AU2005222732B2 - Alpha helical mimics, their uses and methods for their production - Google Patents

Abstract

This invention discloses short chain peptides that have been constrained to adopt an alpha helical conformation and their use as alpha helical scaffolds for directing amino acid side chains into positions analogous to those found in longer chain alpha helical peptides and for attaching peptidic or non-peptidic appendages in order to mimic side chains of longer alpha helical peptides. More particularly the invention discloses alpha helical cyclic pentapeptides and their use as alpha helical scaffolds or macrocyclic alpha helical modules, either alone, or within longer chain peptides or attached to other macrocyclic peptides or attached to non-peptidic structures, for the purpose of mimicking naturally occurring peptides or proteins, and as agonists or antagonists of the biological activity of naturally-occurring peptides or proteins or for the preparation of new materials.

Description

WO 2005/090388 PCT/AU2005/000400 TITLE OF THE INVENTION "ALPHA HELICAL MIMICS, THEIR USES AND METHODS FOR THEIR PRODUCTION" FIELD OF THE INVENTION [0001] This invention relates generally to short chain peptides that have been constrained 5 to adopt an alpha helical conformation and to their use as alpha helical scaffolds for directing amino acid side chains into positions analogous to those found in longer chain alpha helical peptides and for attaching peptidic or non-peptidic appendages in order to mimic side chains of longer alpha helical peptides. More particularly the invention relates to alpha helical cyclic pentapeptides and their use as alpha helical scaffolds or macrocyclic alpha helical modules, either alone, or within longer chain 10 peptides or attached to other macrocyclic peptides or attached to non-peptidic structures, for the purpose of mimicking naturally occurring peptides or proteins, and as agonists or antagonists of the biological activity of naturally-occurring peptides or proteins or for the preparation of new materials. [0002] Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. 15 BACKGROUND OF TIE INVENTION [0003] The alpha helix is a fundamental structural unit in the fabric of proteins, with 30% of all amino acids in proteins occurring in alpha helices.' When helical sequences of amino acids are exposed on an exterior surface of a protein, the helix frequently interacts with another protein, a segment of DNA or of RNA., 3 This biomolecular recognition is central to a large range of biological 20 processes, for example those summarized in Table 1. In most cases however only a few alpha helical turns are actually involved in the molecular recognition. For example, transcriptional regulators (e.g. p53, NF-kBp65, VP16c) 4 6 apoptosis regulators (e.g. Bak) 7 and RNA-transporter proteins (e.g. Rev)' all contain a short alpha helical sequence of only 2-4 turns that mediates function by direct interaction with a receptor. 25 TABLE 1 Some Biological Processes Mediated by Interaction of Alpha-Helices with Other Biomolecules a-Helical Peptide iBiological target Process Mediated Reference Protein-DNA interactions Zif268 G/C rich major DNA transcription 9 groove Protein-RNA interactions HIV Reverse' Rev Response RNA reverse transcription 10 Transcriptase Element (RRE) X-N peptide BoxB RNA Transcriptional anti- 10 - 1- WO 2005/090388 PCT/AU2005/000400 a-Helical Peptide Biological target Process Mediated Reference termination P22 peptides BoxB RNA Transcriptional anti- 10 termination Protein-Protein interactions p53 HDM2 Tumor Suppressor silencing 4 Bak Bcl-XL Apoptosis Regulation 7 VHL peptide Elongin C DNA transcription 11 VP16 activation HTAFr 1 31 DNA transcription 12 domain hPTH hPTHrP Calcium homeostasis 13 Dynorphin A Y,S-Opioid Pain signal transmission 14,15 receptors Apolipoprotein-E LDL receptor Lipid metabolism, 16 cholesterol homeostasis Neuropeptide-Y NPY receptors Multiple functions 17 Galanin Gal receptors Multiple functions 18 Corticotropin CRF receptors Stress responses 19 Releasing Factor Calcitonin Gene CGRP receptors Multiple functions 20 Related Peptide Nociceptin ORL1 receptor Pain transmission Vasointestinal VPACi 8 2 Multiple functions 21 Peptide Nuclear Coactivators Nuclear Receptors DNA Transcription 22,23 (eg. SRC1, GRIP 1) [00041 Short peptide sequences of less than 15 amino acid residues that correspond to these helical protein regions are not thermodynamically stable structures in water when removed from their protein environments.

24 2 5 Short synthetic peptides corresponding to such alpha helical 5 recognition motifs tend not to display appreciable helical structure in water, away from the helix stabilizing hydrophobic environments of proteins. If short peptide alpha helices could be stabilized or mimicked by small molecules, such compounds might be valuable chemical or biological probes and lead to development of novel pharmaceuticals, vaccines, diagnostics, biopolymers, and industrial agents. The goal of structurally mimicking short alpha helices with small molecules that have 10 biological activity comparable to proteins has not yet been realized. [0005] Attempts to stabilize short alpha helical peptides have met with limited success to date. Examples of methods used to stabilize alpha helicity in peptides longer than 15 residues are helix-nucleating templates 2 6

-

2 9 , metals 30 -3 5 , unnatural amino acids 36

'

37 , non-covalent side chain -2- WO 2005/090388 PCT/AU2005/000400 constraints 8

'

39 and covalent side chain linkers (e.g. disulfide- 4"'41, hydrazone 2 , lactam- 4-5", aliphatic linkers 5

'-

3 ). Although mimics of short alpha helical segments have remained elusive, some recent attempts have been reported using non-peptidic oligoamide and terphenyl scaffolds that project 2-3 substituents into similar three dimensional space as the side chains of an alpha helix 54-56. 5 [0006] Helix nucleating templates are organic molecules at the N- or C-terminus of a peptide which can make hydrogen bonds with the first or last four NH or C=O groups in the peptide, and thus nucleate helicity throughout the rest of the peptide. Such a task is not trivial due to the specific position, pitch and orientation of the required NH or C=O groups. Several attempts have had some success, these include Kemp's triacid, cyclic proline molecules, 26 , -61, Mueller's Cage 10 compound 62 , Bartlett's cap 2 8 , and Kahn's cap 6 3 . There have also been some attempts to synthesize capping groups by replacing a hydrogen bond with a covalent link as in the case of Satterthwait's Ca64. cap" [0007] Transition metals 30 -3 5 are often found in proteins serving both catalytic and structural roles. By exploiting the ability of transition metals such as Cu 2 +, Zn 2 +, Cd 2 +, Ru, Pd2+ to 15 bind both acidic and basic residues it has been possible to achieve helix stabilization. Chelation of metals to donor groups generally yields ~1 kcal/mol' in helix stabilization, however stabilization is very dependent on solvent, salt concentration and pH. [00081 Unnatural amino acids have also been reported to favor helix stabilization. In general n-alkyl substitution, a,a- and yy-disubstitution increases helix stability. P,1-Disubstitution 20 reduces helicity, and p-tertiary substitution totally abolishes helix propensity, thus it appears the helix is quite sensitive to steric effects at the beta position 65 . a-Aminoisobutyric acid (Aib) in particular is known to stabilize a- and 3 1 0 -helical conformations and has been used to improve the biological activity of several peptides. Nociceptin analogues containing 1 or 2 Aib residues resulted in 10-15 fold increases in potency and affinity (Ki= 0.02nM) 6 6 . Similarly an analogue of p53 containing Aib and 25 1 -aminocyclopropanecarboxylic acid (Ac 3 c) yielded a peptide 1735 more active than the native peptide 6 7 . Finally when Aib was substituted into deltorphin-C analogues a 10-fold Ki increase in selectivity was obtained for 5 vs p opioid receptor subtypes 68 . 10009] Disulfide bridges have been employed to stabilize helices via two methods. The first involves the use of a modified, unnatural amino acid D,L 2-amino-6-mercaptohexanoic acid 30 placed at the ith (D) and i+ 7 th (L) residues to stabilize two turns of an alpha helix 1 . The second approach involves using a D-cysteine (i) and L-cysteine (i+3) disulfide to stabilize a single alpha helical turn. This approach was successful to a certain extent, however the conformation was quite solvent dependent" 0 . It has recently been reported that this approach was used to constrain the SRC-I peptide, which is known to adopt an alpha helical conformation in the estrogen receptor-a, and inhibit 35 this receptor with a Ki of 25nM 69. -3- WO 2005/090388 PCT/AU2005/000400 [0010] Lactam bridges have often been used to increase helicity and turn conformations in long peptides. They generally involve the covalent amide linkage of the side chains of lysine/ornithine residues with the side chains of aspartic/glutamic acid residues at either i to i+3 or i to i+4 positions. These constraints although initially examined in model peptides have been applied to numerous 5 biological targets in which the bioactive conformation is deemed to be helical. In general this constraint has been employed in relatively long sequences (15-30 residues) generally to create monocyclic analogues, but in some cases, up to three lactam bridges have been included. Some examples of their use include PTH, NPY, CRF, GCN4, Galanin and Dynorphin-A. Despite their inception over 10 years ago, there is still a lack of consensus over which residue combinations are the 10 best, although it appears i to i+4 spacing is optimal for alpha helicity. Early pioneering work by Taylor" suggested Lys-+Asp was the optimal combination, however, later work by Houston identified Glu-+Lys as optimal, although this study totally neglected to use aspartic acid". More recent work by Taylor has involved using overlapping lactam bridges to yield a highly rigid hexapeptide alpha helix, highly resistant to chemical and thermal degradation 45 , and with some templating capability". 15 However, this hexapeptide scaffold is limited for general application as a template since only, two of six residues are available for interaction with a biological target. The synthesis and properties of side chain lactam bridged peptides, their alpha helical nature, functional activity and potential for improved proteolysis resistance has recently been reviewed 4 ". [00111 Modified lactam-type bridges can also be spaced i to i+7, therefore requiring 20 longer linkers, and in this regard, aspartic/glutamic acid, and/or diaminopropionic acid residues provide a convenient functionality to which linkers can be attached. Some of these have included diaminopentane linkers joined to two glutamic acids 53 , 4-(aminomethyl)-phenylacetic acid linked via aspartic acid and 1,3-diaminopropionic acid 4 9 , or alternately 4-(aminomethyl)-phenylazobenzoic acid joined to the N- and C-terminus of an octapeptide. The two former methods resulted in reasonably 25 stable helices, whilst the latter resulted in a 310 helical/random coil conformation depending on the cis/trans isomerization of the azo linkage. [0012] Ring closing metathesis has been used in helix stabilization. Pioneered by Grubbs 72 , this approach has been utilized with allyl-modified serine/homoserine residues in i-+i+4 fashion. It has not been overly successful in stabilizing alpha helicity, although some 310 stabilization 30 was observed. Other approaches have incorporated both S- and R-a-methyl-ax-allylglycine, along with the a-homoallyl and a-homohomoallyl derivatives, positioned at either i->i+4 or i->i+7 51. It was found that the R-isomer at the i position and the S-isomer at the i+7 position, with an 11 carbon link provided 44% helix stability compared to the uncyclized peptide. [0013] Non-peptidic mimicry of alpha helices has been rare, with only a few examples 35 reported. The first reported non-peptidic helix mimetics were 1,1,6-trisubstituted indanes, that when coupled to an amino acid were capable of presenting three side chains in a helical like conformation. When applied as tachykinin mimetics, they had micromolar affinity for NK and NK 3 receptors". -4- WO 2005/090388 PCT/AU2005/000400 These type of molecules were recently applied to magainin mimicry, and whilst they were capable of killing bacterial strains they still maintained high hemolytic activity 74 . Recently Kahne and co-workers developed a pentasaccharide helix mimetic based on GCN4 which bound DNA with micromolar affinity 75 . By far the most successful approach to non-peptidic alpha helix mimicry has been achieved 5 by Hamilton and co-workers who have successfully developed two generic types of molecules terphenyls and oligoamides capable of mimicking the i, i+4, i+7 side chains on one face of an alpha helix. These mimetics have been successfully applied to inhibition of HIV gp41 mediated viral fusion with an IC 50 of 15.7tg/mL 7 6 , and also inhibit Bak/Bcl-XL complex with low micromolar to nanomolar efficiency 77 7 8 . 10 [0014] There have been no previous reports of cyclic pentapeptides adopting alpha helices on their own. Usually cyclic pentapeptides have been used to mimic the smaller beta or gamma turns of peptides and proteins. There are numerous examples of cyclic peptides that mimic beta or gamma turns reported in the literature as demonstrated by several reviews 73 81 . A prime example is synthetic compound 1 which is a cyclic pentapeptide containing the RGD tripeptide sequence. This compound 15 is a potent glycoprotein Ilb/IIla antagonist and orally bioavailable antithrombotic and antitumor agent 73

,

82

'

8 3. Compound 1 provides a demonstration of how the simple insertion into a cyclopeptide of a rigid amino acid as a conformational constraint can result in favorable biological and pharmacological properties; and a number of its derivatives are in advanced clinical trials. For example, in phase III clinical trials, the cyclic RGD-containing heptapeptide drug eptifibatide 20 (Integrilin) has been shown to reduce the incidence of cardiac events in patients at risk of abrupt vessel closure after coronary angioplasty1. H

