lie, Met, Val, Phe, Ala, Cys, Trp, Tyr, Pro, Thr; X₆ is an amino acid selected from the group comprising: Asp, Glu, Asn, His, Lys, Gly, Gin, Arg, Pro, Ser, Thr, Ala;

X₇ is an amino acid selected from the group comprising: Thr, Ser, Asn, Ala, Val, Cys, Asp, Glu, Lys, Met, His, lie, Pro, Gin, Arg, Val, Leu, Gly;

X₈ is an amino acid selected from the group comprising: Gin, Glu, Arg, Lys, His, Asn, Asp, Met, Ser, Pro, Tyr, Trp, Ala, Gly, Thr, Val; and

X₉ is an amino acid selected from the group comprising: Ala, Ser, Gly, Thr, Val, Cys, Glu, He, Lys, Met, Leu, Pro, Gin, Arg, His, Asn. More preferably:

Xt is an amino acid selected from the group comprising: Leu, He, Met, Val, Phe;

X₂ is an amino acid selected from the group comprising: Glu, Asp, Gin, Lys, Gly, His, Arg, Asn;

X₃ is an amino acid selected from the group comprising: Asp, Glu, Asn, His, Lys, Gly, Gin, Arg;

X₄ is an amino acid selected from the group comprising: Lys, Arg, Glu, Gin, Asn, His, Asp, Trp, Tyr;

X₅ is an amino acid selected from the group comprising: Leu, He, Met, Val, Phe;

X₆ is an amino acid selected from the group comprising: Asp, Glu, Asn, His, Lys, Gly, Gin, Arg;

X₇ is an amino acid selected from the group comprising: Thr, Ser, Asn, Ala;

X₈ is an amino acid selected from the group comprising: Gin, Glu, Arg, Lys, His, Asn, Asp; and

X₉ is an amino acid selected from the group comprising: Ala, Ser, Gly, Thr.

In a second aspect, the invention provides a peptide comprising the amino acid sequence:

Gin Gin Leu Glu Glu Asp Leu Lys Gly Tyr Leu Asp Trp He Thr Gin Ala Glu (SEQ ID No: 2).

Thus, whilst the prior art has been focused on the alpha subunit as the target of a number of therapies including antibodies, antagonists, blockers, and reducing expression of the alpha subunit to reduce reperfusion injury, the inventor made the surprising observation that restricting the movement of the β₂ subunit of the L-type Ca channel with a peptide derived against AID prevented interaction of β₂ and ai subunits and attenuated myocardial damage associated with reperfusion after ischemia. Although not wishing to be bound by any particular mechanism, it is believed that the β-interaction domain (BID) interacts with the AID through a conserved hydrophobic cleft, termed the alpha-binding pocket (ABP). The l-ll loop of the ai subunit contains an endoplasmic reticulum retention signal that restricts cell surface expression. The β₂ subunit reverses the inhibition imposed by the retention signal and is able to modulate the biophysical properties of the L-type Ca²⁺ channels CH subunit, producing a leftward shift of the current-voltage relationship, which is consistent with the involvement of the S4 region of the ai subunit voltage-sensor region. Therefore, a peptide of the invention can interact with the BID of a L-type Ca²⁺ channel β₂ subunit in a heart muscle cell, thereby preventing interaction of that β₂ subunit with the AID of an L-type Ca²⁺ channel subunit. The peptide-bound β₂ subunit is unable to reverse the inhibition on the di subunit imposed by the endoplasmic reticulum retention signal which restricts cell surface expression of the ai subunit. The result being a restriction on the activation of the L-type Ca²⁺ channel by a peptide of the invention which, when used in combination with reperfusion therapy, can protect cardiac muscle of a subject after a period of ischemia from calcium overload and reperfusion injury.

In a third aspect, the invention provides a peptide comprising: a peptide portion comprising the amino acid sequence of SEQ ID NO: 1 wherein X_1-9 is a naturally occurring amino acid, or SEQ ID NO: 2; and a peptide portion comprising the amino acid sequence of:

Arg Lys Lys Arg Arg Gin Arg Arg Arg Zaa wherein Zaa is a 6-amino hexanoic acid (SEQ ID NO: 3).

In a fourth aspect, the invention provides a method for modulating movement of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3. In a fifth aspect, the invention provides a method for modulating binding of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3. Preferably, the peptide prevents interaction between the beta subunit of the L-type Ca2+ channel and an alpha subunit of the L-type Ca2+ channel in a cardiac cell of a subject and therefore activation of the channel.

In a sixth aspect, the invention provides a method for modulating a L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3.

In a seventh aspect, the invention provides a method for reducing myocardial damage and/or oxidative stress in the heart of a subject during reperfusion, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ' ID NO: 3. Preferably the myocardial damage comprises cardiac hypertrophy. The peptide of the invention may be administered to the subject before, during or after reperfusion therapy. The peptide of the invention is preferably administered to the subject during reperfusion. Preferably, cardiac hypertrophy and/or oxidative stress is reduced. More preferably, intracellular Ca²⁺ levels in a cardiac cell in the heart of the subject are reduced or substantially maintained in the heart of the subject. The subject is preferably a mammal, and is more preferably a human.

In an eighth aspect, the invention provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for modulating movement of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell in the heart of a subject.

In a ninth aspect, the invention provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for modulating a L-type Ca²⁺ channel in the heart of a subject.

In a tenth aspect, the invention provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for reducing myocardial damage and/or oxidative stress in the heart of a subject during reperfusion.

In an eleventh aspect, the invention provides a polynucleotide encoding a peptide of the invention described herein.

BRIEF DESCRIPTION OF THE FIGURES

Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which: Figure 1 : Illustration of the primary structure of the pore-forming L-type Ca²⁺ channel alpha-1 (a subunit which is composed of 4 homologous repeating motifs (I— IV), each of which consists of 6 putative transmembrane segments (S1-S6).

Figure 2: 1 μΜ AID-TAT peptide of the invention does not alter calcium influx through the L-type calcium channel assessed as changes in Fura 2 fluorescence in guinea pig myocytes after activation of the channel with BayK(-). BayK(+) = inactive analogue of Bay (-).

Figure 3: AID-TAT peptide of the invention decreases infarct size (damage) assessed as area that did not take up nitroblue tetrazolium dye. Image at left: heart perfused with AID-TAT peptide. Image at right: heart perfused with AID-TAT (S). N = no. of hearts.

Figure 4: Application of AID-TAT peptide of the invention during reperfusion decreases myocardial damage assessed as release of creatine kinase. AID-TAT (S) = scrambled control peptide. Pre = prior to ischemia; Post = post ischemia during reperfusion; n = no. of hearts.

Figure 5: Application of AID-TAT peptide of the invention during reperfusion decreases myocardial damage assessed as release of lactate dehydrogenase. AID- TAT (S) = scrambled control peptide. Pre = prior to ischemia; Post = post ischemia during reperfusion; n = no. of hearts.

Figure 6: Application of AID-TAT peptide of the invention during reperfusion decreases myocardial oxidative stress assessed as ratio of reduced glutathione/oxidized glutathione. N = no. of hearts.

Figure 7: Contractility data from rats that have undergone myocardial infarction plus saline injection (saline), myocardial infarction plus AID peptide or myocardial infarction plus scrambled peptide. The contractility studies are performed 6 weeks after myocardial infarction. Peptide A refers to the active AID peptide and Peptide B refers to the scrambled peptide. The number above each bar represents number of rats for each group.