NTNH

2 NH HN N O H k N 1 cN2H O NH I 2 HO

H

2 N 1 2 3 [00151 Constraints do not need to be complex, as shown in compound 2 where an 25 ornithine (or lysine) side chain is used to form the macrocycle. This constraint, in conjunction with proline and D-cyclohexylalanine constraints, induces intramolecular hydrogen bonding that confers potent antagonism (IC 50 10 nM) against human C~a receptors on polymorphonuclear leukocytes both in vitro and in vivo 85 . C~a antagonists are expected to be useful for combating inflammatory diseases. [0016] Cyclotheonamide A (Compound 3) is a 1 9-membered cyclic pentapeptide 30 possessing a-keto amide and trans-4-aminobutenoyl constraints. It was isolated from the marine -Q5 - WO 2005/090388 PCT/AU2005/000400 sponge Theonella sp. and was shown to inhibit the serine proteases thrombin (Ki 180 nM) and trypsin (Ki 23 nM). The NMR solution structure of compound 3 was recently found to be the same in water as those found in the solid state when bound to trypsin and thrombin", suggesting that this natural product is pre-organized for enzyme binding, and that selectivity is associated with the positioning of 5 the D-Phe side chain. 100171 Lactam bridges (i-+ i+3, i-+ i+4, i-* i+7) have previously been reported to increase alpha helicity in longer peptides, although the literature is very inconsistent about their capacity to do so~ . There have been no reports of cyclic pentapeptides adopting alpha helical structures. [00181 The synthesis and conformation of multicyclic alpha helical peptides comprising 10 three repeats of a heptapeptide constrained by a side-chain to side-chain lactam bridge in (i)-+ (i+4) positions has been reported,1u 4 . These studies showed that spaced cyclic moieties in a peptide can induce or stabilize alpha helicity. [00191 Conformational restrictions in the form of (i)-+ (i+4) lactam bridges incorporated into known peptide sequences to induce helical conformation have also been reported' 1 5 . Three . 15 constrained helical 31-residue peptides derived from human parathyroid hormone and containing 1, 2 or 3 cyclic moieties were shown to be potent agonists of the parathyroid hormone and parathyroid hormone-related protein receptor 10020] There are few studies that report alpha helicity for the theoretical minimum (pentapeptide) sequence needed to define a beta turn, the existence and properties of which are not 20 well defined despite the likelihood that only one or a few turns of a protein helix need to be mimicked for agonist/antagonist biological activity. [0021] There are many commercially important peptides that are known to adopt alpha helical structures that would benefit from improved structural stabilization and improved resistance to proteolysis. Some examples include calcitonin which has been launched for the treatment of 25 osteoporosis, the parathyroid hormone which is in phase II clinical trials for the treatment of osteoporosis, a substance-P/saporin conjugate which is in preclinical trials for the treatment of pain and conantokin-G which is under development for the treatment of epilepsy (Pharmaprojects, 2004). [00221 Accordingly, there is a need for stabilized short peptide alpha helices that can mimic biological molecules or that can be incorporated into non-peptidic or semi-peptidic compounds 30 to mimic biological molecules. Such peptides could potentially be valuable as chemical and biological probes, pharmaceuticals, biotechnology products such as vaccines, or diagnostic agents, new components of biopolymers and industrial agents. -6- WO 2005/090388 PCT/AU2005/000400 SUMMARY OF THE INVENTION 100231 This invention is predicated in part on the unexpected discovery that certain short chain peptides, which comprise at least one macrocyclic pentapeptide unit, are highly alpha helical in their own right in water even when subjected to denaturing conditions (e.g., 8M guanidine.HCI; 5 trypsin; human plasma). Based on this discovery, the present invention resides in novel alpha helical compounds and non peptidic structures, which use one or more such cyclic pentapeptides or their analogues as alpha helical scaffolds that can project additional peptidic, cyclic, and non-peptidic appendages into positions typical of side chains of alpha helical peptides and protein segments. The present invention is also directed to methods for their preparation and use, as described hereinafter. 10 DETAILED DESCRIPTION OF THE INVENTION [00241 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. 15 For the purposes of the present invention, the following terms are defined below. [00251 The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one.element. [00261 Throughout this specification and the claims which follow, unless the context 20 requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. [0027] Advantageously, at least one embodiment of the present invention provides compounds comprising at least one macrocyclic moiety, particularly a cyclic pentapeptide moiety, 25 which has surprising alpha helicity in water, even under strong protein denaturing conditions such as high temperature (e.g., 40 to 800 C), or the presence of up to 8M guanidine hydrochloride, or the presence of proteolytic enzymes such as trypsin. [0028] According to one aspect of the present invention there is provided a compound comprising at least one alpha helical cyclic peptide, wherein the peptide consists essentially of a 30 sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other, with the proviso that when the compound comprises a single cyclic peptide it is selected from a compound that consists essentially of the single peptide or a compound that comprises the single peptide and a non-peptide moiety or a 35 compound that comprises the single peptide and at least one other peptide that comprises at least one -7- WO 2005/090388 PCT/AU2005/000400 amino acid whose side chain has been derivatized and that when the compound comprises two or more cyclic peptides, at least two of these are located immediately adjacent to each other. [0029] As used herein "alpha helical" refers to a three dimensional structural conformation which is analogous to those found in proteins and polypeptides. The alpha helix conformation found in 5 naturally occurring proteins and polypeptides has its side chains extending to the outside of the structure, has a complete turn every 3.6 amino acids, is right-handed and typically has hydrogen bonding between the carbonyl groups of the amide bond and an amide N-H group 4 amino acids further on in the sequence. The cyclic peptides of the present invention have a helicity calculated from molar elipticities obtained from circular dichroism spectroscopy (CD spectroscopy) and are expressed 10 as a percentage of the theoretical helicity obtainable for that peptide or a relative helicity compared to a reference standard or standard helix. [00301 As used herein, the term "amino acid" refers to compounds having an amino group and a carboxylic acid group. An amino acid may be a naturally occurring amino acid or non-naturally occurring amino acid and may be a proteogenic amino acid or a non-proteogenic amino acid. The 15 amino acids incorporated into the amino acid sequences of the present invention may be L-ct-amino acids, D-a-amino acids or mixtures thereof. [0031] In some embodiments, the cyclic peptides of the invention are linked directly or indirectly to non-peptide moieties. Such moieties include, but are not limited to, aldehydes, toxins; drugs; polysaccharides; nucleotides; oligonucleotides; labels such as radioactive substances (e.g. mIn, 20 B, 1 99 mTc 2 1 2 B 9 0 Y ' 86 Rh); biotin; fluorescent tags; imaging reagents (e.g., those described in U.S. Pat. No. 4,741,900 and U.S. Pat. No. 5,326,856); hydrocarbon linkers (e.g., an alkyl group or derivative thereof) conjugated to a moiety providing for attachment to a solid substratum, or to a moiety providing for easy separation or purification (e.g., a hapten recognized by an antibody bound to a magnetic bead), etc. Linkage of the peptide to the non-peptide moiety may be by any of several well 25 known methods in the art. [0032] Suitable naturally occurring proteogenic amino acids are shown in Table 2 together with their one letter and three letter codes. TABLE 2 Amino Acid one letter code three letter code L-alanine A Ala L-arginine R Arg L-asparagine N Asn L-aspartic acid D Asp L-cysteine C Cys L-glutamine Q Gln L-glutamic acid E Glu -8- WO 2005/090388 PCT/AU2005/000400 Amino Acid one letter code three letter code glycine G Gly L-histidine H His L-isoleucine. I Ile L-leucine L Leu L-lysine K Lys L-methionine M Met L-phenylalanine F Phe L-proline P Pro L-serine S Ser L-threonine T Thr L-tryptophan W Trp L-tyrosine Y Tyr L-valine V Val [00331 Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis. Examples of side chain modifications contemplated by the present invention include, but are not limited to modifications of amino groups such as by 5 reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 . 10 [0034] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. [0035] The carboxyl group may be modified by carbodiimide activation via 0-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide. [0036] Sulfhydryl groups may be modified by methods such as carboxymethylation with 15 iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4 chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. 20 [0037] Tryptophan residues may be modified by, for example, oxidation with N bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides. Tyrosine residues on the other hand, may-be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. -9- WO 2005/090388 PCT/AU2005/000400 [0038] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate. [0039] Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, t-butylglycine, norvaline, phenylglycine, 5 ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. Examples of suitable non proteogenic or non-naturally occurring amino acids contemplated herein is shown in Table 3. TABLE 3 Non-conventional amino acid Code Non-conventional amino acid Code a-aminobutyric acid Abu L-N-methylalanine Nmala a-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- carboxylate Cpro L-N-methylasparagine Nmasn L-N-methylaspartic acid Nmasp L-N-methylcysteine Nmcys aminonorbornyl-carboxylate Norb L-N-methylglutamine Nmgln cyclohexylalanine Chexa L-N-methylglutamic acid Nmglu cyclopentylalanine Cpen L-N-methylhistidine Nnhis D-alanine Dal L-N-methylisolleucine Nmile D-arginine Darg L-N-methylleucine Nmleu D-aspartic acid Dasp L-N-methyllysine Nmlys D-cysteine Dcys L-N-methylmethionine Nmmet D-glutamine Dgln L-N-methylnorleucine Nmnle D-glutamic acid Dglu L-N-methylnorvaline Nmnva D-histidine Dhis L-N-methylornithine Nmorn D-isoleucine Dile L-N-methylphenylalanine Nmphe D-leucine Dleu L-N-methylproline Nmpro D-lysine Dlys L-N-methylserine Nmser D-methionine Dmet L-N-methylthreonine Nmthr D-ornithine Dorn L-N-methyltryptophan Nmtrp D-phenylalanine Dphe L-N-methyltyrosine Nmtyr D-proline Dpro L-N-methylvaline Nmval D-serine Dser L-N-methylethylglycine Nmetg D-threonine Dthr L-N-methyl-t-butylglycine Nmtbug D-tryptophan Dtrp L-norleucine Nle D-tyrosine Dtyr L-norvaline Nva D-valine Dval a-methyl-aminoisobutyrate Maib D-a-methylalanine Dmala a-methyl- -aminobutyrate Mgabu D-a-methylarginine Dmarg a-methylcyclohexylalanine Mchexa D-a-methylasparagine Dmasn a-methylcylcopentylalanine Mcpen D-a-methylaspartate Dmasp a-methyl -a-napthylalanine Manap D-a-methylcysteine Dmcys a-methylpenicillamine Mpen D-a-methylglutamine Dmgln N-(4-aminobutyl)glycine Nglu -10- WO 2005/090388 PCT/AU2005/000400 Non-conventional amino acid Code Non-conventional amino acid Code D-a-methylhistidine Dmhis N-(2-aminoethyl)glycine Naeg D-a-methylisoleucine Dmile N-(3-aminopropyl)glycine Norn D-a-methylleucine Dmleu N-amino-a-methylbutyrate Nmaabu D-ai-methyllysine Dmlys a-napthylalanine Anap D-a-methylmethionine Dmmet N-benzylglycine Nphe D-a-methylornithine Dmorn N-(2-carbamylethyl)glycine Ngln D-a-methylphenylalanine Dmphe N-(carbamylmethyl)glycine Nasn D-a-methylproline Dmpro N-(2-carboxyethyl)glycine Nglu D-a-methylserine Dmser N-(carboxymethyl)glycine Nasp D-a-methylthreonine Dmthr N-cyclobutylglycine Ncbut D-a-methyltryptophan Dmtrp N-cycloheptylglycine Nchep D-a-methyltyrosine Dmty N-cyclohexylglycine Nchex D-a-methylvaline Dmval N-cyclodecylglycine Ncdec D-N-methylalanine Dnmala N-cylcododecylglycine Ncdod D-N-methylarginine Dnmarg N-cyclooctylglycine Ncoct D-N-methylasparagine Dnmasn N-cyclopropylglycine Ncpro D-N-methylaspartate Dnmasp N-cycloundecylglycine Ncund D-N-methylcysteine Dnmcys N-(2,2-diphenylethyl)glycine Nbhm D-N-methylglutamine Dnmgln N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamate Dnmglu N-(3-guanidinopropyl)glycine Narg D-N-methylhistidine Dnmhis N-(1 -hydroxyethyl)glycine Nthr D-N-methylisoleucine Dnmile N-(hydroxyethyl))glycine Nser D-N-methylleucine Dnmleu N-(imidazolylethyl))glycine Nhis D-N-methyllysine Dnmlys N-(3-indolylyethyl)glycine Nhtrp N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyl-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen L-t-butylglycine Tbug N-(p-hydroxyphenyl)glycine Nhtyr L-ethylglycine Etg N-(thiomethyl)glycine Ncys L-homophenylalanine Hphe penicillamine Pen L-a-methylarginine Marg L-ai-methylalanine Mala L-a-methylaspartate Masp L-a-methylasparagine Masn L-a-methylcysteine Mcys L-a-methyl-t-butylglycine Mtbug L-ai-methylglutamine Mgln L-methylethylglycine Metg L-a-methylhistidine Mhis L-a-methylglutamate Mglu - 11 - WO 2005/090388 PCT/AU2005/000400 Non-conventional amino acid Code Non-conventional amino acid Code L-a-methylisoleucine Mile L-a-methylhomophenylalanine Mhphe L-a-methylleucine Mleu N-(2-methylthioethyl)glycine Nmet L-a-methylmethionine Mmet L-a-methyllysine Mlys L-a-methylnorvaline Mnva L-a-methylnorleucine Mnle L-a-methylphenylalanine Mphe L-a-methylornithine Morn L-a-methylserine Mser L-a-methylproline Mpro L-a-methyltryptophan Mtrp L-a-methylthreonine Mthr L-a-methylvaline Mval L-a-methyltyrosine Mtyr N-(N-(2,2-diphenylethyl) Nnbhm L-N-methylhomophenylalanine Nmhphe carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl Nmbc N-(N-(3,3-diphenylpropyl) Nnbhe ethylamino)cyclopropane carbamylmethyl)glycine [0040] As used herein, "amino acid side chain" or "side chain" refers to the characterizing substituent of the amino acid. This term refers to the substituent bound to the c-carbon of either a natural or non-natural a-amino acid. For example, the characterizing substituents of some naturally 5 occurring amino acids are shown in Table 4. TABLE 4 The Proteinogenic Amino Acids H / R

NH

2 C0 2 H Amino acid -R Alanine -CH3 Arginine -(CH 2

)

3 NHC(=NH)NH2 Asparagine -CH 2

CONIH

2 Aspartic acid -CH 2

CQ

2 H Cysteine -CH 2 SH Glutamine -(CH 2

)

2

CONH

2 Glutamic acid -(CH 2

)

2 CO2H Glycine -H Histidine

-CH

2 (4-imidazolyl) Isoleucine -CH(CH 3

)CH

2

CH

3 Leucine -CH 2

CH(CH

3

)

2 Lysine

-(CH

2

)

4

NH

2 Methionine

-(CH

2

)

2

SCH

3 Phenylalanine -CH 2 Ph - 12 - WO 2005/090388 PCT/AU2005/000400 Amino acid -R Serine -CH 2 OH Threonine -CH(CH 3 )OH Tryptophan -CH 2 (3-indolyl) Tyrosine -CH 2 (4-hydroxyphenyl) Valine -CH(CH 3

)

2 [0041] Another naturally occurring amino acid is proline. CO2 H N H in which the a-side chain terminates in a bond with the amino acid amine nitrogen atom. Some non 5 limiting examples of characterizing substituents of non-naturally occurring amino acids are shown in Table 5: TABLE 5 Non-Natural Amino Acids H / R

NH

2

CO

2 H Amino acid -R a-aminobutyric acid -CH 2

CH

3 ornithine -(CH 2

)

3 N1 2 cyclohexylalanine -CH 2

C

6

H

10 cyclopentylalanine -CH 2

C

5

H

8 norvaline -CH 2

CH

2 CH3 norleucine -(CH 2

)

3

CH

3 10 [00421 In some embodiments, the cyclic peptide is a macrocycle formed by consecutively linking at least 18 to 22 atoms, wherein the first and last atoms are bonded to one another to form a ring. In a preferred embodiment the macrocycle is formed from 19 to 21 atoms, especially preferred are macrocycles formed from 20 atoms. In some embodiments, the first terminal residue and second 15 terminal residue of the pentapeptide are alpha amino acids. In these embodiments, the resulting macrocycle ring size is preferably 18-22 atoms, more preferably 20 atoms. In particular, where one of the first terminal residue and second terminal residue of the pentapeptide is Lys and the other is Asp, the resulting macrocycle ring size is preferably 18-22 atoms, more preferably 20 atoms. It will be - 13 - WO 2005/090388 PCT/AU2005/000400 apparent to persons skilled in the art that modifications to the substituents at the first and second terminal residues of the pentapeptide will result in a slightly different optimal macrocycle requirements. [00431 The two amino acid side chains of the first and second terminal residues defined 5 above may be linked in any suitable manner to form a cyclic pentapeptide. In some embodiments, the side chains are linked by a covalent bond either directly or through a linker. In an illustrative example, the side chains are covalently linked to one another without an intervening linker, for example, by formation of a lactam bridge between a side chain carboxylic acid group and a side chain amino group or a disulfide bond between two side chain thiol groups. In a preferred embodiment, a carboxylic acid 10 in the side chain of one amino acid residue is reacted with an amine in the side chain of a second amino acid residue to form an amide bond or lactam bridge. [00441 In some embodiments, one of the amino acid residues having a side chain participating in the linkage is selected from L-aspartic acid, L-glutamic acid, D-aspartic acid, D glutamic acid, L-ax-methyl-aspartic acid, L-a-methylglutamic acid, D-ca-methylaspartic acid and D-a 15 methyl-glutamic acid, and the other amino acid residue having a side chain participating in the linkage is selected from L-lysine, L-ornithine, D-lysine, D-ornithine, L-ca-methyllysine, D-a-methyllysine, L a-methylornithine and D-a-methylornithine. Preferably the amide bond is formed by reaction of an L aspartic acid or L-glutamic acid with an L-lysine or L-omithine. [00451 In a preferred embodiment of the invention the amino acid residues in the sequence 20 are D- or L-c-amino acids, especially L-c-amino acids. {00461 In another aspect of the invention there is provided a compound having the following formula (I): (I) (CR'R')m-L (CR'R')n H I _ H

R

1 -N-C(R)-C-Xaa-Xaa-Xaa--N-C(R)-C-R 2 25 [0047] wherein each Xaa is independently selected from any amino acid residue; [0048] R 1 is selected from H, an N-terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain; [0049] R 2 is selected from H, a C-terminal capping group, a group that mimics an amino 30 acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; -14- WO 2005/090388 PCT/AU2005/000400 [0050] each R' and R" are independently selected from H, C 1

-C

10 alkyl, C 2

-C

10 alkenyl, C 2 C 10 alkynyl, C 3

-C

1 Ocylcoalkyl, C 5 -Ciocycloalkenyl, -OH, -OC 1 -Cio alkyl, -NH 2 , -NH(C 1 -Cioalkyl), N(C 1 -Cioalkyl) 2 , C 6

-C

1 2 aryl, C 3

-C

1 Oheterocyclyl, C 5 -Cloheteroaryl and halo; [00511 L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH 2 -, CH 2 CH(OH)-, 5 -CH=CH-, -CH 2

-CH

2 -, -NH-CH 2 - -CH 2 -NH-, -CH 2 -S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 -C(O)-, -S(O)t-NH -NH-S(O)t-, CH 2 -P(=O)(OH)- and -P(=0)(OH)-CH 2 -; [0052] m is an integer from 1 to 4, [0053] n is an integer from 1 to 4, and [0054] t is 0, 1 or 2, 10 [00551 wherein m + n = 4, 5 or 6 and wherein when m is 2, n is not 3 and when m is 3, n is not 2. [0056] As used herein, the term "alkyl" refers to a saturated, straight or branched chain hydrocarbon group, preferably having 1 to 10 carbon atoms. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, 2 15 methylbutyl, 3-methylbutyl, 4-methylbutyl, hexyl, 2-ethylbutyl, heptyl, octyl, nonyl and decyl. Preferred alkyl groups have 1 to 6 carbon atoms. Especially preferred alkyl groups have 1 to 3 carbon atoms. [00571 As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbons containing at least one carbon-carbon double bond. Suitable alkenyl groups having 2 20 to 10 carbon atoms and include, but are not limited to, vinyl, allyl, 1 -methylvinyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl. Preferred alkenyl groups have 2 to 6 carbon atoms. Especially preferred alkenyl groups have 2 or 3 carbon atoms. [0058] As used herein, the term "alkynyl" refers to straight chain hydrocarbons containing at least one carbon-carbon triple bond. Suitable alkynyl groups having 2 to 10 carbon atoms include, 25 but not limited to, ethynyl, 1-propynyl, 2-propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl. Preferred alkynyl groups have 2 to 6 carbon atoms. Especially preferred alkynyl groups have 2 or 3 carbon atoms. [00591 As used herein, "halo" is intended to include fluoro, chloro, bromo and iodo. [0060] As used herein, the term "cycloalkyl" refers to saturated mono- or poly- cyclic 30 hydrocarbon groups. Suitable cycloalkyl groups having 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. Preferred cycloalkyl groups include cyclopentyl and cyclohexyl. [0061] As used herein, the term "cycloalkenyl" refers to saturated mono- or poly- cyclic hydrocarbon groups containing at least one carbon-carbon double bond. Suitable cycloalkenyl groups - 15 - WO 2005/090388 PCT/AU2005/000400 having 5 to 10 carbon atoms include, but are not limited to, cyclopentenyl, 1-methyl-cyclopentenyl, cyclohexenyl, cyclooctenyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1, 3 -cyclohexadienyl, 1,4 cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl. Preferred cycloalkenyl groups include cyclopentenyl and cyclohexenyl. 5 [0062] The term "aryl" used either alone or in compound words denotes single, polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples of aryl include, but are not limited to, phenyl, biphenyl, naphthyl, tetrahydronaphthyl. Preferred aryl groups include phenyl and naphthyl. [0063] The term "heteroaryl" refers to aromatic heterocyclic ring systems, wherein one or 10 more carbon atoms (and where appropriate, hydrogen atoms attached thereto) of a cyclic hydrocarbon residue are replaced with a heteroatom to provide an aromatic residue. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable heteroatoms include 0, N, S and Se. Examples of heteroaryl include, but are not limited to, pyridyl, thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, 15 quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. Preferred heteroaryl groups include pyridyl, thienyl, furyl, pyrrolyl. [0064] The term "heterocyclyl" when used alone or in compound words includes monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C 3 10 , preferably C 3 6 , 20 wherein one or more carbon atoms (and where appropriate, hydrogen atoms attached thereto) are replaced by a heteroatom so as to provide a non-aromatic residue. Suitable heteroatoms include, 0, N, S, and Se. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable examples of heterocyclic groups may include pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholino, indolinyl, imidazolidinyl, pyrazolidinyl, 25 thiomorpholino, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl etc. [0065] In preferred embodiments, any one of the following may apply: [0066] R, is selected from H, an N-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a negative charge at the N-terminus to match with the helix dipole, a non-peptidic group or a mimic of an amino acid side 30 chain. Suitable N-terminal capping groups include acyl and N-succinate. Suitable groups that mimic an amino acid side chain are any natural or unnatural amino acid side chain that is attached to the N terminal amino group of the peptide through a carbonyl group derived from a carboxylic acid by formation of an amide bond. Suitable mimics of amino acid side chains include, but are not limited to: [0067] CH 3

CH

2

C(O)(CH

2 )uC(O)-, NH 2

(NH=)CNHC(O)(CH

2 )uC(O)-, 35 H 2

NC(O)(CH

2

)

2

C(O)(CH

2 )uC(O)-, HOC(O)(CH 2

)

2

C(O)(CH

2 )uC(O)-, HS(CH 2

)

2

C(O)(CH

2 )uC(O)-,

H

2

NC(O)(CH

2

)

3

C(O)(CH

2 )uC(O)-, HOC(O)(CH 2

)

2

C(O)(CH

2 )uC(0)-, (4 - 16 - WO 2005/090388 PCT/AU2005/000400 imidazolyl)(CH 2

)C(O)(CH

2 )uC(O)-, CH 3

CH

2

CH(CH

3

)CH

2

C(O)(CH

2 )uC(O)-,

(CH

3

)

2

CH(CH

2

)

2

C(O)(CH

2 )uC(O)-, H 2

N(CH

2

)

5

C(O)(CH

2 )uC(O)-, CH 3

S(CH

2

)

3

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

2

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

4

C(O)(CH

2 )uC(Q)-, HO(CH 2

)

2

C(O)(CH

2 )uC(O)-,

HOCH(CH

3

)CH

2

C(O)(CH

2 )uC(O)-, (3-indolyl)(CH 2

)

2

(CH

2 )uC(O)-, (4 5 hydroxyphenyl)(CH 2

)

2

C(O)(CH

2 )uC(O)-, (4-hydroxyphenyl)(CH 2

)

3

C(O)(CH

2 )uC(O)-,

(CH

3

)

2

CHCH

2

C(O)(CH

2 )uC(O)-, CH 3

CH

2

CH

2

C(O)(CH

2 )uC(O)-, C 6 HmoCH 2

C(O)(CH

2 )uC(O)-, CsH 8

CH

2

C(O)(CH

2 )uC(O)-, CH 3

C(O)(CH

2 )uC(O)-, CH 3

(CH

2

)

4

C(O)(CH

2 )uC(O)-,

CH

3

(CH

2

)

5

C(O)(CH

2 )uC(O)-, HOC(O)CH 2

C(O)(CH

2 )uC(O)-, HS(CH 2

)C(O)(CH

2 )uC(O)-,

H

2

N(CH

2

)

4

C(O)(CH

2 )uC(O)- and HOCH 2

C(O)(CH

2 )uC(O)- wherein u is 0 or an integer from 1 to 10. 10 The preferred non-peptidic groups enhance the stability, bioavailability or activity of the peptides. Suitable non-peptidic groups include, but are not limited to hydrophobic groups such as carbobenzoxyl, dansyl, t-butyloxycarbonyl, acetyl, 9-fluorenylmethoxycarbonyl, groups which stabilize or mimic alpha-helices, groups which mimic the secondary structure of peptides, particularly alpha helical peptides, such as those disclosed in WO 03/018587, groups which improve 15 bioavailability, such as hydrophilic groups which aid aqueous solubility, for example, cyclodextrans; groups which are recognized by transport receptors to allow or improve transport of the peptides to the site of activity, for example, transport across cell walls or through an epithelial layer such as skin or the gut wall. [0068] R 2 is selected from H, a C-terminal capping group that stabilizes the terminus of a 20 helix, usually having hydrogen atoms able to form hydrogen bonds or having a positive charge at the C-terminus to match with the helix dipole, a peptide of 1, 2, 3, 4 or 5 amino acid residues optionally capped with a C-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a positive charge at the C-terminus to match with the helix dipole, a mimic of an amino acid side chain or a group which activates the terminal 25 carboxylic acid carbonyl group to -nucleophilic substitution. A suitable C-terminal capping group is

NH

2 . Suitable mimics of amino acid side chains are any common or unnatural amino acid side chain that is attached to the C-terminal carbonyl group of the peptide through an amine group by formation of an amide bond. Suitable mimics of amino acid side chains include but are not limited to: [0069] -NH(CH 2 )uNHCH 2

CH

3 , -NH(CH 2 )uNH(CH 2

)

4

NHC(=NH)NH

2 , 30 NH(CH 2 )uNH(CH 2

)

2

C(O)NH

2 , -NH(CH 2 )uNH(CH 2

)

2

CO

2 H, -NiH(CH 2 )uNH(CH 2

)

2 SH, NH(CH 2 )uNH(CH 2

)

3

C(O)NH

2 , -NH(CH 2 )uNH(CH 2

)

3

CO

2 H, -NH(CH 2 )uNH(CH 2

)

2 (4-imidazolyl), NH(CH 2 )uNHCH 2

CH(CH

3

)CH

2

CH

3 , -NH(CH 2 )uNH-(CH 2

)

2

CH(CH

3

)

2 , -NH(CH 2 )uNIH(CH 2 )sNH 2 , NH(CH 2 )uNH(CH 3

)

3

SCH

3 , -NH(CH 2 )uNH(CH 2

)

2 (3-indolyl), -NH(CH 2 )uNH(CH 2

)

2 (4-hydroxyphenyl),

-NH(CH

2 )uNH(CH 2

)

3 (4-hydroxyphenyl), -NH(CH 2 )uNH-CH 2

CH(CH

3

)

2 , -(NHCH 2 )uNHCH 2

CH

2

CH

3 , 35 NH(CH 2 )uNH-CH 2

C

6 HmO, -NH(CH 2 )uNHCH 2

C

5 Hs, -NH(CH 2 )uNHCH 3 , -NH(CH 2 )uNH(CH 2

)

4

CH

3 , NH(CH 2 )uNH(CH 2 )sCH 3 , -NH(CH 2 )uNHCH 2

CO

2 H, -NH(CH 2 )uNHCH 2 SH, -NH(CH 2 )uNH(CH 2

)

2 OH,

-NH(CH

2 )uNH(CH 2

)

5

NH

2 and -NH(CH 2 )uNHCH 2 OH; wherein u is 0 or an integer from 1 to 10. - 17 - WO 2005/090388 PCT/AU2005/000400 [00701 Suitable groups which activate the C-terminal carboxylic to nucleophilic attack include converting the carboxylic acid to an acid chloride, an acid anhydride, an acyl azide, an 0 acylisourea, a phosphonium derivative or an activated ester, especially those known in the art for activating carboxylic acids for peptide bond formation. 5 1 . [0071] In some embodiments, non-peptidic groups enhance the stability and circulating time, or decrease immunogenicity, or increase solubility, bioavailability or activity of the peptides (see U.S. Patent No. 4,179,337). Suitable non-peptidic groups include but are not limited to hydrophobic groups such as t-butyl, groups which stabilize or mimic alpha-helices, groups which mimic the secondary structure of peptides, particularly alpha helical peptides, such as those disclosed in WO 10 03/018587, groups which improve bioavailability, such as hydrophilic groups which aid aqueous solubility, for example, cyclodextrans; groups which are recognized by transport receptors to allow or improve transport of the peptides to the site of activity, for example, transport across cell walls or through an epithelial layer such as skin or the gut wall. In some embodiments, PEG (polyethylene glycol) groups are conjugated to the peptide compounds to make those compounds more easily 15 formulated and orally available. The amphiphilic nature of PEG helps protect the parent peptide from enzymatic breakdown and positions the drug for absorption across the gastrointestinal tract into the plasma. The terms "pegylated" and "pegylation" refer to the process of reacting a poly(alkylene glycol), suitably an activated poly(alkylene glycol), with a facilitator such as an amino acid, e.g. lysine, to form a covalent bond. Although "pegylation" is often carried out using poly(ethylene glycol) 20 or derivatives thereof, such as methoxy poly(ethylene glycol), the term is not intended to be so limited here, but is intended to include any other useful poly(alkylene glycol), such as, for example poly(propylene glycol). The chemical moieties for derivitization may also be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxyrnethylcellulose, dextran, polyvinyl alcohol and the like. The pentapeptide compounds may be 25 modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties. In some embodiments, the modification occurs at a position outside of the cyclic pentapeptide moiety, for example at amino acids preceding the cyclic pentapeptide moiety or at the N-terminus. [0072] The polymer may be of any molecular weight, and may be branched or 30 unbranched. For polyethylene glycol, exemplary examples include micropegylated groups devised specifically to enhance oral delivery in peptides as described in W02004047871. Methods for attaching Peg groups are well described in the patent literature (W0200404787 1, US Patent No. 5,643,575; EP 0 401 384; W003057235A2) For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. 35 Reactive groups are those to which an activated polyethylene glycol molecule may be bound. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the polypeptide or to more - 18 - WO 2005/090388 PCT/AU2005/000400 than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein or polypeptide. Polyethylene glycol may be attached to the protein or polypeptide either directly or by an intervening linker. Polyethylene glycol can also be attached to polypeptides using a number of different intervening linkers. For example, U.S. Patent No. 5 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins. Protein polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein or polypeptide by a linker can also be produced by reaction of proteins or polypeptides with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1, I'-carbonyldiimidazole, MPEG-2,4,5 trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A 10 number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins and polypeptides are described in WO 03/057235; PCT/GB03/00062; U.S. Patent No. 5,428,128; U.S. Patent No. 6,127,355; and U.S. Patent No. 5,880,131. [0073] Each R' is selected from H, CH 3 , CH 2

CH

3 , vinyl, OH, OCH 3 , NH 2 , NH(CH 3 ),

N(CH

3

)

2 , phenyl, F or Cl; most preferably H or CH 3 , especially H. 15 [0074] Each R" is selected from H, CH 3 , CH 2