Figure 8: AID-TAT peptide decreases reperfusion injury in vivo 6 weeks after myocardial infarction assessed as area that does not take up nitroblue tetrazolium dye. Figure 9: AID-TAT peptide is taken up rapidly into cardiac myocytes. Fluorescent uptake of Rhodamine B labelled -AID-TAT association with cardiac myocytes over changes in time. Minimum n = 12 per time point, per sample.

DESCRIPTION OF EMBODIMENTS

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

The invention described herein may include one or more range of values (for example, size, displacement and field strength etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

Features of the invention will now be discussed with reference to the following non-limiting description and examples.

AID Peptide

The present invention provides a peptide comprising the amino acid sequence:

Gin Gin X, Glu X₂ X₃ Leu X₄ Gly Tyr X₅ X₆ Trp lie X₇ X₈ X₉ Glu wherein Xi._g are naturally occurring amino acids (SEQ ID No: 1 ).

The present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 , wherein 'X' is a naturally occurring amino acid. In this respect, nine of the eighteen amino acids within SEQ ID NO: 1 are designated 'Χ .₉' where each of Xi_-9 can be any one of the twenty naturally occurring amino acids which are also commonly known as proteinogenic or standard amino acids, and each of X_1-9 in SEQ ID NO: 1 may code for the same or a different amino acid to another X. It will be recognized that an amino acid in an X position of a peptide according to SEQ ID NO: 1 can be varied without significantly affecting the structure or function of the peptide of the invention. Thus, different variations of amino acids in the X positions in SEQ ID NO: 1 results in a large number of variant peptides of the invention. Preferably:

XT is an amino acid selected from the group comprising: Leu, lie, Met, Val, Phe, Ala, Cys, Trp, Tyr, Pro, Thr;

X₄ is an amino acid selected from the group comprising: Lys, Arg, Glu, Gin, Asn, His, Asp, Trp, Tyr, Ser, Gly, Met, Pro, Ala, Thr, lie, Val;

X₅ is an amino acid selected from the group comprising: Leu, He, Met, Val, Phe, Ala, Cys, Trp, Tyr, Pro, Thr; X₆ is an amino acid selected from the group comprising: Asp, Glu, Asn, His, Lys, Gly, Gin, Arg, Pro, Ser, Thr, Ala;

X₇ is an amino acid selected from the group comprising: Thr_; Ser, Asn, Ala, Val, Cys, Asp, Glu, Lys, Met, His, He, Pro, Gin, Arg, Val, Leu, Gly;

X₉ is an amino acid selected from the group comprising: Ala, Ser, Gly, Thr, Val, Cys, Glu, lie, Lys, Met, Leu, Pro, Gin, Arg, His, Asn. More preferably:

Xi is an amino acid selected from the group comprising: Leu, lie, Met, Val, Phe;

X₄ is an amino acid selected from the group comprising: Lys^* Arg, Glu, Gin, Asn, His, Asp, Trp, Tyr;

X₅ is an amino acid selected from the group comprising: Leu, lie, Met, Val, Phe;

X₇ is an amino acid selected from the group comprising: Thr, Ser, Asn, Ala;

X₉ is an amino acid selected from the group comprising: Ala, Ser, Gly, Thr.

Preferably, the peptide is provided in a pharmaceutically acceptable form.

The present invention further and more preferably provides a peptide comprising the amino acid sequence of SEQ ID NO: 2.

Gin Gin Leu Glu Glu Asp Leu Lys Gly Tyr Leu Asp Trp He Thr Gin Ala Glu

(SEQ ID No: 2) The present invention further provides an isolated peptide comprising the highly conserved AID motif of the human L-type Ca²⁺ channel αι subunit.

A peptide of the present invention may be recombinant, natural or synthetic. A peptide of the invention may be mixed with diluents, adjuvants or carriers (including nanoparticles) that will not interfere with the intended purpose of the peptide. A peptide of the invention may also be in a substantially purified form, in which case it will generally comprise the peptide in a preparation in which at least 90%, 95%, 98% or 99% of the protein in the preparation is a peptide of the invention. The term 'peptide' as used herein may be used interchangeably with the term 'polypeptide' as referring to a chain of at least two amino acid monomers.

Peptide Variants

Functional variants include peptides of the invention which comprise an AID portion that can bind to the BID of a L-type Ca²⁺ channel β₂ subunit. Such variants include the recited sequences with deletions, insertions, inversions, repeats, and type substitutions. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).

Thus, a variant peptide of the present invention as described herein may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the peptide is fused with another compound, such as a compound to increase the half-life of the peptide (for example, polyethylene glycol or polypropylene glycol), or (iv) one in which the additional amino acids, such as a leader, signal or secretory sequence or a sequence which is employed for purification of the peptide sequence are fused to the mature peptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

As indicated above, variants of the peptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. The particular replacements may be determined by a skilled person as detailed more fully hereunder. However, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the peptide (see for example the table hereunder). Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Amino acids in the peptides of the present invention that are essential for function can be identified by methods known in the art, such as site directed mutagenesis or alanine- scanning mutagenesis. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as interaction with the BID of a L-type Ca²⁺ channel β₂ subunit, and ability to enter cells such as cardiac cells. Nuclear magnetic resonance or photoaffinity labelling may also be used when developing functional variants. Alternatively, synthetic peptides corresponding to candidate functional variants may be produced and their ability to display one or more activities of the peptides assessed in vitro or in vivo.

Peptide variants of the present invention can also be prepared as libraries comprising sequences according to SEQ ID No: 1. Phage display can also be effective in identifying variants useful according to the invention. Briefly, a phage library is prepared (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a biased degenerate array or may completely restrict the amino acids at one or more positions (e.g., for a library based on a peptide of SEQ ID No: 1). One then can select phage-bearing inserts that have a relevant biological activity of the peptide, of the invention such as interacting with the BID of a L-type Ca channel β₂ subunit in a heart muscle cell. This process can be repeated through several cycles of reselection of phage. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that confers the relevant activity can be determined. One can repeat the procedure using a biased library containing inserts containing part or the entire minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof.

Peptides of .the invention, including variant peptides, can be tested for retention of any of the given activity. For example, a peptide can be tested for in vitro properties using transient transfection assays with a responsive reporter that assesses the ability of the peptide to bind the BID of a L-type Ca²⁺ channel β₂ subunit in a cardiac cell.

Preferred variant peptides of the present invention comprise an amino acid sequence that is at least 70-80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to SEQ ID No: 1 or SEQ ID NO: 2, provided the variant peptides can bind the BID of a L-type Ca²⁺ channel β₂ subunit in a cardiac cell.

By a peptide having an amino acid sequence at least, for example, 90% "identical" to a reference amino acid sequence of a peptide of the invention it is intended that the amino acid sequence of the peptide is identical to the reference sequence except that the polypeptide sequence may include up to one amino acid alteration per each 10 amino acids of the reference peptide. In other words, to obtain a peptide having an amino acid sequence at least 90% identical to a reference amino acid sequence, up to 10% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 10% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

In general, a peptide of the present invention can be synthesized directly or obtained by chemical or mechanical disruption of larger molecules, fractioned and then tested for one or more activity of the peptide. Functional variants may also be produced by Northern blot analysis of total cellular RNA followed by cloning and sequencing of identified bands derived from different tissues/cells, or by PCR analysts of such RNA also followed by cloning and sequencing. Thus, synthesis or purification of a large number of functional variants is possible using the information contained herein.

It may be desirable to use derivatives of peptides of the invention that are conformationally constrained. Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide. Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.

The active conformation of the peptide may be stabilized by a covalent modification, such as cyclization or by incorporation of gamma-lactam or other types of bridges. For example, side chains can be cyclized to the backbone to create a L-gamma-lactam moiety on each side of the interaction site. Cyclization also can be achieved, for example, by formation of cysteine bridges, coupling of amino and carboxy terminal groups of respective terminal amino acids, or coupling of the amino group of a Lys residue or a related homolog with a carboxy group of Asp, Glu or a related homolog. Coupling of the alpha-amino group of a peptide of the invention with the epsilon-amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken.

Another approach is to include a metal-ion complexing backbone in the structure of a peptide of the invention. Typically, the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the coordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a coordination number of four to six. The nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulphur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities. In addition, the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino. The peptide construct can be either linear or cyclic, however a linear construct is typically preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly that has four nitrogens (an N₄ complexation system) in the backbone that can complex to a metal ion with a coordination number of four.

SUBSTITUTE SHEET^" (RULE 26) Other methods for identifying variants of peptides of the invention herein rely upon the development of amino acid sequence motifs to which potential epitopes may be compared. Each motif describes a finite set of amino acid sequences in which the residues at each (relative) position may be (a) restricted to a single residue, (b) allowed to vary amongst a restricted set of residues, or (c) allowed to vary amongst all possible residues. For example, a motif might specify that the residue at a first position may be any one of valine, leucine, isoleucine, methionine, or phenylalanine; that the residue at the second position must be histidine; that the residue at the third position may be any amino acid residue; that the residue at the fourth position may be any one of the residues valine, leucine, isoleucine, methionine, phenylalanine, tyrosine or tryptophan; that the residue at the fifth position must be lysine, and so on.

Thus, the present invention also provides methods for identifying functional variants of peptides of the invention. In general, the methods include selecting a peptide of the invention. Then a first amino acid residue of the peptide is mutated to prepare a variant peptide. In one embodiment, the amino acid residue can be selected and mutated as indicated by a computer model of peptide conformation. Peptides bearing mutated residues that maintain a similar conformation (e.g. secondary structure) can be considered potential functional variants that can be tested for function using the assays described herein. Any method for preparing variant peptides can be employed, such as synthesis of the variant peptide, recombinantly producing the variant peptide using a mutated nucleic acid molecule, and the like. The properties of the variant peptide in relation to the peptides described previously are then determined according to standard procedures as described herein.

Variants of peptides of the invention prepared by any of the foregoing methods can be sequenced, if necessary, to determine the amino acid sequence and thus deduce the nucleotide sequence which encodes such variants.

The present invention also includes non-peptide mimetics of peptides of the invention. A wide variety of techniques may be used to elucidate the precise structure of a peptide. These techniques include amino acid sequencing, x-ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, computer-assisted molecular modelling, peptide mapping, and combinations thereof. Structural analysis of a peptide provides a large body of data that comprise the amino acid sequence of the peptide as well as the three-dimensional positioning of its atomic components. From this information, non- peptide peptidomimetics may be designed that have the required chemical functionalities for therapeutic activity but are more stable, for example less susceptible to biological degradation.

Thus, variant peptides of the present invention also include mimetics. Nonpeptide analogs of peptides of the invention, such as those that provide a stabilized structure or lessened biodegradation, are within the scope of the present invention. Peptide mimetic analogs can be prepared based on a selected peptide of the invention by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, conformation such as a conformation able to bind the BID of a L-type Ca²⁺ channel β₂ subunit. Thus, the present invention also provides for the use of a peptide of the invention described herein for designing a mimetic thereof such as a non-peptide peptidomimetic.

Preferably, the peptides of the invention are non-hydrolyzable in that the bonds linking the amino acids of the peptides are less readily hydrolyzed than peptide bonds formed between L-amino acids. To provide such peptides, one may select isolated peptides from a library of non-hydrolyzable peptides, such as peptides containing one or more D-amino acids or peptides containing one or more non-hydrolyzable peptide bonds linking amino acids.

Alternatively, one can select peptides that are optimal for a preferred function in suitable assay systems and then modify such peptides as necessary to reduce the potential for hydrolysis by proteases. For example, to determine the susceptibility to proteolytic cleavage, peptides may be labelled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing peptides and proteolytic fragments. Alternatively, potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of an isolated peptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds that are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide.

Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Non-hydrolyzable bonds include - psi[CH.sub.2 NH]- reduced amide peptide bonds, -psi[COCH.sub.2 ]- ketomethylene peptide bonds, -psi[CH(CN)NH]~ (cyanomethylene)amino peptide bonds, -psi[CH.sub.2 CH(OH)]- hydroxyethylene peptide bonds, -psi[CH.sub.2 OJ- peptide bonds, and - psi[CH.sub.2 S]~ thiomethylene peptide bonds. Likewise, various changes may be made including the addition of various side groups that do not affect the manner in which the peptides function, or which favourably affect the manner in which the peptides function, Such changes may involve adding or subtracting charge groups, substituting amino acids, adding lipophilic moieties that do not affect binding but that affect the overall charge characteristics of the molecule facilitating a specific outcome with a physiological benefit. For each such change, no more than routine experimentation is required to test whether the molecule functions according to the invention. One simply makes the desired change or selects the desired peptide and applies it in a fashion as described in detail herein.

The peptides of the invention may also be linked to a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG). Replacement of naturally occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify peptides. Another approach is to use bifunctional crosslinkers, such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-(2 pyridyldithio) propionamido] hexanoate, and sulfosuccinimidyl 6-[3-(2 pyridyldithio) propionamido]hexanoate.

The present peptide or analogues, such as those recited infra may be derivatized by the attachment of one or more chemical moieties to the peptide sequence. Chemical modification of biologically active peptides provides advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic peptides, decreasing immunogenicity and to enhance bioavailability and/or to enhance efficacy and/or specificity. See, U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review, see Abuchowski et al., in Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp. 367 383 (1981)).

Fusion Peptides

Peptide variants of the present invention also include fusion to further peptides, for example, where an additional peptide sequence is fused to a peptide of the invention to aid in extraction and purification. Examples of additional fusion peptide partners include glutathione-S-transferase (GST), hexahistidine, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the additional peptide partner and the peptide of the invention to allow removal of additional peptide sequences. Preferably the additional peptide will not hinder binding of the peptide of the invention to the, BID of a L-type Ca²⁺ channel β₂ subunit. A peptide of the present invention may also include conjugated peptides. In this regard, a peptide may be modified by attachment of a moiety (e.g. a fluorescent, radioactive, or enzymatic label, or an unrelated sequence of amino acids) that does not correspond to a portion of the peptide in its native state. Thus, a peptide of the present invention may comprise chimeric peptides comprising an additional fusion of a peptide of the invention with another peptide. For example, a peptide capable of targeting the peptide of the invention to a cell type or tissue type, enhancing stability of the peptide of the invention under assay conditions, or providing a detectable moiety, such as green fluorescent protein. A moiety fused to a peptide of the invention or a fragment thereof also may provide means of readily detecting the peptide of the invention, for example, by immunological recognition or by fluorescent labelling such as green fluorescent protein. Purified peptides of the invention include peptides isolated by methods including, but are not limited to, immunochromotography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.

A peptide of the invention can be conjugated by well-known methods, including bifunctional linkers, formation of a fusion peptide, and formation of biotin/streptavidin or biotin/avidin complexes by attaching either biotin or streptavidin/avidin to the peptide and the complementary molecule. Depending upon the nature of the reactive groups in a peptide of the invention and a targeting agent, a conjugate can be formed by simultaneously or sequentially allowing the functional groups of the above-described components to react with one another. Numerous art-recognized methods for forming a covalent linkage can be used. See, e.g., March, J., Advanced Organic Chemistry, 4th Ed., New York, N.Y., Wiley and Sons, 1985), pp.326-1120.