CH

3 or vinyl, especially H. [0075] m is 1 and n is 3 or 4, m is 2 and n is 4, mis 3 and n is 1 or m is 4 and n is 1 or 2, especially where m is 1 and n is 4. [0076] Each Xaa may be any amino acid residue selected to mimic the amino acid residues in a known alpha helical peptide of interest or to prepare an unknown peptide having new properties. 20 An individual Xaa can be the same or different as another Xaa and is preferably selected from a D- or L- alpha amino acid residue. Especially preferred peptides of formula (I) have at least one Xaa which is a D- or L- alpha amino acid residue that is favorable to helix formation. Even more preferred are peptides in which 2 or 3 of Xaa are D- or L- alpha amino acid residues that are favorable to helix formation, for example, alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, 25 cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid, especially alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine. [00771 L is preferably -NH-C(O)- or -C(O)-NH-. [0078] Surprisingly, the cyclic pentapeptides of the invention display tolerance of variation of Xaa residues, with most amino acid substitutions of these residues retaining a high degree 30 of helicity. The range of amino acid substitutions that could be made at a specific Xaa residue would be readily apparent to a person of skill in the art. [0079] Representative peptides of the invention include, but are not limited to: [0080] Ac-cyclo-1,5-[KXaaXaaXaaD]-NH 2 [SEQ ID NO. 1] [0081] Ac-cyclo-1,5-[DXaaXaaXaaK]-NH 2 [SEQ ID NO. 2] 35 [0082] Ac-cyclo-1,5-[KXaaXaaXaaE]-NH 2 [SEQ ID NO. 3] - 19 - 100831 Ac-cyclo-I1,5-[EXaaXaaXaaK]-NH 2 [SEQ ID NO. 4] [00841 Ac-cyclo- 1,5.-[OXaaXaaXaaD]-NH 2 [SEQ ID NO. 5] [00851 Ac-cyclo-1,5-[DXaaXaaXaaO]-NH 2 [SEQ ID NO. 6] [00861 Ac-Xaa-cyclo-2,6-[KXaaXaaXaaD]-NH 2 [SEQ ID NO. 7] 5 [00871 Especially preferred peptides are those of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO, 6 and SEQ ID NO. 7, more especially SEQ ID NO. I and SEQ ID NO. 7. [00881 Illustrative examples of amino acid sequences represented by the above peptides include: [00891 Ac-(cyclo-1,5)-[KARAD]-NH 2 [SEQ ID NO. 8] 10 100901 Ac-(cyclo-I,5)-[DARAK]-NH 2 [SEQ ID NO. 9] [00911 Ac-(cyclo-1,5)-[KARAE]-NH 2 [SEQ ID NO. 10] [00921 Ac-(cyclo-1,5)-[EARAK]-NH 2 [SEQ ID NO. I I] [00931 Ac-(cyclo- ,5)-[OARAD]-NH 2 [SEQ ID NO. 12] [00941 Ac-(cyclo-I,5)-[DARAO]-NH 2 [SEQ ID NO. 13] 15 [00951 Ac-cyclo-2,6-R[KLLLD]-NH 2 [SEQ ID NO. 15] [00961 Ac-cyclo-2,6-R[KLALD]-NH 2 [SEQ ID NO. 16] [00971 Ac-cyclo-2,6-R[KLFAD]-NH 2 [SEQ ID NO. 17] 100981 Ac-(cyclo-1,5)-[OARAE]-NH 2 [SEQ ID NO. 18] [00991 Ac-(cyclo-1,5)-[EARAO]-NH 2 [SEQ ID NO. 19] 20 101001 Ac-(cyclo-1,5)-[KARAD]-OH [SEQ ID NO. 20] 101011 H-(cyclo-1,5)-[KARAD]-NH 2 [SEQ ID NO. 21] 10102] H-(cyclo-I,5)-[KARAD]-OH [SEQ ID NO. 22] [0103] Ac-(cyclo-2,6)-R[KAAAD]-NH2 [SEQ ID NO. 23] [0104] Ac-(cyclo-2,6)-R[KA LAD]-N H2 [SEQ ID NO. 24] 25 [01051 Ac-(cyclo-2,6)-R[KAMA DI-N H2 [SEQ ID NO. 25] [01061 Ac-(cyclo-2,6)-R[KAQAD]-NH2 [SEQ ID NO. 26] [01071 Ac-(cyclo-2,6)-R[KAFAD]-NH2 [SEQ ID NO. 27] [0108] Ac-(cyclo-2,6)-R[KAGAD]-NH2 [SEQ ID NO. 28] - 20 - WO 2005/090388 PCT/AU2005/000400 [01091 Ac-(cyclo-2,6)-R[KGSAD]-NH2 [SEQ ID NO. 29] [0110] Ac-(cyclo-2,6)-R[KSSSD]-NH2 [SEQ ID NO. 30] [0111] Ac-(cyclo-2,6)-R[KGGGD]-NH2 [SEQ ID NO. 31] [0112] In some embodiments, the peptide compound comprises at least one cyclic 5 pentapeptide of the invention and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues adjacent thereto. In specific embodiments, the peptide compound comprises a single cyclic pentapeptide of the invention and another amino acid residue located immediately upstream or downstream thereof. [01131 In another aspect, the present invention provides a method for constructing a 10 constrained helical peptide comprising the steps of: (1) synthesizing a peptide, wherein the peptide comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the individual side chains of the first and second terminal residues are linkable to each other; and (2) cyclizing the peptide by linking the side chain of the first terminal residue with the side chain of the 15 second terminal residue, thereby yielding a constrained helical peptide. In certain embodiments, the first terminal residue has a side chain containing an amide bond-forming substituent and the second terminal residue has a side chain containing a functional group capable of forming an amide linkage with the side chain amide bond-forming substituent of the first terminal residue and the peptide is cyclised by reacting the side chain amide bond-forming substituent of the first terminal residue with 20 the functional group of the second terminal residue to form an amide bond linkage, thereby yielding a constrained helical peptide. During peptide synthesis, reactive groups on the side chains, including the amide forming substituents are suitably protected, for example, carboxy groups can be suitably protected as esters such as methyl, ethyl, allyl, benzyl, t-butyl or phenyl esters and amino groups can be suitably protected with alkyloxy carbonyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Z), t 25 butoxycarbonyl (Boc), 2-(4-biphenylyl)-isopropoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), triphenylmethyl (trityl) or 2-nitrophenylsulphenyl (Nps) groups, which may be removed after synthesis of the peptide and before reaction to form the amide bond linkage. Suitable methods for selectively protecting and deprotecting functional groups can be found in Green & Wutz' 4 and Taylor (2002)4". 30 [0114] The peptides of the present invention may be prepared using techniques known in the art. For example, peptides can be synthesized using various solid phase techniques' or using an automated synthesis and standard Fmoc chemistry 92 . These techniques are also suitable for incorporating non-naturally occurring amino acid residues into the amino acid sequence. [0115] Alternatively, non-naturally occurring amino acids may be incorporated into the 35 sequence by manipulation of a residue in the sequence. For example, the hydroxy group or thiol group of threonine, serine or cysteine may be alkylated to provide an ether or thioether, or substituents may -21- WO 2005/090388 PCT/AU2005/000400 be introduced into the phenyl ring of phenylalanine or tyrosine using known substitution reactions such as Friedel-Crafts alkylation or acylation. [0116] Once the peptides of the present invention have been prepared, they may be substantially purified using preparative HPLC. The composition of the peptides can be confirmed by 5 amino acid analysis or by sequencing, for example, using the Edman Degradation procedure. [01171 Suitable protecting groups for use during solid phase synthesis or solution phase of the amino acid sequences, together with suitable protecting and deprotecting methods for reactive functional groups such as amines and carboxylic acids, are known in the art, for example, as found in Green & Wutz' 4 . 10 [0118] Once the peptide is prepared and deprotection of the side chains is effected, cyclization to form a cyclic peptide may be achieved by methods known in the art. For example, an amide bond may be formed between a side chain carboxylic acid and a side chain amine by activation of the carboxylic acid, for example, as an acid chloride, acid anhydride, an acyl azide, a carbodiimide, an acyloxyphosphonium or uronium compound or an active ester, and allowing nucleophilic attack 15 from the amine nitrogen atom. A particularly preferred method of activating the carboxylic acid to nucleophilic attack is preparation of an acyloxyphosphonium or uronium derivative of the carboxylic acid, for example, by reaction with the carboxylic acid with benzotriazolyloxy-tri (dimethylamino)phosphonium hexafluorophosphate (BOP) or benzotriazolyloxy-tris (pyrrolidinyl)phosphonium hexafluorophosphate (Py-BOP) in the presence of a tertiary amine such as 20 triethylamine or diisopropylethylamine (DIPEA) or similar reaction using Benzotriazol-1-yl-1,1,3,3 tetramethyluronium ion (HBTU). [0119] A representative solid phase synthesis is shown in Scheme 1:

H

2 N- Ac-K A R A D-N Alloc Allyl Pd(PPh 3

)

4 NDMBA

CH

3 COOH BOP HDIPEA H~ Ac-K A R A D-NE Ac-KARAD [NH

H

2 N COOH 0 TFA, TIPS

H

2 0 Ac-K A R A D-NH 2

-NH

1 2 - 22 - WO 2005/090388 PCT/AU2005/000400 Scheme 1 [0120] The peptides of the invention are designed to mimic binding determinants from alpha helical binding domains of known proteins. Such peptides have a number of uses, including the determination of whether a binding determinant in an alpha helical binding domain of a known protein 5 can serve as a structural model for the design of peptidomimetics or small molecules capable of mimicking or antagonizing the binding activity of the intact protein. In using the peptides of the invention for this purpose, the practitioner may select a binding protein with an alpha helical domain that interacts with a ligand, and then identify a candidate binding determinant situated within a sequence of (e.g., three or more) contiguous amino acid residues in the helical binding domain. The 10 candidate binding determinant can be identified by using mutagenesis (e.g., alanine scanning mutagenesis) to determine whether the candidate sequence contains one or more amino acid residues that are critical for ligand binding. Subsequently, a constrained peptide containing the candidate sequence is designed by selecting two residues in the candidate sequence (designated i and i+4) which are separated by an intervening sequence of n-1 (e.g., 3) amino acid residues and which do not 15 substantially interact with ligand (as determined by mutagenesis in the previous step) for substitution with amino acid residues having side chains that can be linked to each other. The peptide is synthesized and the side chains of the foreign i and i+4 residues are used to tether the peptide in an alpha helical conformation according to the methods of the invention described herein. Finally, the peptide's binding activity with the ligand is assayed, e.g., in a binding competition assay with the 20 intact binding protein, and the results of the assay can be used to determine whether a peptidomimetic or small molecule antagonist could be developed using the binding determinant as a structural model. [01211 Thus, in a further aspect, the invention contemplates the use an alpha helical cyclic peptide, wherein the peptide comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino 25 acid residues, and wherein the side chains of the first and second terminal residues are linked to each other as a scaffold for presenting the side chains of at least some of the five amino acid residues in a (three dimensional) conformation that is analogous to the conformation of amino acid side chains of at least a portion of an alpha helical domain of a known protein. In some embodiments, the side chains of at least 1 or 2 or all 3 of the intervening amino acid residues are so analogously presented. In other 30 embodiments, the side chains of at least 1 or 2 or all 3 of the intervening amino acid residues and at least'one terminal amino acid residue are so analogously presented. Suitably, at least part of the conformationally constrained secondary structure defined by the five amino acid residues (i.e., pentapeptide) mimics a member of a ligand-receptor binding pair. Illustrative examples of ligand receptor binding pairs include protein-DNA binding partners (e.g., Zif268 and G/C rich major groove), 35 protein-RNA binding partners (e.g., HIV reverse transcriptase and Rev response element (RRE); X-N peptide and BoxB RNA; p22 peptides and BoxB RNA) and protein-protein binding partners (e.g., p53 and HDM2; Bak and Bcl-XL;-VHL peptide and Elongin C; VP16 activation domain and -23 - WO 2005/090388 PCT/AU2005/000400

HTAF

11 3 1; hPTH and hPTHrP; Dynorphin A and ,8-Opioid receptors; Apolipoprotein-E and LDL receptor; Neuropeptide-Y and NPY receptors; Galanin and Gal receptors; Corticotropin Releasing Factor and CRF receptors; Calcitonin Gene Related Peptide and CGRP receptors; Nociceptin and ORL1 receptor; Vasointestinal Peptide and VPACI &2; and Nuclear Coactivators (eg. SRC1, GRIP 1) 5 and Nuclear Receptors. [01221 While not wishing to be limited by any one particular theory or mode of operation, the constrained helical peptides of the present invention are believed to derive their activity by interaction of the face of the helix opposing the i->i+4 constraint. However, when two or more tandemly arrayed constrained helical peptides are present, as part of an extended helix polypeptide 10 backbone or super helix, the positions i-*i+4 of a first constrained helical pentapeptide will be offset by approximately one third of a turn relative to positions i->i+4 of a second constrained helical pentapeptide. In other words, the i->i+4 faces of the two helices will not be aligned directly in the same plane and will be out of register by approximately one third of a turn. Thus, in certain embodiments where an extended helix polypeptide backbone or super helix is required for interaction 15 with a biomolecule of interest, it may be desirable to take this offset into account when designing a helical peptide so that one face of its helix is substantially free of any cyclizing linkages that may occlude or otherwise interfere with this interaction. In illustrative examples, the helical peptide may simply comprise two or three consecutive constrained helical pentapeptides. In other illustrative examples, the helical peptide may comprise two consecutive constrained helical pentapeptides spaced 20 from a third constrained helical pentapeptide by about 1, 2, 5, 8 or 9 natural or unnatural helix-forming amino acid residues. In still other illustrative examples, the helical peptide may comprise three consecutive constrained helical pentapeptides spaced from a fourth constrained helical pentapeptide by about 0, 3, 4, 6 or 7 natural or unnatural helix-forming amino acid residues; or alternatively 1, 2, 5, 6 or 9 natural or unnatural helix-forming amino acid residues, depending on which face is required to be 25 kept substantially free of any cyclizing linkages. In still other illustrative examples, the helical peptide may comprise four consecutive constrained helical pentapeptides spaced from a fifth constrained helical pentapeptide by about 1, 2 or 3 natural or unnatural helix-forming amino acid residues. In still other illustrative examples, the helical peptide may comprise five consecutive constrained helical pentapeptides spaced from a sixth constrained helical pentapeptide by about 2, 7, 12 or 17 natural or 30 unnatural helix-forming amino acid residues. The optimal spacing between cyclic pentapeptide modules is determined on a case-by-case basis and would be readily apparent to a person skilled in the art through simple molecular modeling experiments using commercially available programs (e.g., InsightII) 104 . [0123] In certain embodiments which require mimicking multiple turns of an alpha helical 35 binding domain, the conformationally constrained peptide comprises a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) of pentapeptides as broadly described above. Accordingly, in yet another aspect, the present invention provides the use of a conformationally constrained peptide having a plurality of -24- WO 2005/090388 PCT/AU2005/000400 alpha helical pentapeptide sequences, wherein the pentapeptide sequences comprise a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other, as a scaffold for presenting the side chains of at least 5 some of the amino acid residues of the pentapeptide sequences in a (three-dimensional) configuration that is analogous to the configuration of amino acid side chains of at least a portion of an alpha helical domain of a known protein. [0124] As used herein, the term "scaffold" is used in its broadest sense and includes a region or domain that has a conserved tertiary structural motif that can be modified to display one or 10 more specific amino acid residues in a fixed conformation. [0125] In some embodiments, the side chains of at least 1 or 2 or all 3 of the intervening amino acid residues of each pentapeptide sequence are so analogously presented. In other embodiments, the side chains of at least 1 or 2 or all 3 of the intervening amino acid residues and at least one terminal amino acid residue of each pentapeptide sequence are so analogously presented. 15 Suitably, at least part of the conformationally constrained secondary structure defined by the pentapeptide sequences mimics a member of a ligand-receptor binding pair. In illustrative examples, some or all of the pentapeptides are located adjacent to each other. Alternatively, at least one of the pentapeptides is spaced from a pair of adjacent pentapeptides. [0126] In certain embodiments, the conformationally constrained peptides of the invention 20 are designed to mimic epitopes in proteins and are used to selectively raise polyclonal or monoclonal antibodies against such individual epitopes. Since the peptides will frequently be too small to generate an immune response, the peptides can be conjugated to carriers known to be immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatising agent, for example, maleimidobenzoyl 25 sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R' N=C=NR, where R and R 1 are different alkyl groups. [01271 Advantageously, the macrocyclic moiety of the pentapeptide is stable in water to temperatures of up to about 80*C and stable to denaturants such as 8M guanidine hydrochloride, and 30 to the degradative effects of proteolytic enzymes such as trypsin or those present in human serum. The alpha helical short-chain peptides are therefore suitable for use as chemical or biological probes, pharmaceuticals, biotechnology products such as vaccines or diagnostic agents, new components of novel biopolymers and as industrial agents. [0128] The alpha helical pentapeptides of the invention can be used alone to mimic a 35 specific peptide motif of a protein or polypeptide or may be incorporated into a larger polymeric or non polymeric non-peptidic molecules or into hybrids of peptidic and non-peptidic components. -25- WO 2005/090388 PCT/AU2005/000400 [01291 In another aspect of the present invention there is provided a use of at least one alpha helical cyclic peptide, wherein the peptide comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal 5 residues are linked to each other, as a macrocyclic module for incorporation into a non-peptidic molecular structure, or for constructing a multi-macrocyclic structure that mimics multiple turns of an alpha helix. [0130] Multi-macrocyclic structures may provide new or unknown three dimensional positioning of side chains in an alpha helix or may mimic a portion of, or an entire, alpha helical motif 10 from a known protein or polypeptide. [0131] In a preferred embodiment, the alpha helical cyclic peptide, which is used as the scaffold or macrocyclic module, has the formula (II): '(II) (CR'R')m- L (CR'R')n _ H __ _,_ H I

R

3 -N-C(R)-C-Xaa-Xaa-XaaN-C(R")-C-R 4 15 [0132] wherein each Xaa is independently selected from any amino acid; [0133] each R' and R" are independently selected from H, CI-Cio alkyl, C2-CI alkenyl, C2 C10 alkynyl, C 3 -Ciocylcoalkyl, C 5

-C

1 Ocycloalkenyl, -OH, -OC1-C10 alkyl, -NH 2 , -NH(C 1 -Cioalkyl), N(C 1

-C

1 oalkyl) 2 , C 6 -Cioaryl, C 3

-C

1 oheterocyclyl, C 5 -Cioheteroaryl and halo; 20 [0134] L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH 2 -, CH 2 CH(OH)-, CH=CH-, -CH 2

-CH

2 -, -NH-CH 2 - -CH 2 -NH-, -CH 2 -S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 -C(O)-, -S(O)-NH-, -NH-S(O)-, CH 2 -P(=O)(OH)- and -P(=O)(OH)-CH 2 -; [0135] R 3 is selected from H, an N-capping group or a mimic of an amino acid side chain, [0136] R 4 is selected from H, a C-terminal capping group, a mimic of an amino acid side 25 chain or a group which activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; [0137] m is an integer from 1 to 4, [0138] n is an integer from 1 to 4, and [0139] t is 0, 1 or 2, -26 - WO 2005/090388 PCT/AU2005/000400 [01401 wherein m +n = 4, 5 or 6 and, wherein when m is 2, n is not 3 and when m is 3, n is not 2. [0141] In preferred embodiments, any one of the following may apply: [0142] R 1 is selected from H, an N-terminal capping group that stabilizes the terminus of a 5 helix, usually having hydrogen atoms able to form hydrogen bonds or having a negative charge at the N-terminus to match with the helix dipole, or a mimic of an amino acid side chain. Suitable N terminal capping groups include acyl and N-succinate. Suitable groups that mimic an amino acid side chain are any natural or unnatural amino acid side chain that is attached to the N-terminal amino group of the peptide through a carbonyl group derived from a carboxylic acid by formation of an amide 10 bond. Suitable mimics of amino acid side chains include, but are not limited to: [0143] CH 3

CH

2

C(O)(CH

2 )uC(O)-, NH 2

(NH=)CNHC(O)(CH

2 )uC(O)-,

H

2

NC(O)(CH

2

)

2

C(O)(CH

2 )uC(O)-, HOC(O)(CH 2

)

2

C(O)(CH

2 )uC(O)-, HS(CH 2

)

2

C(O)(CH

2 )uC(O)-,

H

2

NC(O)(CH

2

)

3

C(O)(CH

2 )uC(O)-, HOC(O)(CH 2

)