In general, conjugated fusion peptides of the invention can be prepared by using well- known methods for forming amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective conjugated peptide components. As would be apparent to one of ordinary skill in the art, reactive functional groups that are present in the amino acid side chains of the fusion peptide preferably are protected, to minimize unwanted side reactions prior to coupling the fusion peptide to the derivatizing agent and/or to the extracellular agent. As used herein, "protecting group" refers to a molecule which is bound to a functional group and which may be selectively removed therefrom to expose the functional group in a reactive form. Preferably, the protecting groups are reversibly attached to the functional groups and can be removed therefrom using, for example, chemical or other cleavage methods. Thus, for example, fusion peptides of the invention can be synthesized using commercially available side-chain-blocked amino acids (e.g., FMOC- derivatized amino acids from Advanced Chemtech Inc., Louisville, Ky.). Alternatively, the peptide side chains can be reacted with protecting groups after peptide synthesis, but prior to the covalent coupling reaction. In this manner, conjugated fusion peptides of the invention can be prepared in which the amino acid side chains do not participate to any significant extent in the coupling reaction of the peptide to the other agent, such as a cell- type-specific targeting agent.

If a peptide of the invention does not have a free amino- or carboxyl-terminal functional group that can participate in a coupling reaction, such a group can be introduced, e.g., by introducing a cysteine (containing a reactive thiol group) into the peptide by synthesis or site directed mutagenesis. Disulfide linkages can be formed between thiol groups in, for example, the peptide and the targeting compound. Alternatively, covalent linkages can be formed using bifunctional cross linking agents, such as bismaleimidohexane (which contains thiol-reactive maleimide groups and which forms covalent bonds with free thiols). See also the Pierce Co. Immunotechnology Catalogue and Handbook Vol. 1 for a list of exemplary homo- and hetero-bifunctional cross linking agents, thiol-containing amines and other molecules with reactive groups.

For peptides of the invention that exhibit reduced activity in a conjugated form, the covalent bond between the peptide of the invention and its conjugate is selected to be sufficiently labile (e.g., to enzymatic cleavage) so that it is cleaved following transport to its target, thereby releasing the free peptide at the target. Biologically labile covalent linkages, e.g., imino bonds, and "active" esters can be used to form prodrugs where the covalently coupled peptides are found to exhibit reduced activity in comparison to the activity of the peptide of the invention alone.

It will be appreciated that the amino acids in a peptide of the invention that are required for activity may be incorporated into larger fusion proteins and still maintain their function.

AID-TAT Fusion Peptide

In a preferred form, the invention further provides a peptide comprising: a peptide portion comprising the amino acid sequence of SEQ ID NO: 1 wherein Xi_-9 is a naturally occurring amino acid, or SEQ ID NO: 2; and a peptide portion comprising the amino acid sequence:

Arg Lys Lys Arg Arg Gin ^rg Arg Arg Zaa wherein Zaa is a 6-amino hexanoic acid (SEQ ID NO: 3).

The peptide portion of the peptide of the invention comprising the amino acid sequence of SEQ ID NO: 3 encodes a TAT peptide. Trans-activating transcriptional activator (TAT) from Human Immunodeficiency Virus 1 is a cell-penetrating peptide which is known in the art to deliver attached molecules such as peptides into cells. Thus, not wishing to be bound by any particular mechanism, it is believed the TAT peptide portion in the peptide of the invention facilitates transport of the peptide into cardiac cells via endocytosis or by direct translocation across the plasma membrane. The nuclear localisation signal found within the domain, GRKKR, mediates further translocation of Tat into the cell nucleus. The biological role of this domain and exact mechanism of transfer is currently unknown. The amino acid sequence of the protein transduction domain is YGRKKRRQRRR.

However, the peptide of the invention may comprise other or additional peptide portions which assist or facilitate in the transport of the peptide into cardiac or other cells, or provide some other benefit, for example, amongst others, identifying the location of a peptide of the invention within a cell.

Selective Binding Agents

As used herein, the term "selective binding agent" refers to a molecule that has specificity for peptides of the invention described herein. Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides and small molecules. Suitable selective binding agents may be prepared using methods known in the art. An exemplary selective binding agent is capable of binding a portion of a peptide of the invention. Such binding agents can be utilised to determine the presence of peptides of the invention in tissue or individual cells and determine binding activity and/or localisation with other molecules.

Selective binding agents such as antibodies and antibody fragments that bind peptides of the invention include monospecific polyclonal, monoclonal (MAbs), recombinant, chimeric, humanized such as CDR-grafted, human, single chain, and/or bispecific, as well as fragments, variants or derivatives thereof. Antibody fragments include those portions of the antibody that bind to an epitope on the peptide. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions. Polyclonal antibodies generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of the peptide and an adjuvant. It may be useful to conjugate the peptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for antibody titre.

Monoclonal antibodies are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybrid ma methods of Kohler et al. , Nature, 256:495-497 (1975) and the human B-cell hybridoma method, Kozbor, J. Immunol., 133:3001 (1984);(1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). Also provided by the invention are hybridoma cell lines that produce monoclonal antibodies reactive with peptides herein.

In another embodiment, a monoclonal antibody that binds a peptide of the invention is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using methods described in the art (Jones et al. , Nature 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting at least a portion of a rodent complementarity-determining region (CDR) for the corresponding regions of a human antibody.

Using transgenic animals (e.g. , mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunization with a peptide antigen (i.e., having at least 6 contiguous amino acids), optionally conjugated to a carrier. In one method, such transgenic animals are produced by incapacitating the endogenous loci encoding the heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof. Partially modified animals, that is, those having less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies with human (rather than e.g., murine) amino acid sequences, including variable regions that are immunospecific for these antigens. See PCT application nos. PCT/US96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT application nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP 546073A1. Human antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

In an alternative embodiment, human antibodies can be produced from phage-display libraries (Hoogenboom ef a/., J. Mol. Biol., 227:381 (1991 );(1991) and Marks et al., J. Mol. Biol., 222:581 (1991)). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT Application No. PCT/US98/17364, which describes the isolation of high affinity and functional agonistic antibodies.

Chimeric, CDR grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

The antibodies to peptides of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)) for the detection and quantitation of peptides. The antibodies will bind peptides with an affinity that is appropriate for the assay method being employed.

For diagnostic applications, in certain embodiments, antibodies may be labelled with a detectable moiety. The detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C, ^3ZP, ³⁵S, or ¹²⁵l; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase. Competitive binding assays rely on the ability of a labelled standard (e.g., a peptide described herein or an immunologically reactive portion thereof) to compete with the test sample (a candidate polypeptide) for binding with a limited amount of antibody. The amount of the candidate polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibody. To facilitate determining the amount of standard that becomes bound, the antibodies typically are insplubilized before or after the competition, so that the standard and candidate polypeptide that are bound to the antibodies may conveniently be separated from the standard and candidate polypeptide which remain unbound.

Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the peptide to be detected and/or quantitated. In a sandwich assay, the test sample (analyte) is typically bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. The second antibody may itself be labelled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labelled with a detectable moiety (indirect sandwich assays). For example, one type of sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.

The selective binding agents, including antibodies, are also useful for in vivo imaging of administered peptides of the invention. An antibody labelled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labelled antibody in the host is assayed. The antibody may be labelled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

The invention also relates to a kit comprising selective binding agents (such as antibodies) and other reagents useful for detecting the levels and localisation of the peptides described herein in biological samples. Such reagents may include, a detectable label, blocking serum, positive and negative control samples, and detection reagents.