2

C(O)(CH

2 )uC(O)-, (4 imidazolyl)(CH 2

)C(O)(CH

2 )uC(O)-, CH 3

CH

2

CH(CH

3

)CH

2

C(O)(CH

2 )uC(O)-, 15 (CH 3

)

2

CH(CH

2

)

2

C(O)(CH

2 )uC(O)-, H 2

N(CH

2

)

5

C(O)(CH

2 )uC(Q)-, CH 3

S(CH

2

)

3

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

2

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

4

C(O)(CH

2 )uC(O)-, HO(CH 2

)

2

C(O)(CH

2 )uC(O)-,

HOCH(CH

3

)CH

2

C(O)(CH

2 )uC(O)-, (3-indoly)(CH 2

)

2

(CH

2 )uC(O)-, (4 hydroxyphenyl)(CH 2

)

2

C(O)(CH

2 )uC(O)-, (4-hydroxyphenyl)(CH 2

)

3

C(O)(CH

2 )uC(O)-,

(CH

3

)

2

CHCH

2

C(O)(CH

2 )uC(O)-, CH 3

CH

2

CH

2

C(O)(CH

2 )uC(O)-, C 6

H

1 oCH 2

C(O)(CH

2 )uC(O)-, 20 C 5

HSCH

2

C(O)(CH

2 )uC(O)-, CH 3

C(O)(CH

2 )uC(O)-, CH 3

(CH

2

)

4

C(O)(CH

2 )uC(O)-,

CH

3

(CH

2 )sC(O)(CH 2 )uC(O)-, HOC(O)CH 2

C(O)(CH

2 )uC(Q)-, HS(CH 2

)C(O)(CH

2 )uC(O)-,

H

2

N(CH

2

)

4

C(O)(CH

2 ).C(O)- and HOCH 2

C(O)(CH

2 )uC(O)- wherein u is 0 or an integer from 1 to 10; [0144] R 2 is selected from H, a C-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a positive charge at the 25 C-terminus to match with the helix dipole, a mimic of an amino acid side chain or a group which activates the terminal carboxylic acid carbonyl group to nucleophilic substitution. 'A suitable C terminal capping group is NH 2 . Suitable mimics of amino acid side chains are any common or unnatural amino acid side chain that is attached to the C-terminal carbonyl group of the peptide through an amine group by formation of an amide bond. Suitable mimics of amino acid side chains 30 include but are not limited to: [0145] -NH(CH 2 )uNHCH 2

CH

3 , -NH(CH 2 )uNH(CH 2

)

4

NHC(=NH)NH

2 , NH(CH 2 )uNH(CH 2

)

2

C(O)NH

2 , -NH(CH 2 )uNH(CH 2

)

2

CO

2 H, -NH(CH 2 )uNH(CH 2

)

2 SH, NH(CH 2 )uNH(CH 2

)

3

C(O)NH

2 , -NH(CH 2

)UNH(CH

2

)

3

CO

2 H, -NH(CH 2 )uNH(CH 2

)

2 (4-imidazolyl), NH(CH 2 )uNHCH 2

CH(CH

3

)CH

2

CH

3 , -NH(CH 2 )uNH(CH 2

)

2

CH(CH

3

)

2 , -NH(CH 2 )uNH(CH 2 )sNH 2 , 35 NH(CH 2 )uNH(CH 3

)

3

SCH

3 , -NH(CH 2 )uNH(CH 2

)

2 (3-indolyl), -NH(CH 2 )uNH(CH 2

)

2 (4-hydroxyphenyl),

-NH(CH

2 )uNH(CH 2

)

3 (4-hydroxyphenyl), -NH(CH 2 )uNHCH 2

CH(CH

3

)

2 , -NIH(CH 2 )uNHCH 2

CH

2

CH

3 , -27 - WO 2005/090388 PCT/AU2005/000400

NH(CH

2 )uNHCH 2

CH

10 , -NH(CH 2 )uNHCH 2

C

5

H

8 , -NH(CH 2

)INHCH

3 , -NH(CH 2 )uNH(CH 2

)

4

CH

3 , NH(CH 2 )uNH(CH 2

)

5

CH

3 , -NH(CH 2 )uNHCH 2

CO

2 H, -NH(CH 2 )uNHCH 2 SH, -NH(CH 2 )uNH(CH 2

)

2 OH,

-NH(CH

2 )uNH(CH 2

)

5

NH

2 and -NH(CH 2 )uNHCH 2 OH; wherein u is 0 or an integer from 1 to 10. [01461 Suitable groups which activate the C-terminal carboxylic to nucleophilic attack 5 include converting the carboxylic acid to an acid chloride, an acid anhydride, an acyl azide, an 0 acylisourea, a phosphonium derivative or an activated ester, especially those known in the art for activating carboxylic acids for peptide bond formation; [0147] Each R' is selected from H, CH 3 , CH 2

CH

3 , vinyl, OH, OCH 3 , NH 2 , NH(CH 3 ),

N(CH

3

)

2 , phenyl, F or Cl; most preferably H or CH 3 , especially H; 10 [0148] Each R" is selected from H, CH 3 , CH 2

CH

3 or vinyl, especially H; [0149] m is 1 and n is 3 or 4, m is 2 and n is 4, m is 3 and n is 1 or m is 4 and n is 1 or 2, especially where m is 1 and n is 4; [0150] Each Xaa may be any amino acid residue selected to mimic the amino acid residues in a known alpha helical peptide of interest or to prepare an unknown peptide having new properties. 15 Xaa is preferably a D- or L- alpha amino acid residue. Especially preferred peptides of formula (II) have at least one Xaa which is a D- or L- alpha amino acid residue that is favorable to helix formation. Even more preferred are peptides in which 2 or 3 of Xaa are D- or L- alpha amino acid residues that are favourable to helix formation, for example, alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid, 20 especially alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine; and [01511 L is selected from -NH-C(O)- and -C(O)-NH-. [0152] Scaffolds or macrocyclic modules of formula (II) can be prepared as described for peptides of formula (I). [0153] N-terminal capping groups may and groups which mimic -an amino acid side chain 25 may be introduced by methods kmown in the art. For example the N-terminal amino group may be reacted with a carboxylic acid derivative of the capping group or mimic or an activated carboxylic acid derivative to form an amide bond. [0154] C-terminal capping groups and groups which mimic an amino acid side chain may be introduced by methods known in the art. For example the C-terminal carboxylic acid may be 30 activated and reacted with an amine derivative, preferably a primary amine derivative of the C terminal capping group or group that mimics an amino acid side chain. [0155] C-terminal carboxylic acid groups or any other carboxylic acid groups that require activation toward nucleophilic substitution can be activated by methods known in the art 5 . For example the carboxylic acid may be activated by conversion to an acyl chloride using PC1 5 or SOCl 2 , 35 conversion to an acyl azide by hydrazinolysis of a protected amino acid or peptide ester followed by -28- WO 2005/090388 PCT/AU2005/000400 treatment with NaNO 2 in aqueous acid, conversion to a symmetrical or mixed anhydride using two equivalents of an amino acid and a dicyclohexylcarbodiimide or by reaction with an acid chloride in a dry solvent in the presence of a mild base, conversion to an O-acylisourea by reaction with dicyclohexylcarbodiimide or by conversion to an acyloxyphosphonium or uronium species by reacting 5 a carboxylate anion with a phosphonium or uronium cation, for example, BOP, PyBOP or HBTU. [0156] A representative example of an alpha helical pentapeptide as a scaffold for projecting attached substituents into positions normally occupied the side chains of longer peptides than pentapeptides is given by formula (III): 0 00 N NH 10 (III) [0157] The pentapeptide of formula (III) is an example of a peptide of formula (II) in which the three variable amino acid residues that represent Xaa are all alanine, the macrocycle is formed by amide bond formation between a lysine residue and an aspartic acid residue, R 3 is an amide formed from the reaction of phenylbutanoic acid and the N-terminal amino group and mimics a 15 phenylalanine side chain, and R 4 is an amide formed by the reaction of isobutyl amine with an activated C-terminal carboxylic acid and mimics a valine side chain. [0158] The scaffold or macrocyclic module may also be incorporated into a multi macrocyclic structure or may be incorporated into a non-peptidic molecule. [0159] As used herein, the term "macrocyclic module" refers to a cyclic pentapeptide 20 which may be unsubstituted at the N and C termini or may be activated for incorporation into a larger structure. For example, a pentacyclic peptide of formula II in which R 3 is H and R 4 is H or a group which activates the terminal carboxylic acid carbonyl group to nucleophilic substitution is a macrocyclic module. [0160] Preparation of a non-peptidic molecule incorporating a scaffold or macrocyclic 25 module may be prepared by reacting the N-terminal and/or activated C-terminal of the macrocyclic module with desired non-peptidic moieties. [0161] Alternatively, a number of modules, which may be the same or different, may be prepared as described herein and then consecutively linked to form a multi-macrocyclic peptide that -29- WO 2005/090388 PCT/AU2005/000400 mimics a number of turns of an alpha helix. The multi-macrocyclic peptide may then be used to mimic a protein or polypeptide or part thereof, or may be incorporated into a longer peptide sequence. [01621 Accordingly, in a further aspect of the invention there is provided a conformationally constrained peptide having a plurality of alpha helical pentapeptide sequences, 5 wherein the pentapeptide sequences comprise a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other. [0163] In a preferred embodiment at least one of the alpha helical pentapeptide sequences 10 is a pentapeptide module of formula (II). [0164] The number of macrocyclic modules in the peptide or polypeptide will depend on the length of the alpha helical portion of the polypeptide required. If the peptide is intended to mimic an alpha helical portion of a known protein or polypeptide, the number of macrocyclic modules will be determined by the number of turns in the alpha helical portion of the known protein or polypeptide. 15 For example, two cyclic pentapeptide modules of Formula (II) could be linked such that the N terminal nitrogen atom is directly bonded to the C-terminal carbonyl group, to form a 2.8-turn alpha helix. In a similar manner, three consecutively linked cyclic pentapeptide modules would form a 4.2 turn alpha helix, four consecutively linked cyclic pentapeptide modules would form a 5.6-turn alpha helix, five consecutively linked cyclic pentapeptide modules would form a 6.9-turn alpha helix, six 20 consecutively linked cyclic pentapeptide modules would form a 8.3-turn alpha helix and larger alpha. helices may be obtained in a similar fashion. In this manner multi-macrocyclic assemblies which are alpha helical in nature can be obtained. [0165] In a preferred embodiment the conformationally constrained peptide having a plurality of alpha helical pentapeptide sequences, is a compound of formula (IV): 25 (IV) |(CR'Rk)m- L (CR'R')n H H

R

1 -- N- C(R")-C- Xaa- Xaa- Xaa-N--C(R")- C--R2 O O - .. p [0166] wherein each Xaa is independently selected from any amino acid residue; -30- WO 2005/090388 PCT/AU2005/000400 [0167] R 1 is selected from H, an N-terminal capping group, a peptide of 1 to 20 amino acid residues optionally capped by an N-terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain; [0168] R 2 is selected from H, a C-terminal capping group, a peptide of 1 to 20 amino acids 5 optionally capped by a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; [01691 each R' and R" are independently selected from H, C 1

-C

10 alkyl, C 2

-C

10 alkenyl, C 2 C 10 alkynyl, C 3 -Ciocylcoalkyl, Cs-C 1 ocycloalkenyl, -OH, -OC 1 -C0 alkyl, -NH 2 , -NH(C 1 -Coalkyl), N(CI-Cioalkyl) 2 , C 6 -Cioaryl, C 3

-C

1 oheterocyclyl, C 5 -Cioheteroaryl and halo; 10 [0170] L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH 2 -, CH 2 CH(OH)-, CH=CH-, -CH 2

-CH

2 -, -NH-CH 2 - -CH 2 -NH-, -CH 2 -S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 -C(O)-, -S(O)t-NH-, -NH-S(O)t-, ICH 2 -P(=O)(OH)- and -P(=O)(OH)-CH 2 -; [0171] m is an integer from 1 to 4, [0172] n is an integer from 1 to 4, and 15 [0173] t is 0, 1 or 2, [0174] wherein m + n = 4, 5 or 6 and wherein when m is 2, n is not 3 and when m is 3, n is not2; and [0175] p is an integer from 2 to 12; with the proviso that bicyclo (Lys - Asp 7 , Lys 18 Asp 22 ) [Ala', Nlc 8 , Lys 8 , Asp 22 , Leu 27 ] hPTH (1-31) NH 2 is excluded. 20 [0176] In preferred embodiments, any one of the following may apply: [0177] R 1 is selected from H, an N-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a negative charge at the N-terminus to match with the helix dipole, a peptide of 1 to 15, 1 to 10 or 1 to 5 amino acid residues optionally capped with an N-terminal capping group that stabilizes the terminus of a helix, usually 25 having hydrogen atoms able to form hydrogen bonds or having a negative charge at the N-terminus to match with the helix dipole, or a mimic of an amino acid side chain. Suitable N-terminal capping groups include acyl and N-succinate. Suitable groups that mimic an amino acid side chain are any natural or unnatural amino acid side chain that is attached to the N-terminal amino group of the peptide through a carbonyl group derived from a carboxylic acid by formation of an amide bond. 30 Suitable mimics of amino acid side chains include, but are not limited to: [0178] CH 3

CH

2 C(0)(CH 2 )uC(0)-, NH 2

(NH=)CNHC(O)(CH

2 )uC(O)-,

H

2

NC(O)(CH

2

)

2

C(O)(CH

2 )uC(O)-, HOC(O)(CH 2

)

2

C(O)(CH

2 ).C(O)-, HS(CH 2

)

2

C(O)(CH

2 )uC(O)-, H2NC(O)(CH2)3C(O)(CH2)uC(O)-, HOC(0)(CH2)2C(O)(CH2)uC(O)-, (4 imidazolyl)(CH2)C(O)(CH2)uC(O)-, CH3CH2CH(CH3)C H2C(O)(CH2)uC(O)-, - 31 - WO 2005/090388 PCT/AU2005/000400

(CH

3

)

2

CH(CH

2

)

2

C(O)(CH

2 )uC(O)-, H 2

N(CH

2

)

5

C(O)(CH

2 )uC(O)-, CH 3

S(CH

2

)

3

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

2

C(O)(CH

2 )uC(O)-, Ph(CH 2

)

4

C(O)(CH

2 )uC(O)-, HO(CH 2

)

2

C(O)(CH

2 )uC(O)-,

HOCH(CH

3

)CH

2

C(O)(CH

2 )uC(O)-, (3-indolyl)(CH 2

)

2

(CH

2 )uC(O)-, (4 hydroxyphenyl)(CH 2

)

2

C(O)(CH

2 )uC(O)-, (4-hydroxyphenyl)(CH 2

)

3

C(O)(CH

2 )uC(O)-, 5 (CH 3

)

2

CHCH

2

C(O)(CH

2 )uC(O)-, CH 3

CH

2

CH

2

C(O)(CH

2 )uC(O)-, C 6

HOCH

2

C(O)(CH

2 )uC(O)-,

C

5 HsCH 2

C(O)(CH

2 )uC(O)-, CH 3

C(O)(CH

2 )uC(O)-, CH 3

(CH

2

)

4

C(O)(CH

2 )uC(O)-,

CH

3

(CH

2

)

5

C(O)(CH

2 )uC(O)-, HOC(O)CH 2

C(O)(CH

2 )uC(O)-, HS(CH 2

)C(O)(CH

2 )uC(O)-,

H

2

N(CH

2

)

4

C(O)(CH

2 )uC(O)- and HOCH 2

C(O)(CH

2 )uC(O)- wherein u is 0 or an integer from 1 to 10. The preferred non-peptidic groups enhance the stability, bioavailability or activity of the peptides. 10 Suitable non-peptidic groups include, but are not limited to hydrophobic groups such as carbobenzoxyl, dansyl, t-butyloxycarbonyl, acetyl, 9-fluorenylmethoxycarbonyl, groups which stabilize or mimic alpha-helices, groups which mimic the secondary structure of peptides, particularly alpha helical peptides, such as those disclosed in WO 03/018587, groups which improve bioavailability, such as hydrophilic groups which aid aqueous solubility, for example, cyclodextrans; 15 groups which are recognized by transport receptors to allow or improve transport of the peptides to the site of activity, for example, transport across cell walls or through an epithelial layer such as skin or the gut wall; [0179] R 2 is selected from H, a C-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a positive charge at the 20 C-terminus to match with the helix dipole, a peptide of 1 to 15, 1 to 10 or 1 to 5 amino acid residues optionally capped with a C-terminal capping group that stabilizes the terminus of a helix, usually having hydrogen atoms able to form hydrogen bonds or having a positive charge at the C-terminus to match with the helix dipole, a mimic of an amino acid side chain or a group which activates the terminal carboxylic acid carbonyl group to nucleophilic substitution. A suitable C-terminal capping 25 group' is NH 2 . Suitable mimics of amino acid side chains are any common or unnatural amino acid side chain that is attached to the C-terminal carbonyl group of the peptide through an amine group by formation of an amide bond. Suitable mimics of amino- acid side chains include but are not limited to: [0180] -NH(CH 2 )uNHCH 2

CH

3 , -NH(CH 2 )uNH(CH 2

)

4

NHC(=NH)NH

2 , NH(CH 2 )uNH(CH 2

)

2

C(O)NH

2 , -NH(CH 2 )uNH(CH 2

)

2

CO

2 H, -NH(CH 2 )uNH(CH 2

)

2 SH, 30 NH(CH 2 )uNH(CH 2

)

3

C(O)NH

2 , -NH(CH 2 )uNH(CH 2

)

3

CO

2 H, -NH(CH 2 )uNH(CH 2

)

2 (4 imidazolyl), -NH(CH 2 )uNHCH 2

CH(CH

3

)CH

2

CH

3 , -NH(CH 2 )uNH(CH 2

)

2

CH(CH

3

)

2 , NH(CH 2 )uNH(CH 2 )sNH 2 , -NH(CH 2 )uNH(CH 3

)

3

SCH

3 , -NH(CH 2 )uNH(CH 2

)

2 (3-indolyl), NH(CH 2 )uNH(CH 2

)

2 (4-hydroxyphenyl), -NH(CH 2 )uNH(CH 2

)

3 (4-hydroxyphenyl), NH(CH 2 )uNHCH 2

CH(CH

3

)

2 , -NH(CH 2 )uNHCH 2

CH

2

CH

3 , -NH(CH 2 )uNHCH 2

C

6

H

0 , 35 NH(CH 2 )uNHCH 2

C

5

H

8 , -NH(CH 2 )uNHCH 3 , -NH(CH 2 )uNH(CH 2

)

4

CH

3 , NH(CH 2 )uNH(CH 2

)

5

CH

3 , -NH(CH 2 )uNHCH 2

CO

2 H, -NH(CH 2 )uNHCH 2 SH, -32- WO 2005/090388 PCT/AU2005/000400

NH(CH

2 )uNH(CH 2

)

2 OH, -NH(CH 2 )uNH(CH 2

)

5

NH

2 and -NH(CH 2 )uNHCH 2 OH; wherein u is 0 or an integer from I to 10. [01811 Suitable groups which activate the C-terminal carboxylic to nucleophilic attack include converting the carboxylic acid to an acid chloride, an acid anhydride, an acyl azide, an 0 5 acylisourea, a phosphonium derivative or an activated ester, especially those known in the art for activating carboxylic acids for peptide bond formation; [0182] The preferred non-peptidic groups enhance the stability, bioavailability or activity of the peptides. Suitable non-peptidic groups include but are not limited to hydrophobic groups such as t-butyl, groups which stabilize or mimic alpha-helices, groups which mimic the secondary structure of 10 peptides, particularly alpha helical peptides, such as those disclosed in WO 03/018587, groups which improve bioavailability, such as hydrophilic groups which aid aqueous solubility, for example, cyclodextrans; groups which are recognized by transport receptors to allow or improve transport of the peptides to the site of activity, for example, transport across cell walls or through an epithelial layer such as skin or the gut wall; 15 [01831 Each R' is selected from H, CH 3 , CH 2

CH

3 , vinyl, OH, OCH 3 , NH 2 , NH(CH 3 ),

N(CH

3

)

2 , phenyl, F or Cl; most preferably H or CH 3 ,.especially H; [0184] Each R" is selected from H, CH 3 , CH 2

CH

3 or vinyl, especially H; [01851 m is 1 and n is 3 or 4, m is 2 and n is 4, m is 3 and n is 1 or m is 4 and n is 1 or 2, especially where m is 1 and n is 4; 20 [01861 Each Xaa may be any amino acid residue selected to mimic the amino acid residues in a known alpha helical peptide of interest or to prepare an unknown peptide having new properties. Xaa is preferably a D- or L- alpha amino acid residue. Especially preferred peptides of formula (IV) have at least one Xaa which is a D- or L- alpha amino acid residue that is favorable to helix formation. Even more preferred are peptides in which 2 or 3 of Xaa are D- or L- alpha amino acid residues that 25 are favorable to helix formation, for example, alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid, especially alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine; [0187] L is -NH-C(O)- or -C(O)-NH-; [01881 Preferably p is selected to provide the appropriate number of turns in the alpha 30 helix. Especially preferred are those peptides where p is 2 to 11, 2 to 10, 2 to 9 or 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, especially 2 to 5. [0189] Preferred peptides containing more than one consecutive macrocyclic module include those of formula (V): 35 R 1 -[1,5-cyclo(Zaa-XaaXaaXaa-Yaa)]q-R 2 (V) -33 - WO 2005/090388 PCT/AU2005/000400 [01901 wherein each 1,5-cyclo(Zaa-XaaXaaXaa-Yaa) is independently selected from: [01911 cyclo-1,5-KXaaXaaXaaD [SEQ ID NO: 32] [0192] cyclo-1,5-DXaaXaaXaaK [SEQ ID NO: 33] 5 [0193] cyclo-1,5-KXaaXaaXaaE [SEQ ID NO: 34] [0194] cyclo-1,5-EXaaXaaXaaK [SEQ ID NO: 35] [0195] cyclo-1,5-OXaaXaaXaaD [SEQ ID NO: 36 ]and [0196] cyclo-1,5-DXaaXaaXaaO [SEQ ID NO: 37] [0197] q is an integer from 2 to 12 and R 1 and R 2 are as defined above. 10 [00101] Illustrative examples of 1,5-cyclo(Zaa-XaaXaaXaa-Yaa) sequences include: [00102] cyclo-1,5-KARAD [SEQ ID NO: 38] [00103] cyclo-1,5-DARAK [SEQ ID NO: 39] [00104] cyclo-1,5-KARAE [SEQ ID NO: 40] [00105] cyclo-1,5-EARAK [SEQ ID NO: 41] 15 [00106] cyclo-1,5-OARAD [SEQ ID NO: 42] [00107] cyclo-1,5-DARAO [SEQ ID NO: 43] [00108] cyclo-1,5-KAAAD [SEQ ID NO: 44] and [00109] cyclo-1,5-KGSAD [SEQ ID NO: 45]. [00110] In another embodiment, individual macrocyclic modules in the peptide are 20 different. [0198] In yet another embodiment, individual macrocyclic modules in the peptide are the same. [0199] Examples of peptides containing more than one consecutive cyclic pentapeptide module which are very stable alpha helices in water include: 25 [0200] cyclo(1-5, 6-10)-Ac-[KARADKARAD]-NH 2 [SEQ ID NO: 46] and [0201] cyclo(1-5, 6-10, 11-15)-Ac-[KARADKARADKARAD]-N1 2 [SEQ ID NO: 47]. [0202] Peptides comprising more than one macrocyclic module can be prepared by conventional solid phase synthesis as described for single macrocycles above, where cyclization occurs while the peptide is still attached to the solid phase resin by incorporation of amino acid 30 residues with suitably protected side chains such as allyl protected aspartic acid or Alloc protected lysine, deprotection and cyclization. Further amino acid residues may be added to the resin bound -34- WO 2005/090388 PCT/AU2005/000400 macrocycle including other amino acid residues with suitable protected side chains, after the addition of five further amino acids, further cyclization may be effected to provide two consecutively linked macrocycles. This may be continued until the desired number of macrocycles is present and then the peptide can be cleaved from the resin. 5 [0203] Alternatively, a single cyclic macrocyclic module may be prepared using solid phase synthesis as hereinbefore described. The single macrocyclic module may be cleaved from the resin and undergo either N-terminal protection or deprotection or C-terminal protection or deprotection. A macrocycle having N-terminal protection and a macrocycle having C-terminal protection may then be reacted with one another by activating the unprotected carboxylic acid to 10 nucleophilic attack by the unprotected amine nitrogen, to provide a multi-macrocyclic structure. Further N-terminal and/or C-terminal protection and deprotection of a single macrocyclic module and a multi-macrocyclic module followed by coupling will allow the preparation of a multi-macrocyclic peptide. [0204] Two macrocyclic modules may be coupled using conventional peptide coupling 15 chemistry. For example, the C-terminal carboxylic acid may be activated by formation of an acid chloride, acid anhydride, an acyl azide, a carbodiimide, an acyloxyphosphonium compound or an active ester, and allowing nucleophilic attack from the N-terminal nitrogen atom. A particularly preferred method of activating the carboxylic acid to nucleophilic attack is preparation of an acyloxyphosphonium derivative of the carboxylic acid, for example, by reaction with the carboxylic 20 acid with BOP, Py-BOP or HBTU in the presence of a tertiary amine such as triethylamine or diisopropylethylamine. [02051 A representative synthesis of a multi-macrocyclic peptide where each macrocyclic module is the same is shown in Scheme 2. -35- WO 2005/090388 PCT/AU2005/000400 1. Fmoc SPPS 2. Pd(0), Et 2 NHF ] 3. Piperidine Boc-KARAD 4. BOP, DIPEA R = CI (TCP) 95% TFA

NH

2 (RAM) 1% TFA (RAM) (TCP) . Boc-KARAD-OH

H-KARAD-NH

2 BOP, DIPEA TFA | | Boc-KARAD-KARAD-NH 2 1. BOP, DIPEA 2. TFA 1.TFA 3. AcOH, HBTU 2. AcOH, HBTU Ac-KARAD-KARAD-KARAD-NH 2 Ac-KARAD-KARAD-NH 2 Scheme 2 [0206] The helically constrained peptides described herein can be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the 5 -stability, bioavailability, and/or inhibitory activity of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups, may be added to the amino termini. An acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the amino termini. A hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to carboxy termini. Furthermore, the peptides of the invention can be synthesized such that their steric 10 configuration is altered. For example, the D-isomer of one or more of the amino acid residues of the peptide can be used, rather than the usual L-isomer. The compounds can contain at least one bond linking adjacent amino acids that is a non-peptide bond, and is preferably not helix breaking. Non peptide bonds for use in flanking sequences include an imino, ester, hydrazine, semicarbazide, oxime, or azo bond. Still further, at least one of the amino acid residues of the peptides of the invention can be 15 substituted by one of the well known non-naturally occurring amino acid residues, that is preferably not helix breaking. Desirably, the non-natural amino acid or non-amide bond linking adjacent amino acids, when present, is present outside of the internal sequence, and is, preferably, not helix breaking. Still further, at least one of the amino acid residues of the peptides of the invention can be substituted by one of the well known non-naturally occurring amino acid residues. Alterations such as these can 20 serve to increase the stability, bioavailability, immunogenicity, and/or inhibitory action of the peptides of the invention. [0207] A representative example of alpha helical cyclic pentapeptides incorporated in a modular fashion into biologically active sequences is described. The opioid receptor-like 1 (ORL-1) is -36- WO 2005/090388 PCT/AU2005/000400 the most recently identified member of the opioid receptor family 1. Unlike the other three types of opioid receptor (pi, 5, K), the ORL-1 receptor does not display affinity for the naturally occurring opioid peptide ligands or for many synthetic opiates that selectively bind p-, 6-, x-receptors 7. In 1995 the endogenous ligand for the ORL-1 receptor was identified and called nociceptin (NC). Like 5 other opioid receptor peptide ligands nociceptin consists of an N-terminal tetrapeptide which is referred to as the "message" sequence and is primarily responsible for triggering stimulation of the receptor, whilst the remaining C-terminal portion is referred to as the "address" sequence and is involved in binding and receptor specificity 13 7 .

H

2 N-FGGFTGARKSARKLANQ-COOH "Address" "Message" SEQ ID NO: 48 10 [0208] Recent NMR structures of NC and related peptides revealed a highly helical structure in the address domain and suggested amphipathicity 9

.

1 39 . Another recent report successfully substituted Aib residues into the NC address sequence resulting in increased potency and affinity in 13-residue peptide sequences. Structure-activity relationship (SAR) studies suggest the minimal sequence is NC 1-13. An alanine scan showed the first five residues (FGGFT) are critical, whilst G6 15 and A7 appear to tolerate substitution, R8 is highly crucial, whilst the remaining residues are necessary but tolerate alanine substitution 1". Another recent report identified a pure, selective peptide antagonist of the ORL-1 receptor which involved replacing the first residue in the native sequence with Nphe 1 37 . [0209] Since the present invention establishes a general method for constraining short 20 peptides into alpha helical conformations, nociceptin is an ideal target to show that constraining biologically important helices into an alpha helical conformation can improve activity and affinity. Thus the peptides of SEQ ID NOs: 49 to 51 were designed using the available SAR. The peptide of SEQ ID NO: 49 is designed to be a nociceptin mimetic for agonism, whilst the peptide of SEQ ID NO: 50 is based on the recently reported antagonist [Nphel]NC (1-15). The peptide of SEQ ID NO: 51 25 consists of just the address sequence and the inventors consider that if this peptide has sufficiently high affinity for the receptor it may function as an antagonist. There are no studies to date on peptides incorporating only the address sequence. -37- +H2NO HN H Q NH2 Phe H2 NH 2 FGFT[1,5-cycl(KARKD)][1,5-cyclo(KRKLD)]-NH 2 SEQ ID NO: 49 9 iH 2 N OH2 O NH 2 PhCH 2 -GGGFT[1,.5-cyclo(KARKD)][1 ,5-cyclo(KRKLD)]-NH 2 SEQ ID NO: 50 m NH2H HH 0 N( NH 2