In particular, antibodies may be used to detect peptides of the invention present in biological samples. Suitable samples are preferably from heart tissue but may also include extracts of tissues such as brain, skin, breast, ovary, lung, colon, pancreas, testes, liver, muscle, prostate and bone tissues. Such antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

Polynucleotides

The present invention also provides an isolated polynucleotide encoding a peptide of the present invention as described herein including peptides comprising SEQ ID No: 1 , SEQ ID No: 2, and SEQ ID No: 3. It will be understood by a skilled person that due to the degeneracy of the amino acid code, numerous different polynucleotides can encode the same peptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the peptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single- stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. They may also be polynucleotides that include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.

Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.

Reference to "isolated" polynucleotide(s) means a polynucleotide, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.

Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated peptides of the present invention further include such molecules produced synthetically.

The present invention also provides isolated polynucleotides that selectively hybridize with at least a portion of a polynucleotide of the present invention. As used herein to describe nucleic acids, the term "selectively hybridize" excludes the occasional randomly hybridizing nucleic acids under at least moderate stringency conditions. Thus, selectively hybridizing polynucleotides preferably hybridize under at least moderate stringency conditions and more preferably under high stringency conditions. The hybridising polynucleotides may be used, for example, as probes or primers for detecting the presence of polynucleotides encoding peptides of the invention, for example, cDNA or mRNA.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_m of 55°C, can be used, e.g., 5x SSC, 0.1 % SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, e.g. , 40% formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest T_m, e.g., 50% formamide, 5x or 6x SCC.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived and are known to those skilled in the art. For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity. Preferably a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20, 30 or 40-70 nucleotides.

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as a 3' terminal poly(A) tail of a polynucleotide of the present invention), or to a complementary stretch of T (or U) residues, would not be included as a selectively hybridizable polynucleotide of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

Using the nucleic acid sequences taught herein and relying on cross-hybridization, one skilled in the art can identify polynucleotides in other species that encode peptides of the invention. If used as primers, the invention provides compositions including at least two nucleic acids that selectively hybridize with different regions of the target nucleic acid so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity.

The selectively hybridisable polynucleotides described herein or more particularly portions thereof can be used to detect the nucleic acid of the present invention in samples by methods such as the polymerase chain reaction, ligase chain reaction, hybridization, and the like. Alternatively, these sequences can be utilized to produce an antigenic protein or protein portion, or an active protein or protein portion.

In addition, portions of the selectively hybridisable polynucleotides described herein can be selected to selectively hybridize with homologous polynucleotides in other organisms. These selectively hybridisable polynucleotides can be used, for example, to simultaneously detect related sequences for cloning of homologues of the peptides of the present invention.

As indicated above, the polynucleotides of the present invention that encode a peptide of the present invention include, but are not limited to, those peptides encoded by the amino acid sequences of SEQ ID No: 1 and 2. Rather the polynucleotides of the present invention may comprise the coding sequence for the peptides and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the peptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Preferably, the additional sequence comprises the peptide encoded by the amino acid sequences of SEQ ID No: 3. Polynucleotides according to the present invention also include those encoding a peptide lacking the N terminal methionine.

Thus, polynucleotides of the present invention include those with a sequence encoding a peptide of the invention fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused peptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode variants of the peptides of the present invention.

Such variants include those produced by nucleotide substitutions, deletions or additions that may involve one or more nucleotides. Non-naturally occurring variants may be produced using mutagenesis techniques known to those in the art. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the encoded peptide. Also especially preferred in this regard are conservative substitutions.

It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a peptide of the invention having one or more properties of the full polypeptide such as being able to interact with the BID of a L-type Ca²⁺ channel β₂ subunit. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). Screening Methods

A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. cDNA or genomic libraries of various types may be screened as natural sources of the polynucleotides encoding the BID-interacting peptides of SEQ ID NO: 1 or 2, and TAT peptide of SEQ ID NO: 3. Such polynucleotides may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR. The choice of cDNA libraries normally corresponds to a tissue source that is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.

Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 : "Molecular Cloning: a laboratory manual. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Coldspring Harbour Laboratory Press, Coldspring Harbour, NY or Ausubel et al., 1992 Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.G. and Struhl, K. (1987). John Wiley and Sons, NY. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce peptides of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.

Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridisation conditions are well known in the art.

The probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g. Sambrook et al., 1989 : "Molecular Cloning: a laboratory manual. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Coldspring Harbour Laboratory Press, Coldspring Harbour, NY or Ausubel et al., 1992 Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.G. and Struhl, K. (1987). John Wiley and Sons, NY. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of a peptide of the invention, perhaps to change ligand- binding affinities, interchain affinities, or the polypeptide degradation or turnover rate.

Probes comprising synthetic oligonucleotides or other polynucleotides encoding peptides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labelled by nick translation, Klenow fill-in reaction, or other methods known in the art.

Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding a peptide according to the present invention or fragment thereof are preferred as probes. The probes may also be used to determine whether mRNA encoding the peptide is present in a recombinant cell.

Vectors and Host Cells

A nucleic acid molecule encoding the amino acid sequence of a peptide of the invention may be inserted into an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell employed (i.e. , the vector is compatible with the host cell machinery such that amplification of the nucleic acid molecule and/or expression of the nucleic acid molecule can occur). A nucleic acid molecule encoding the amino acid sequence of a peptide of the invention may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems), and/or eukaryotic host cells. Selection of the host cell will depend in part on whether the peptide is to be post- translationally modified (e.g. , glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable. Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in certain embodiments, will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for secretion of the peptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the peptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a "tag"-encoding sequence, i.e. , an oligonucleotide molecule located at the 5' or 3' end of the peptide coding nucleic acid sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another "tag" such as FLAG, HA (hemaglutinin influenza virus) or myc for which commercially available antibodies exist. This tag is typically fused to the peptide upon expression of the peptide, and can serve as a means for affinity purification of the peptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified peptide by various means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e. , from the same species and/or strain as the host cell), heterologous (i.e. , from a species other than the host cell species or strain), hybrid (i.e. , a combination of flanking sequences from more than one source) or synthetic, or the flanking sequences may be native sequences that normally function to regulate polypeptide expression. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein other than the gene flanking sequences will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning. Where all or only a portion of the flanking sequence is known, it may be obtained using PGR and/or by screening a genomic library with suitable oligonucleotide and/or flanking sequence fragments from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for the optimal expression of the peptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, MA) is suitable for most Gram- negative bacteria, and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).

A transcription termination sequence is typically located 3' of the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the nucleic acid molecule that will be expressed. Amplification is the process wherein genes that are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to the amplification of both the selection gene and the DNA that encodes a peptide of the invention. As a result, increased quantities of the peptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by ^' a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the peptide to be expressed. The Shine-Dalgarno sequence is varied but is typically a polypurine {i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth herein and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct the peptide out of the host cell. Typically, a nucleotide sequence encoding the signal sequence is positioned in the coding region of the nucleic acid molecule encoding the peptide, or directly at the 5' end of the polypeptide coding region. Many signal sequences have been identified, and any of those that are functional in the selected host cell may be used in conjunction with the nucleic acid molecule. Therefore, a signal sequence may be homologous (naturally occurring) or heterologous to the gene or cDNA encoding the peptide. Additionally, a signal sequence may be chemically synthesized using methods described herein. In most cases, the secretion of the peptide from the host cell via the presence of a signal peptide will result in the removal of the signal peptide from the secreted peptide. The signal sequence may be a component of the vector, or it may be a part of the nucleic acid molecule that is inserted into the vector. Included within the scope of this invention is the use of either a nucleotide sequence encoding a native signal sequence joined to a peptide coding region or a nucleotide sequence encoding a heterologous signal sequence joined to a peptide coding region. The heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell. For prokaryotic host cells that do not recognize and process the native polypeptide signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders. For yeast secretion, the native polypeptide signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders. In mammalian cell expression the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various presequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add presequences, which also may affect glycosylation. The final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incidental to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the N-terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.

The expression and cloning vectors of the present invention will each typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding a peptide of the invention. Promoters are untranscribed sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes, inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, initiate continual polynucleotide product production; that is, there is little or no control over expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the peptide of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. A native gene promoter sequence may be used to direct amplification and/or expression of a nucleic acid molecule encoding a fusion protein of the invention. A heterologous promoter is preferred, if it permits greater transcription and higher yields of the expressed peptide as compared to the native promoter, and if it is compatible with the host cell system that has been selected for use.

Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been^published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence(s), using linkers or adapters as needed to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, e.g., heat-shock promoters and the actin promoter.

Additional promoters which may be of interest in controlling transcription of the polynucleotide encoding a peptide of the invention include, but are not limited to: the SV40 early promoter region; the CMV promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus; the herpes thymidine kinase promoter, the regulatory sequences of the metallothionine gene, prokaryotic expression vectors such as the beta- lactamase promoter; or the tac promoter. Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region which is active in pancreatic acinar cells; the insulin gene control region which is active in pancreatic beta cells; the immunoglobulin gene control region which is active in lymphoid cells; the mouse mammary tumour virus control region which is active in testicular, breast, lymphoid and mast cells; the albumin gene control region which is active in liver; the alphafetoprotein gene control region which is active in liver; the alpha 1 -antitrypsin gene control region which is active in the liver; the beta-globin gene control region which is active in myeloid cells; the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain; the myosin light chain-2 gene control region which is active in skeletal muscle; and the gonadotropic releasing hormone gene control region which is active in the hypothalamus.

An enhancer sequence may be inserted into the vector to increase the transcription of a polynucleotide encoding the peptide of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (for example, globin, elastase, albumin, alpha-feto- protein and insulin). Typically, however, an enhancer from a virus will be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically located at a site 5' from the promoter.

Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the desired flanking sequences are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.

Preferred vectors for practicing this invention are those that are compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, Carlsbad, CA), pBSII (Stratagene Company, La Jolla, CA), pET15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacll; Invitrogen), pDSR-alpha (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco/BRL, Grand Island, NY).

Additional suitable vectors include, but are not limited to, cosmids, plasmids or modified viruses, but it will be appreciated that the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as

Bluescript plasmid derivatives (a high copy number ColE1 -based phagemid, Stratagene Cloning Systems Inc., La Jolla CA), PCR cloning plasmids designed for cloning Taq-Taq- amplified PCR products (e.g. , TOPO™ TA Cloning® Kit, PCR2.1® plasmid derivatives, Irwitrogen, Carlsbad, CA), and mammalian, yeast, or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).

After the vector has been constructed and a polynucleotide molecule encoding a peptide of the invention has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or fusion protein expression. The transformation of an expression vector for a peptide of the invention into a selected host cell may be accomplished by well-known methods including transfection, infection, calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al., supra.

Host cells may be prokaryotic host cells (such as £. coli) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell). The host cell, when cultured under appropriate conditions, synthesizes the peptide that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, peptide modifications that are desirable or necessary for activity, such activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

A number of suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10-2209. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61 ); CHO DHFR-cells (Urlaub et a/., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)); human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573); or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and screening, product production and purification are known in the art. Other suitable mammalian cell lines, are the monkey COS-1 (ATCC No. CRL1650) and COS-7cell lines (ATCC No. CRL1651) cell lines, and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which are available from the ATCC. Each of these cell lines is known by and available to those skilled in the art of protein expression.

Similarly useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101 , (ATCC No. 33694) DH5a, DH10, and MC1061 (ATCC No. 53338)) are well-well known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas spp. , other Bacillus spp. , Streptomyces spp. , and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are also available as host cells for the expression of peptides of the present invention. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in the methods of the present invention. Such systems are described for example in Kitts ef a/., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. (J.al., J. Virol., 67:4566-4579 (1993). Preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).

One may also use transgenic animals to express glycosylated peptides of the invention. For example, one may use a transgenic milk-producing animal (a cow or goat, for example) and obtain the present glycosylated peptide in the animal milk. One may also use plants to produce peptides of the invention. However, in general, the glycosylation occurring in plants is different from that produced in mammalian cells, and may result in a glycosylated product which is not suitable for human therapeutic use.

Therapeutic Compositions

Therapeutic compositions are within the scope of the present invention. Peptides of the invention may be combined with various components to produce compositions of the invention. Such compositions may comprise a therapeutically effective amount of a peptide or nucleotide of the invention in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Pharmaceutical compositions may also comprise a therapeutically effective amount of one or more peptide of the invention in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically 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 incorporated into the compositions. See, e.g., Remington's Pharmaceutical Sciences, 19th Ed. (1995, Mack Publishing Co., Easton, Pa.) which is herein incorporated by reference.

The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colouring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.

The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the peptide of the invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application. Pharmaceutical compositions prepared according to the invention may be administered by any means that leads to the peptides of the invention coming in contact with a causative agent of a disease or disorder as herein described including cardiac hypertrophy or oxidative stress.

The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. In one embodiment of the present invention, pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the peptide product may be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be capable of parenteral delivery. Alternatively, the compositions may be capable of inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired peptide of the invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the active agent is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid, acid or polygiycolic acid), or beads or liposomes, that provides for the controlled or sustained release of the product which may then be delivered as a depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition may be formulated for inhalation. For example, a peptide may be formulated as a dry powder for inhalation. The peptide inhalation solution may also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions may be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, peptides of the present invention that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the active agent. Diluents, flavourings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity of a peptide of the invention in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving a peptide of the invention in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 that describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained- sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, for example, films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl- L-glutamate, ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Sustained- release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

The pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is Iyophilized, sterilization using these methods may be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration may be stored in Iyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or Iyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., Iyophilized) requiring reconstitution prior to administration.

The effective amount of the active agent in the pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the active agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titre the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.1 g kg to up to about 00 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parameters of the active agent and the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose- response data.

The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intracoronary, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implants. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

In some cases, it may be desirable to use the pharmaceutical compositions herein in an ex vivo manner. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.

In another form, nanoparticles may be employed as carriers for delivery of peptides of the invention. Nanoparticles have been shown to overcome some limitations of conventional therapeutic delivery such as nonspecific biodistribution and targeting, and lack of water solubility, amongst others. Thus, nanoparticles may be used for delivering peptides of the invention to cardiac cells for treatment of a patient with the peptides.

The routes of administration described herein are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient.

Uses and Methods for Peptides of the Invention

The invention provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for modulating binding to a L-type Ca²⁺ channel alpha-interacting domain. This preferably occurs intracellular^ in a cardiac cell.

The invention provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for modulating movement of a L-type Ca²⁺ channel ₂'subunit in a cardiac cell such as a myocyte in the heart of a subject.

In this respect, the invention also provides a method for modulating movement of a L-type Ca²⁺ channel β₂ subunit in a cardiac cell such as a myocyte in the heart of a subject, comprising administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3.