NH

2 0 Ac-T-[1,5-cyclo(KARKD)][1,5-cyclo(KRKLD)]-NH 2 SEQ ID NO: 51 - 38 - WO 2005/090388 PCT/AU2005/000400 [0210] The reaction scheme for the synthesis of the Nociceptin mimetics of SEQ ID NOs: 49 to 51 is shown in Scheme 3. The 2-hydroxy-4-methoxybenzyl protecting group is used during synthesis of multi-macrocyclic compounds, however this group is removed during deprotection and cleavage of the peptides from the resin. 5 SPIPS H2N- ON.S Fmoc-KRKLD Alloc Allyl 1. Allyl Deprotection Hmb 2. Cyclisation HO [ KRKLI--j .introduction F 1~RK D-~ ~Fmoc-KRKLID OMe SPPDS 1. Allyl Deprotection 2. Cyclisation Alloc Allyl I 1I7 Fmo HRK KRKLD Fmoc-KARK KRKLD HOHO~ OMe OMe 1. SPPS 2. Cleavage SEQ ID NOs: 49-51 Scheme 3 [0211] The present invention also provides compositions which comprise one or more compounds of the invention. The compounds themselves may be present in the compositions in any of 10 a wide variety of forms. For example, two or more compounds may be merely mixed together or may be more closely associated through complexation, crystallization, or ionic or covalent bonding. [0212] Those of skill in the art will appreciate that a wide variety of prophylactic, diagnostic, and therapeutic treatments may be prepared from the compounds and compositions of the present invention, due in large part to the cross-reactivity - i.e., agonism or antagonism - of the 15 macrocyclic moieties of the compounds with one or more naturally-occurring peptides or polypeptides. Thus, a compound of the present invention finds utility as a molecular mimic or antagonist of a member of a ligand-receptor binding pair that underlies or is otherwise associated with -39- WO 2005/090388 PCT/AU2005/000400 the development of a particular disease or condition, wherein the ligand-receptor interaction is mediated at least in part by one or more alpha helical motifs present in the ligand or the receptor. Accordingly, in some embodiments, a compound of the present invention having one or more macrocyclic moieties that antagonize the interaction of a ligand and a receptor will be useful in the 5 prevention or treatment of a disease or condition that results from inappropriate activation of the receptor by the ligand. In other embodiments, a disease or condition may arise through inadequate activation of a receptor, in which case the disease or condition may be treated or prevented by means of a compound having one or more macrocyclic moieties that mimic the binding determinants of the ligand or the receptor. Illustrative diseases or conditions mediated by alpha-helix associated ligand 10 receptor interactions include diseases or conditions related to DNA transcription, diseases related to RNA reverse transcription, diseases or disorders related to transcriptional antitermination, diseases related to apoptosis regulation and tumor suppression, for example, cancers such as brain tumors, breast cancer, lung cancer, bone cancer, colon cancer, ovarian cancer, testicular cancer, renal cancer, liver cancer, lymphoma and leukemia; diseases or disorders related to calcium homeostasis, diseases 15 or disorders related to pain transmission, diseases or disorders associated with lipid metabolism and cholesterol homeostasis, diseases and disorders related to stress response, or to anxiety, appetite, alcohol withdrawal, opiate withdrawal or epilepsy. [0213] Thus, a further aspect of the invention contemplates a method for treating or preventing a disease or condition associated with a ligand-receptor interaction that is mediated'at least 20 in part by an alpha helical domain present in the ligand or the receptor, comprising administering an effective amount of a compound comprising at least one alpha helical cyclic peptide, wherein each peptide comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other and wherein 25 the side chains of at least some of the amino acid residues of the or each peptide are in a (three dimensional) configuration that is analogous to the configuration of amino acid side chains of at least a portion of the alpha helical domain of the ligand or the receptor. Preferably the compound is a compound of any one of formula (I), (II) or (IV). [0214] As used herein, the term "effective amount" relates to an amount of compound 30 which, when administered according to a desired dosing regimen, provides the desired mediation of the disease or disorder, therapeutic activity or disease prevention. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. A therapeutic, or treatment effective amount is an amount of the compound which, when administered according to a desired dosing regimen, is sufficient to at least partially attain the desired therapeutic 35 effect, or delay the onset of, or inhibit the progression of or halt or partially or fully reverse the onset or progression of the disease or disorder. A prevention effective amount of compound which when -40 - WO 2005/090388 PCT/AU2005/000400 administered to the desired dosing regimen is sufficient to at least partially prevent or delay the onset of a particular disease or condition. [0215] Yet another aspect of the invention provides a use of a compound comprising an alpha helical cyclic peptide, wherein the peptide comprises a sequence of five amino acid residues 5 having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other, in the preparation of a medicament for the treatment or prevention of a disease or disorder mediated by the interaction of alpha helical peptides with biomolecules. [0216] Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 10 1 g per kg of body weight per dosage. The dosage is preferably in the range of 1 gg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another preferred embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body 15 weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1ptg to 1mg per kg of body weight per dosage. [0217] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the subject. 20 [02181 The active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. [0219] According to a further aspect, the invention contemplates a pharmaceutical composition comprising a compound comprising an alpha helical cyclic peptide, wherein the peptide 25 comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second terminal residues are linked to each other, or a conformationally constrained peptide having a plurality of alpha helical pentapeptide sequences, wherein the pentapeptide sequences comprise a sequence of five amino acid residues having a first terminal 30 residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side-chains of the first and second terminal residues are linked to each other, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent. [0220] Suitable pharmaceutically acceptable salts include, but are not limited to, salts of 35 pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids -41- WO 2005/090388 PCT/AU2005/000400 such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. 5 [02211 Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium such as salts formed from triethylamine, alkoxyammonium such as those formed with ethanolamine and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine. 10 [02221 Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. [0223] The formulation of such compositions is well known to those skilled in the art. The composition may contain pharmaceutically acceptable additives such as carriers, diluents or 15 excipients. These include, where appropriate, all conventional solvents, dispersion agents, fillers, solid carriers, coating agents, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents. [02241 The carrier must be pharmaceutically acceptable in the sense of being compatible 20 with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, inhalational, nasal, transdermal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intraspinal, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of 25 bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. [02251 Depending on the disease or condition to be treated, it may or may not be desirable 30 for a compound of Formula (I) or (IV) to cross the blood/brain barrier. Thus the compositions for use in the present invention may be formulated to be water or lipid soluble. [0226] Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non 35 aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. -42 - WO 2005/090388 PCT/AU2005/000400 [0227] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg inert diluent, preservative, disintegrant (eg. sodium starch glycolate, cross-linked polyvinyl 5 pyrrolidone, cross-linked sodium carboxymethyl cellulose)) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release 10 profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. [0228] Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and 15 acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier. [0229] The compounds of Formula (I) or (IV) may also be administered intranasally or via inhalation, for example by atomizer, aerosol or nebulizer means. [0230] Compositions suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, 20 gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal devices, such as patches, may also be used to administer the compounds of the invention. [02311 Compositions for rectal administration may be presented as a suppository with a 25 suitable carrier base comprising, for example, cocoa butter, gelatin, glycerin or polyethylene glycol. - [0232] Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. [0233] Compositions suitable for parenteral administration include aqueous and non 30 aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried lyophilizedd) condition requiring only the addition of the 35 sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous -43 - WO 2005/090388 PCT/AU2005/000400 injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. [02341 Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient. 5 [0235] It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavoring agents, disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable 10 sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavoring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavoring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include 15 sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. BRIEF DESCRIPTION OF THE FIGURES [0236] Figure 1 depicts: left, CD spectra of cyclic pentapeptides SEQ ID NOs: 10 (pink), 20 11 (blue), 8 (black), 9 (red), 12 (light blue), 13 (purple), 18 (red), 19 (yellow) in 10 mM phosphate buffer (pH 7.4, 25 C) and; right, schematic demonstrating the positions of the three hydrogen bonds (dotted lines) important for stabilization of the pentapeptide helix. [0237] . Figure 2 depicts CD spectra of compounds SEQ ID NOS: 23 (black), 24 (grey), 25 (red), 26 (blue), 27 (yellow), 28 (purple), 29 (green), 30 (light blue), 31 (orange) in 10 mM phosphate 25 buffer (pH 7.4, 250 C) demonstrating the variation of helicity by varying the residues within the lactam cycle. [02381 Figure 3 depicts: left, a ROE Summary Diagram (left) and 20 lowest energy calculated structures for Ac-(cyclo-2,6)-R[KAAAD]-NH 2 (23) in 90% H20: 10% D 2 0 at 20' C. Thickness of bars reflects intensity of ROEs; right, lactam bridge in purple 30 [0239] Figure 4 depicts CD spectra of 8 in 10mM phosphate buffer (black) (pH 7.4, 25* C) and 50% TFE (red). [0240] Figure 5 is a graphical representation showing the variation in molar elipticity of 8 at 215nm with increasing [guanidine.HCI] at 25* C. [0241] Figure 6 depicts a CD spectrum comparing the helicity of SEQ ID NOs: 46 and 47 35 with their acyclic linear analogues SEQ ID NOs: 54 and 55. -44 - WO 2005/090388 PCT/AU2005/000400 [02421 Figure 7 depicts the sequential and medium ROEs, temperature coefficients, and coupling constants for SEQ ID NO: 46 in 90% H 2 0: 10% D 2 0. [0243] Figure 8 depicts (a) Helical wheel for dimer SEQ ID NO: 46, cyclo(1-5,6-10)-Ac

[KARADKARAD]-NH

2 showing side chain distribution; (b) side view of SEQ ID NO: 46 with helical 5 backbone (yellow), bridging lactam restraints (white), exposed side chains (green spheres); and (c) SEQ ID NO: 46 viewed end on. [02441 Figure 9 depicts CD spectra in 10 mM phosphate buffer, pH 7.4, 250 C for 32 44 mM solutions of (a) SEQ ID NO: 46 (-), SEQ ID NO: 47 (- - - -) and acyclic analogues SEQ ID NO: 54 (--) and SEQ ID NO: 55 (- - -); (b) SEQ ID NO: 46 (-) versus SEQ ID NO: 47(---), SEQ 10 ID NO: 54 (--) and SEQ ID NO: 55 (---) in 50% TFE. [02451 Figure 10 is a illustration of the crystal structure of Bad (grey) bound to Bcl-xL protein, NMR structures of monocycle(purple) and bicycle (green) overlay closely with the Bad helix and can display the side chains required for binding in the correct position. (PDBID: I g5j) [02461 Figure 11 is an illustration of the crystal structure of p53 (grey) bound to MDM2 15 oncoprotein (PDBID: lycq), with monocycle (Ac-R[KAAAD-NH 2 [SEQID NO: 23]) overlayed showing it can position the binding residues in the required position [02471 Figure 12 depicts CD spectra of constrained nociceptin mimetics SEQ ID NOs: 79 and 77, known peptidic agonist (FGGFTGARKSARK-NH 2 ; Kj:0.3nM), and linear address sequence (AcTGARKSARK-NH 2 ). 20 [0248] 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 which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any 25 two or more of said steps or features. [0249] The invention will now be described with reference to the following examples which are included for the purpose of illustration only and are not intended to limit the generality of the invention hereinbefore described. -45 - WO 2005/090388 PCT/AU2005/000400 EXAMPLES EXAMPLE 1 PEPTIDE SYNTHESIS (PENTAPEPTIDES AND HEXAPEPTIDES) [0250] Peptides represented by SEQ ID NO. 8 to SEQ ID NO: 31 were prepared on 5 0.25mmol scale by manual stepwise solid phase peptide synthesis using HCTU/DIPEA activation for Fmoc chemistry on Rink Amide MB3HA resin (substitution 0.78mmol.g 1 ), or Tentagel S RAM resin (substitution 0.25mmol.g~1), or Trityl chloride resin (substitution 1.0 mmol.g-). Four equivalents of amino acid and eight equivalents of diisopropylethylamine (DIPEA) were employed in each coupling step (45mins), except for Fmoc-Asp(OAllyl)-OH and Fmoc-Lys(Alloc)-OH where only 2 equivalents 10 were used. Fmoc deprotections were achieved with 3 x 5 min treatments with excess 1:1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay" 1 and double couplings were employed for yields below 99.6%. After the assembly was complete, the allyl ester of aspartic acid and allyl carbamate of lysine were removed by treating the peptide resin with Pd(PPh 3

)

4 (0. leq) and N.N-dimethylbarbituric acid (4eq), in DCM, under argon and in the dark for 2hrs, this 15 procedure was repeated once. After which the peptide was washed with DCM, DMF and 0.5% diethyldithiocarbamate in DMF. 2mg of resin was subjected to cleavage and the progress of the reaction monitored by MS. This process was repeated if necessary. [0251] Cyclization was effected on-resin using 1.5 eq BOP, 2eq DIPEA in DMSO/NMP (1:4). The reaction was monitored by cleavage of -2mg resin and subjecting the residue to MS, total 20 reaction time was <24 hours. The peptides were simultaneously deprotected and cleaved from the resin by 2hr treatment of the washed and dried resin in 95% TFA, 2.5% TIPS, 2.5% H 2 0, or 1% TFA in DCM (15 pl per 10mg resin). The solution was then filtered, the filtrate concentrated in vacuo and the peptide precipitated with cold diethyl ether. The peptide precipitate was filtered washed With copious amounts of diethyl ether, redissolved in 1:1 acetonitrile/water and lyophilised. The crude 25 peptides were purified by rp-HPLC (Rul:Vydac C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA in H20, Solvent B = 0.1% TFA, 10% H 2 0 in Acetonitrile. Gradient: 0%B to 100%B over 30 mins. Ru:Phenomenex C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA in H 2 0,. Solvent B = 0.1% TFA, 10% H20 in Acetonitrile. Gradient: O%B to 100%B over 30 mins.). 'H NMR was carried out in H 2 0:D 2 0 (9:1) at 298K. 30 EXAMPLE 2 CYCLIC PENTAPEPTIDES WITH NON-PEPTIDIC CAPPING GROUPS [0252] Synthesis of the peptide of formula (II) was achieved by standard Fmoc SPPS protocols using trityl chloride polystyrene resin. The peptide was capped with phenyl butanoic acid, cleaved from the resin using 1% TFA in dichloromethane (DCM) leaving side chain protecting groups 35 intact. Isobutylamine was then coupled on using BOP, DIPEA, with CuCl 2 - an additive known to -46 - WO 2005/090388 PCT/AU2005/000400 minimize racemisation of the C-terminal residue. Following this final deprotection was effected with 95% TFA, 2.5% TIPS, 2.5% H20. EXAMPLE 3 N-TERMINAL CYCLIC PENTAPEPTIDE BUILDING BLOCK 5 [0253] NH 2 -(cyclo-1-5)-KARAD-NH 2 (SEQ ID NO. 52) was prepared by manual stepwise solid phase peptide synthesis using HBTU/DIPEA activation for Fmoc chemistry 07 on Rink Amide MBHA resin (substitution 0.78mmol.g', 1.56mmol, 2000mg). Four equivalents of amino acid and eight equivalents of diisopropylethylamine (DIPEA) were employed in each coupling step (45mins), except for Fmoc-Asp(OAllyl)-OH and Boc-Lys(Fmoc)-OH where only 2 equivalents were used. Fmoc 10 deprotections were achieved with 3x5min treatments with excess 1:1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay 0 8 and double couplings were employed for yields below 99.6%. After the assembly was complete, the allyl ester of aspartic acid was removed by treating the peptide resin with Pd(PPh 3

)