Binding of the peptide to the alpha-interacting domain can prevent movement of the β₂ subunit during activation and inactivation of the L-type Ca²⁺ channel. Since the β₂ subunit is proposed to facilitate inactivation of the alpha subunit, this can result in a delay in inactivation of the current.

A subject that can be treated with a peptide of the invention will include humans as well as other mammals and animals.

Use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for modulating a L-type Ca²⁺ channel in a cardiac cell such as a myocyte in the heart of a subject.

In this respect, the invention also provides a method for modulating a L-type Ca²⁺ channel in a cardiac cell such as a myocyte in the heart of a subject, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3.

Use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; for reducing myocardial damage and/or oxidative stress in the heart of a subject during reperfusion.

In this respect, the invention also provides a method for reducing myocardial damage and/or oxidative stress in the heart of a subject during reperfusion, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3. The myocardial damage may include cardiac hypertrophy. Preferably, cardiac hypertrophy is reduced but intracellular Ca²⁺ levels are reduced or substantially maintained in the heart of the subject. Substantially maintained indicates Ca²⁺ levels which are the same or close to what is observed normally in the subject such as before cardiac hypertrophy. The binding of a peptide of the invention to a L-type Ca²⁺ channel results in a decrease in heart muscle damage during reperfusion. Since this can occur at a concentration that does not alter calcium influx (1pM), although not wishing to be bound by any particular mechanism, it is believed that the reduction in muscle damage may occur as a result of a decrease in mitochondrial oxygen consumption or metabolism. This is preferable post ischemia reperfusion because any agent that decreases calcium influx may decrease contractility.

Thus, the invention provides uses and methods for the peptides of the invention as a treatment to reduce damage and/or oxidative stress to ischemic myocardial cells following a myocardial infarction in a subject during and after reperfusion by modulating L-type Ca²⁺ channel activity.

In this respect, the invention also provides a use of a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3; in the preparation of a medicament for preventing myocardial damage to a patient during reperfusion.

It may be preferable to administer the peptides of the invention in combination with other therapeutic agents that are useful for treating myocardial infarction in a subject, reperfusion, or other agents which assist in reducing myocardial damage and/or oxidative stress before, during or following reperfusion. Such combinations could use conjugates comprising the peptides or the therapy could be concomitant or involve the sequential administration of the agents. Examples of such therapeutic agents may include, as some non-limiting examples, Antioxidants such as N-acetylcysteine, reduced glutathione, TAT-conjugated catalase or TAT-conjugated superoxide dismutase.

The peptides may be administered via a variety of methods, for example, as a therapeutic depending on the particular circumstances and as deemed appropriate by a medical practitioner.

In one non-limiting example, a peptide of the invention may be administered via the coronary arteries by a cardiologist/physician at the time of angiography or angioplasty in a hospital after admission with chest pain and diagnosis of coronary occlusion (myocardial infarction).

The effect of the administered therapeutic composition can be monitored by standard diagnostic procedures.

For example, effectiveness of the peptide may be monitored by echocardiography (ultrasound analysis of cardiac function) in one example. Size of damage could be assessed by release of muscle enzymes into the blood and by changes on electrocardiography (ECG).

Methods of Treatment

In yet another aspect, the present invention provides a method for modulating movement of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3.

The present invention further provides a method for modulating binding of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO. 3. Preferably, the peptide prevents interaction between the beta subunit of the L-type Ca2+ channel and an alpha subunit of the L-type Ca2+ channel in a cardiac cell of a subject.

The present invention further provides a method for modulating a L-type Ca²⁺ channel in a cardiac cell in the heart of a subject, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3.

The present invention further provides a method for reducing myocardial damage and/or oxidative stress in the heart of a subject during reperfusion, comprising the step of administering to the subject a peptide comprising: the amino acid sequence of SEQ ID NO: 1 ; the amino acid sequence of SEQ ID NO: 2; or a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3. The peptide of the invention may be administered to the subject before, during and/or after reperfusion. The peptide of the invention is preferably administered to the subject during reperfusion. The myocardial damage can lead to cardiac hypertrophy.

The method of the invention may reduce cardiac hypertrophy but substantially maintain intracellular Ca²⁺ levels in the heart of the subject.

A used herein the term "subject" generally includes mammals such as: humans; farm animals such as sheep, goats, pigs, cows, horses, llamas; companion animals such as dogs and cats; primates; birds, such as chickens, geese and ducks; fish; and reptiles. The subject is preferably human.

Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, and other medications administered. Treatment dosages need to be titrated to optimize safety and efficacy.

EXAMPLES

Example 1

Measurement of intracellular calcium

Intracellular calcium was monitored in guinea pig myocytes in the presence of the dihydropyridine agonist BayK(-) and AID-TAT peptide of the invention using the fluorescent indicator Fura-2 acetoxymethyl ester as described by Viola et al.(Circ Res, 2007; 100: 1036- 1044). Fluorescence at 340/380 nm excitation and 510 nm emission were measured at 1 min intervals with an exposure of 50 ms on a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope. Ratiometric 340/380 nm signal of individual myocytes were quantified using Metamorph 6.3 to measure signal intensity of manually traced cell regions (Figure 2). An equivalent region not containing cells was used for background and was subtracted. Fluorescence ratios recorded for 5 min just prior to and 10 min after exposure to treatments were averaged and alterations in fluorescent ratio were reported as a percentage of baseline average. Experiments were performed at 37 °C.

Ischemia-reperfusion of guinea-pig heart

Guinea-pig hearts were excised and perfused on a Langendorff apparatus with calcium- containing Krebs-Henseleit Buffer (KHB) solution containing (in mM): 110 K Glutamate, 25 KCI, 10 KH₂P0₄, 2 MgSO-4, 20 Taurine, 5 Creatine Base, 5 HEPES, 20 Glucose, 1.5 CaCI₂, 0.5 EGTA, pH 7.4 at 37 °C. Control (non-ischemic) hearts were perfused with calcium- containing KHB solution at a rate of 7 mL/min for 90 min. Ischemia-reperfused (l/R) hearts were perfused for 30 min at a rate of 7 mL/min, followed by a 30 min no-flow ischemia, then a 30 min reperfusion. Prior to removal from the Langendorff apparatus hearts were perfused for 10 min with calcium-free KHB solution. Perfusates were collected at 25 min (pre-control and pre-l/R) and 10 min after ischemia (post- control and post-l/R) for CK and LDH assays. Infarct size (damage) was assessed as area that did not take up nitroblue tetrazolium dye (Figure 3). It was found that 1 μΜ AID-TAT did not decrease contractility post ischemia reperfusion, measured as changes in developed pressure ex vivo in a heart perfused on a Langendorff apparatus. Higher concentrations of the peptide of the invention are expected to be more effective at reducing damage but may also influence contractility. 10 μΜ AID-TAT was found to decrease calcium influx approximately 50% in myocytes.

Hearts were perfused retrogradely via the aorta on a Langendorff apparatus for 30 min at 7 mL/min with normal Krebs solution (normoxia). Ischemia was induced with no-flow for 30 minutes. Reperfusion was induced by perfusing hearts for 30 minutes at 7mL/min with normal Krebs solution. Myocardial damage was assessed as release of creatine kinase and lactate dehydrogenase from the muscle. Infarct size was determed as area of heart muscle that did not take up nitroblue tetrazolium dye. The ratio of reduced to oxidised glutathione in the muscle is determined as a measure of oxidative stress.

Creatine kinase assay

Creatine kinase (CK) activity was determined from perfusate from control and ischemia- reperfused hearts using a Randox CK NAC-activated diagnostic kit (Randox Laboratories). The rate of increase in absorbance was measured at 340 nm (PowerWave XS, BioTek) over 15 min at 30 °C. CK activity was calculated using the equation: CK activity (U/L) = 4127 x Δ Abs 340 nm/min (Figure 4).

Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) activity was determined from perfusate from control and ischemia-reperfused hearts. Briefly, 150 pL of perfusate sample was mixed with 50pL LDH assay buffer (50 mM imidazole, 375 μΜ NADH, 4 mM pyruvate, 0.05% BSA, pH 7.0) and the rate of decrease in absorbance was measured at 340 nm (PowerWave XS, BioTek) over 15 min at 25 °C. LDH activity was calculated using the following equation: LDH activity (U/ml) = ((Δ Abs 340 nm/min TEST - Δ Abs 340 nm/min BLANK)(3)(dilution factor)) / ((6.22X0.1 )) (Figure 5).

Glutathione assay

GSH/GSSG ratio was determined in control and ischemia reperfused hearts as described by Rahman et al.(Nature Protocols. 2008;1 :3159-3165). Tissue homogenates were centrifuged at 5200 g for 15 min at 4 °C and GSH/GSSG ratio was measured from the supernatant. Fluorescence was detected using a RF 2000 fluorescence detector (Dionex) at excitation wavelength of 340 nm and emission at 525 nm. Verification of peak identity was confirmed using 15 mM standard solutions of glutamate-glutamate, GSH and GSSG. Some GSH and GSSG measurements were confirmed using HPLC Samples that were run on an Acclaim^® 120, C18, 3 pm, 120A, 2.1 x 100 mm column on an UltiMate 300 HPLC (Dionex) as described by Jones et al.( Clin Chim Acta. 1998;275. 75-184) (Figure 6).

Example 2 In vivo studies

Animal studies were performed in conformance with the principles described in the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of health (NIH publication no. 85-23, revised 1996) and the Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia (2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc 2001 ;218:669), and were approved by the UCLA Institute for Animal Care and Use Committee. Rats were randomly assigned to 3 groups: 1 . Active AID-peptide, 2. Scrambled control peptide, 3. Saline only. For the induction of myocardial infarction, 250-300g Sprague Dawley rats were anesthetized, intubated ventilated.and a left thoracotomy was created as described previously (Journal of Surgical Research 153, 217-223 (2009)). A length of 7-0 Prolehe suture was placed around the left anterior descending artery (LAD) and tightened to occlude the vessel. Blanching of the myocardium and ECG ST-segment elevation were indicative of successful occlusion.

After 30 min of LAD occlusion, the ligature was loosened and subsequently removed. All animals were allowed to stabilize for approximately 5 min before 0.1 mL of 1 or 10 uM AID- peptide in saline or scrambled peptide in saline or saline only were injected into the LV using a 26G needle as a slow bolus. The surgeon was blinded .to active and scrambled peptides that were labelled as peptide A or peptide B. The thoracic cavity was then closed and the animals recovered. All rats underwent morphometric and functional assessment prior to the Ml and again 6 wk after Ml, using echocardiography (echo) with a Siemens Acuson Sequoia C256 instrument (Siemens Medical Solutions, Mountain View, CA). Prior to sacrifice, hemodynamic measurements were recorded using a Millar catheter inserted into the left ventricle. Left ventricular systolic pressure, maximal pressure over time (dP/dT) and heart rate were recorded.

The data shown in Figure 7 represents heart weight/body weight ratio, ventricular systolic pressure, max pressure over time (dP/dT) and heart rate. Overall there are no deleterious effects of the peptides. At 6 weeks rats are hemodynamically compromised in the control group (Peptide B) demonstrated as a decrease in developed pressure over time (dP/dT) However dP/dT is improved after administration of the active peptide (peptide A). The AID- TAT peptide was found to decrease reperfusion injury in vivo 6 weeks after myocardial infarction assessed as area that does not take up nitroblue tetrazolium dye (Figure 8).

Publication reference: Schenke-Layland K, Strem BM, Jordan MC, DeEmedio MT, Hedrick MH, Kenneth Roos KP, Fraser JK and MacLellan WR. .Adipose Tissue-Derived Cells Improve Cardiac Function Following Myocardial Infarction. Journal of Surgical Research 153, 217-223 (2009).

Example 3

In vitro studies: Uptake of AID-TAT into cardiac myocytes

Uptake of AID-TAT was monitored in freshly isolated myocytes by assessing alterations in Rhodamine B fluorescence (ex 535nrp, , ern 58Qnm) prior to and following addition of Rhodamine B labelled AID-TAT, at 37°C using a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope. Fluorescent images were taken at 5 min intervals with 200 ms exposure. Metamorph 6.3 was used to quantify the signal by manually tracing myocytes. An equivalent region not containing cells was used as background and was subtracted. Fluorescent values for each time point were plotted relative to associated basal (Pre) fluorescent value assigned a value of 1.0 (Figure 9).

Claims

1. A peptide comprising the amino acid sequence:

Gin Gin X, Glu X₂ X₃ Leu X4 Gly Tyr X₅X₆Trp lie X₇ X₈ X₉ Glu wherein X_1-9 are naturally occurring amino acids.

2. A peptide comprising the amino acid sequence:

Gin Gin Leu Glu Glu Asp Leu Lys Gly Tyr Leu Asp Trp lie Thr Gin Ala Glu.

3. A peptide comprising: a peptide portion comprising the amino acid sequence of claim 1 or claim 2; and a peptide portion comprising the amino acid sequence:

Arg Lys Lys Arg Arg Gin Arg Arg Arg Zaa wherein Zaa is a 6-amino hexanoic acid.

4. A method for modulating movement of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell of a subject, comprising administering to the subject a peptide according to any one of claims 1 to 3.

5. A method for modulating binding of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell of a subject, comprising administering to the subject a peptide according to any one of claims 1 to 3.

6. A method according to claim 5, wherein the peptide prevents interaction between the beta subunit of the L-type Ca²⁺ channel and an alpha subunit of the L-type Ca ⁺ channel in a cardiac cell of a subject.

7. A method for modulating a L-type Ca²⁺ channel in a cardiac cell of a subject, comprising the step of administering to the subject a peptide according to any one of claims 1 to 3.

8. A method for reducing myocardial damage and/or oxidative stress in the heart of a subject during and/or after repeffusion, comprising the step of administering to the subject a peptide according to any one of claims 1 to 3.

9. The method according to claim 8, wherein the myocardial damage comprises cardiac hypertrophy.

10. The method according to claim 9, wherein cardiac hypertrophy is reduced.

11. The method according to claim 8, wherein intracellular Ca ⁺ levels are reduced or substantially maintained in the heart of the subject.

12. Use of a peptide according to any one of claims 1 to 3 for modulating movement of a beta subunit of the L-type Ca²⁺ channel in a cardiac cell of a subject.

13. Use of a peptide according to any one of claims 1 to 3 for modulating binding to a L-type Ca²⁺ channel alpha-interacting domain in a cardiac cell of a subject.

14. Use of a peptide according to any one of claims 1 to 3 for modulating a L-type Ca²⁺ channel in a cardiac cell of a subject.

15. Use of a peptide according to any one of claims 1 to 3 for reducing myocardial damage and/or oxidative stress in the heart of a subject during and/or following reperfusion.

16. A polynucleotide encoding a peptide according to any one of claims 1 to 3.

17. Use of a peptide according to any of claims 1 to 3 in the manufacture of a medicament for the treatment of reperfusion injury in the heart of a subject.