4 (0.05eq) and diethylamine (5eq) in DCM, under argon and in the dark for 2hrs. After which the peptide was washed with DCM, DMF and 0.5% 15 diethyldithiocarbamate in DMF. 2mg of resin was subjected to cleavage and the progress of the reaction monitored by Mass spectrometry (MS). This process was repeated if necessary. Following Allyl ester deprotection the N(C)-Fmoc group was removed by treatment with piperidine (1:1 in DMF). Cyclization was effected on-resin using 1.5 eq BOP, 2eq DIPEA in DMF/Benzene (2:1). The reaction was monitored by cleaving ~2mg resin and subjecting the residue to MS, total reaction time 20 was approximately 48-72 hours. The peptides were simultaneously deprotected and cleaved from the resin by 2hr treatment of the washed and dried resin in 95% TFA, 2.5% TIPS, 2.5% H20 (15pl per 10mg resin). The solution was then filtered, the filtrate concentrated in vacuo and the peptide precipitated with cold diethyl ether. The peptide precipitate was filtered washed with copious amounts of diethyl ether, redissolved in 1:1 acetonitrile/water and lyophilised. The crude peptides were purified 25 by rp-HPLC (Vydac C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA inH20, Solvent B = 0.1% TFA, 10% H20 in Acetonitrile. Gradient: 0%B to 100%B over 30 mins. Yield 30% (isolated). [Rt=12.82min]. MS: [M+H+] (calc.) = calc. 541.31 (expt.)=541.39. EXAMPLE 4 C-TERMINAL CYCLIC PENTAPEPTIDE BUILDING BLOCK 30 [0254] Boc-(cyclo-1-5)-K4R(PbJ)AD-OH (SEQ ID NO. 53) was synthesized in an analogous manner to peptide (SEQ ID NO: 52 above), however using trityl chloride resin (0.95mmol.g', 1.28g, 1.16mmol). Cleavage was achieved using 50mL 10% acetic acid, 20%'2,2,2 trifluoroethanol, 70% DCM for 2hrs. After lyophilization the crude peptide was deemed pure enough by analytical HPLC and used without further purification. Yield 50%. MS: [M+H*] (calc)= 893.43 35 (expt.) = 893.67]. -47- WO 2005/090388 PCT/AU2005/000400 EXAMPLE 5 SYNTHESIS OF cyclo(1-5, 6-10)-Ac-[KARADKARAD]-NH 2 [SEQ ID NO: 46] [0255] To DIPEA (135 pL, 0.38 mmol) was added to a solution of Boc-cyclo(1-5) KAR(Pbf)AD-OH [SEQ ID NO: 53] (154 mg, 0.17 mmol), NH2-cyclo(1-5)-KARAD-NH 2 [SEQ ID 5 NO: 52] (102 mg, 0.19 mmol, and BOP (80 mg, 0.18mmol) in DMF (5 mL). After stirring (2 h, RT), solvent was evaporated in vacuo, the residue dissolved in H 2 0/MeCN (1:1), lyophilized and purified (rpHPLC). The product was treated with TFA/TIPS 19:1 (1 h, 200 C), evaporated, and reacted (2 h, 200 C) with AcOH (15 pL, 0.26 mmol), 0.5M HBTU (500 pL 0.25 mmol) and DIPEA (90 gL, 0.52 mmol). Solvent was removed in vacuo, H 2 0/MeCN (1:1) added, lyophilized and purified (rpHPLC) to 10 yield cyclo(1-5,6-10)-Ac-[KARADKARADJ-NH 2 [SEQ ID NO: 46] (19.1mg, 10% isolated). MS [M+H+] (calc.) 1106.6 (expt.) 1106.97, [M+2H]/2 (calc.)= 554.3 (expt.) = 554.04. Anal. rpHPLC: 14.8 min. (Gradient 0%-100% acetonitrile over 30 min). EXAMPLE 6 SYNTHESIS OF cyclo(1-5, 6-10, 11-15)-Ac-[KARADKARADKARAD]-NH 2 [SEQ ID NO: 45] 15 [02561 DIPEA (135pL, 0.38 mmol) was added to a solution of Boc-(cyclol-5) KAR(Pb)AD-OH [SEQ ID NO: 53] (66mg, 0.077mmol), NH 2 -(cyclo1-5)-KARAD-NH 2 [SEQ TD NO: 52] (42mg, 0.074 mmol, and BOP (52mg, 0.154 mmol) in DMF (5 mL). After stirring (2 h, RT), solvent was evaporated in vacuo, the residue dissolved in H 2 0/MeCN (1:1), lyophilized and purified (rpHPLC). The product (34 mg, 0.024mmol) was treated with TFA/TIPS 19:1 (1 h, 20'C), 20 evaporated, and reacted (2 h, RT) with peptide Boc-(cyclo1-5)-K4R(Pbj)AD-OH ([SEQ ID NO: 53] (20 mg, 0.024 mmol), BOP (15 mg, 0.034 mmol), and lastly DIPEA (50 pL, 0.24 mmol). The solvent was evaporated in vacuo, the residue dissolved in H 2 0/MeCN (1:1), lyophilized and purified (rpHPLC). The product was once again treated with TFA/TIPS 19:1 (1h, 20'C), evaporated, and reacted with AcOH (2 gL, 0.0132 mmol), BOP 7mg, 0.016mmol) and DIPEA (19 gL, 0.138 mmol) 25 for 2 hrs at RT. The solvent was removed in vacuo, H 2 0/MeCN (1:1) added, lyophilized and purified (rpHPLC) to yield cyclo(1-5, 6-10, 1 l-15)-Ac-[KARADKARADKARAD]-NH 2 [SEQ ID NO: 47] (7.8mg, 5.5%(isolated). MS [M+2H*]/2 (calc.)= 815.44 (expt.) = 815.55. [M+3H]/3 (calc.) = 543.97 (expt.) = 544.03. Anal. rpHPLC: 15.09 min. EXAMPLE 7 30 NON-CYCLIC ANALOGUES [02571 Linear Peptides Ac(KARAD)-NH 2 where n=2 (SEQ ID NO: 54) and n=3 (SEQ ID NO: 55) were prepared by manual stepwise solid phase peptide synthesis using HBTU/DIPEA activation for Fmoc chemistry 07 on Rink Amide MBHA resin (substitution 0.78 mmol.g', 0.5 mmol, 648 mg). Four equivalents of amino acid and eight equivalents of diisopropylethylamine (DIPEA) 35 were employed in each coupling step (45 min). Fmoc deprotections were achieved with 3x5min -48 - WO 2005/090388 PCT/AU2005/000400 treatments with excess 1:1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay... and double couplings were employed for yields below 99.6%. After assembly of the first 10 residues, the peptide resin was washed, dried and split into two portions, one portion was acetylated, whilst to the other was added the final 5 residues. N-terminal acetylation was achieved by treating the 5 fully protected peptide with 4 equivalents of glacial acetic acid, 4 equivalents of HBTU, and 8 equivalents of DIPEA. The peptides were simultaneously deprotected and cleaved from the resin by 2-hr treatment of the washed and dried resin in 95% TFA, 2.5% TIPS, 2.5% H20 (15 pL per 10 mg resin). The solution was then filtered, the filtrate concentrated in vacuo and the peptide precipitated with cold diethyl ether. The peptide precipitate was filtered washed with copious amounts of diethyl 10 ether, redissolved in 1:1 acetonitrile/water and lyophilized. The crude peptides were purified by rp HPLC (Vydac C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA in H20, Solvent B = 0.1% TFA, 10% H20 in Acetonitrile. Gradient: 0%B to 100%B over 30 mins. (SEQ ID NO: 54) Yield 20% (isolated). [Ri=12.65min]. MS: [M+H*] (calc.) = 1142.63 (expt.) = 1142.75; [M+2H*]/2 (calc.) 571.85 (expt.) =571.86. (SEQ ID NO: 55) Yield 30% (isolated) [Ri=13.16min]. MS: [M+2H]/2 15 (calc.) = 842.46 (expt.) = 842.64; [M+3H+]/3 (calc.) 561.98 (expt.) = 562.08. EXAMPLE 8 [0258] Synthesis of SEQ ID NOs: 77 to 80 was carried on Tentagel-S-RAM resin (0.25mmol scale) by manual stepwise solid phase peptide synthesis using HCTU/DIPEA activation Tentagel-S-RAM resin using standard Fmoc SPPS (scheme ), Four equivalents of amino acid and 20 eight equivalents of diisopropylethylamine (DIPEA) were employed in each coupling step (45mins), except for Fmoc-Asp(OAllyl)-OH and Fmoc-Lys(Alloc)-OH where only 2 equivalents were used. Fmoc deprotections were achieved with 3 x 5 min treatments with excess 1:1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay and double couplings were employed for yields below 99.6%. After the assembly was complete, the allyl ester of aspartic acid and allyl 25 carbamate of lysine were removed by treating the peptide resin with Pd(PPh 3

)

4 (0.1 eq) and N.N dimethylbarbituric acid (4eq), in DCM, under argon and in the dark for 2hrs, this procedure was repeated once. After which the peptide was washed with DCM, DMF and 0.5% diethyldithiocarbamate in DMF. 2mg of resin was subjected to cleavage and the progress of the reaction monitored by MS. This process was repeated if necessary. 30 [0259] Cyclization was effected on-resin using 1.5 eq BOP, 2eq DIPEA in DMSO/NMP (1:4). The reaction was monitored by cleavage of ~2 mg resin and subjecting the residue to MS, total reaction time was <24. After subsequent piperidine deprotection, the resin was shaken with 2 hydroxy-4-methoxybenzaldehyde in dimethyformamide/ trimethylorthoformate (1:1) for 10hr, the resin was then drained and NaBH(OAc) 3 (10 eq) in dimethyformamide/ trimethylorthoformate (1:1). 35 The ninhydrin test indicated that the 2-hydroxy-4-methoxybenzyl had successfully (>99.4%) been introduced onto Na of lysine. The resin was then acylated overnight with the symmetrical anhydride of Fmoc-Asp(OAll)-OH (generated by stirring 6eq Fmoc-Asp(OAll)-OH, and 3eq -49 - WO 2005/090388 PCT/AU2005/000400 Diisopropylcarbodimide in DCM for 30mins). Cleavage of a small amount of resin and analysis by MS indicated complete Na acylation after 24hrs. The remaining residues were introduced using the standard HCTU/DIPEA activation, and allyl deprotection and macrolactamization was achieved as previously described. After attachment of the final 5 residues the peptide resin was deprotected, 5 washed and dried. Final cleavage of the peptides was achieved with 92.5% TFA, 2.5% TIPS, 2.5% EDT, 2.5% H20. The solution was then filtered, the filtrate concentrated in vacuo and the peptide precipitated with cold diethyl ether. The peptide precipitate was filtered washed with copious amounts of diethyl ether, redissolved in 1:1 acetonitrile/water and lyophilised. The crude peptides were purified by rp-HPLC (Rt:Vydac C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA in H 2 0, 10 Solvent B = 0.1% TFA, 10% H20 in Acetonitrile. Gradient: O%B to 100%B over 30 mins. Ra:Phenomenex C18 column, 300A. 22 x 250mm, 214nm, Solvent A = 0.1% TFA in H 2 0, Solvent B = 0.1% TFA, 10% H20 in Acetonitrile. Gradient: 0%B to 100%B over 30 mins.). EXAMPLE 9 CD AND NMR STUDIES ON CYCLIC PENTA AND HEXAPEPTIDES 15 [0260] Circular Dichroism (CD) was performed on peptides having SEQ ID NOs: 8 to 14 and 18 to 31, using methods described above. The molar elipticities at 222 nm, 208 nm and 190 nm, ratios of elipticities at 222 nm/208 nm and relative helicity are shown in Tables 6 and 7. CD spectra of these peptides are given in Figure 1. TABLE 6 20 CD spectra on Peptides having one cyclic pentapeptide module - effects of varying the bridge partner and the termini Molar elipticities ([6] deg.cn 2 .dinol-'.residue') at A = 215, 207 and 190 nin, ratios of elipticities at 215/207nm, and percentage helicity for peptides in 10mM phosphate buffer (pH 7.4, 25 C). Peptide [0] 21 5 [0]207 [0] 190 0 215 /0 207 Relative helicityc Ac-(cyclo-1,5)-[KARAE]-NH 2 -1068 -3393 -10611 0.31 0.08 [SEQ ID NO: 10] Ac-(cyclo-1,5)-[EARAK]-NH 2 -7430 -12803 20735 0.58 0.58 [SEQ lID NO: 11] Ac-(cyclo-1,5)-[KARAD]-NH 2 -12757 -12211 38300 1.04 1.00 [SEQ ID NO: 8] Ac-(cyclo-1,5)-[DARAK]-NH 2 -7723 -10705 15600 0.72 0.60 [SEQ ID NO: 9] Ac-(cyclo-1,5)-[OARAD]-NH 2 92 -2077 -4613 -0.04 0 [SEQ ID NO: 12] Ac-(cyclo-1,5)-[DARAO]-NH 2 -4671 -9748 -6954 0.48 0.37 [SEQ ID NO: 13] - 50 - WO 2005/090388 PCT/AU2005/000400 Ac-(cyclo-1,5)-[OARAE]-NH 2 741 -3368 -16228 -0.22 0 [SEQ ID NO: 18] Ac-(cyclo-1,5)-[EARAO]-NH 2 2442 -1917 -11256 -1.27 0 [SEQ ID NO: 19] Ac-KARAD-NH 2 -524 -5555 -7372 0.09 0.04 [SEQ ID NO: ZZ14] Ac-(cyclo-1,5)-[KARAD]-OH 207 -5643 -13953 -0.04 0 [SEQ ID NO: 20] a -625 -3195 -2659 0.20 0.06 H-(cyclo-1,5)-[KARAD]-NH 2 -812 -2228 1355 0.36 0.06 [SEQ ID NO: 21] b -2590 -3327 8452 0.78 0.2 H-(cyclo-1,5)-[KARAD]-OH -1033 -5737 -4966 0.18 0.08 [SEQ ID NO: 22] a In 0.01M HCl pH 2. bIn 0.001M NaOH pH 10. C [0]2 (8)/ [0]215 (x) refer to "Quantitation of helicity". [0261] Of interest, the CD spectrum for SEQ ID NO: 8 shows a slight shift in its minima to lower wavelengths compared with longer alpha helical peptides (222 nm -> 215 nm, 208 nm -> 5 207 nm), as has been observed before in short fixed nucleus alanine helices 16 . Given that these are the first CD spectra of very short isolated alpha helices, it is not surprising that their CD spectra differ from those of much longer helices. Theoretical studiesI 17

'

11 8 into the chiroptic properties of the alpha helix have predicted that short alpha helices should have different CD spectra from longer alpha helices. The negative minimum at 215nm is consistent with the long wavelength n-n7u* transition 10 commonly observed for alpha helices and beta sheets in the 215-230 nm wavelength range." 9 The observed positive maximum at 190nm and negative minimum at 207nm characterize the structure as alpha helix rather than beta sheet, as these bands can only arise from exciton splitting of the NVI transition by the interaction of electric dipole transition moments among amides in the well defined geometry of the alpha helix. The relative intensities of these peaks for SEQ ID NO 8 mirror those 15 observed for other alpha helices, therefore we have quoted the intensities at 190 nm, 207 nm and 215 nm in Table 6. Helix Dependence On Sequence [02621 Certain residues are known to favor or disfavor alpha helicity, therefore the residues in this system were altered in an attempt to gain insight into the helicity for these cycles. 20 Initial solubility issues with hydrophobic pentapeptides prompted us to incorporate an additional arginine at the N-terminus. Nine K(i) ->D(i+4) side-chain cyclised hexapeptides were synthesized with the three residues intervening the bridge systematically replaced by alpha helix inducing (alanine, leucine, methionine, glutamine), alpha helix indifferent (phenylalanine, seinee, or alpha helix breaking (glycine) residues (Table 7). -51- WO 2005/090388 PCT/AU2005/000400 TABLE 7 Molar elipticities ([0] deg. cm 2 .dinol-'.residue') at A=215, 207 and 190 nm, ratios of elipticities at 215/207nm, and percentage helicity for cyclic peptides 23-31 in 10mM phosphate buffer pH 7.4 at 25 0 C. Peptide [0]215 [0]207 [0]190 0215/02 Relative 07 Helicitya Ac-(cyclo-2,6)-R[KAAAD]-NH 2 -13537 -13684 39352 0.99 0.91 [SEQ ID NO: 23] Ac-(cyclo-2,6)-R[KALAD]-NH 2 -14798 -15165 46621 0.98 1.00 [SEQ ID NO: 24] Ac-(cyclo-2,6)-R[KAMAD]-NH 2 -11853 -12296 38464 0.96 0.80 [SEQ ID NO: 25] Ac-(cyclo-2,6)-R[KAQAD]-NH 2 -11394 -12279 36865 0.93 0.84 [SEQ ID NO: 26] Ac-(cyclo-2,6)-R[KAFAD]--NH 2 -8644 -9087 27718 0.95 0.76 [SEQ ID NO: 27] Ac-(cyclo-2,6)-R[KAGAD]-NH 2 -4874 -7678 10036 0.63 0.32 [SEQ ID NO: 28] Ac-(cyclo-2,6)-R[KGSAD]-NH 2 -4810 -6975 12831 -0.69 0.32 [SEQ ID NO: 29] Ac-(cyclo-2,6)-R[KSSSD]-NH 2 -4432 -8017 6827 0.55 0.30 [SEQ ID NO: 30] Ac-(cyclo-2,6)-R[KGGGD]-NH 2 -2131 -4868 -1593 0.44 0.14 [SEQ ID NO: 31] 5a 0215 [SEQ ID NO: 24] / 0215 (x) refer to "Quantitation of helicity" section below. [0263] Figure 2 shows that helicity is dependent on which residues intervene between the bridging residues. The helical structure is tolerant of substitution by alpha helix inducing residues like Ala ([SEQ ID NO: 23], Leu [SEQ ID NO: 24], Met [SEQ ID NO: 25], Gln [SEQ ID NO: 26] and Phe [SEQ ID NO: 27], as demonstrated by the deep minima at 215/207nm, high maximum at 190nm, and 10 high ratio 0215/0207. There is some variation in the intensity at these wavelengths which closely mirrors the intrinsic alpha helical propensity of specific amino acids determined in protein environments 5 . The system can be perturbed by replacing the central residue with glycine [SEQ ID NO: 28], which results in a decrease in intensity at 215 nm, 207 nm and 190 nm, along with the appearance of a deeper minimum at 20 1nm that is commonly observed for 3 1 0 -helicity/random coil structures. This reduction 15 in alpha helicity also results from placement of two [SEQ ID NO: 29] or three [SEQ ID NO: 30] helix disfavoring residues between the bridging residues, although based on the shape of their CD spectra there is some bias towards a helical conformation. Not surprisingly, where three alpha helix breaking residues are present [SEQ ID NO: 31], total abolition of helicity was indicated by the single deep minimum at 200 nm characteristic of a random coil. -52 - WO 2005/090388 PCT/AU2005/000400 NMR Evidence For alpha Helicity [02641 Structural characterization was conducted for SEQ ID NO. 8 and SEQ ID NO 23 using 1D and 2D lH-NMR spectroscopy in 90% H 2 0: 10% D 2 0 at 288 K (pH 4.0). 2D-TOCSY spectra at 600 MHz were used to identify resonances for each amino acid. Due to the molecular weight 5 of the macrocycle, ROESY instead of NOESY spectra had to be used to identify sequential connectivity and intra-residue NH-NH and NH-CH cross peaks Spectral overlap in SEQ ID NO. 8 prevented unambiguous identification of key long range ROEs, however SEQ ID NO. 23 gave well defined resonances and was investigated further. There were a number of spectral features that are characteristic of well-defined structure in the cyclic hexapeptide [SEQ ID NO: 23], and specifically 10 characteristic of alpha helicity. [02651 First, there were conspicuously low coupling constants ( 3 JNHcH < 6Hz) for all amide resonances except D6, as normally observed in alpha helical peptides. Second, all of the residues displayed a upfield shift (0.18 to 0.32 ppm) for S(Ha) relative to the corresponding residue in random coil structures, this also being typical of helical peptides. Third, there was a low temperature 15 dependence for amide NH chemical shifts, with temperature coefficients (AS/T) being 4ppb/K for Ala3, Ala4, Ala5, Asp6, and one C-terminal amide NH (Fig. S5, Supporting Information), consistent with their involvement in hydrogen bonds that characterise an alpha helix. Fourthly, the observation in ROESY spectra of non sequential medium range d N(iji4), daN(ij+3), dap(i,i+3) ROEs (Fig. 4) suggest helical structure, and the particularly prominent daN(i,i+4) versus weak daN(i,i+2) ROEs 20 supports a high proportion of alpha helicity rather than 3 i-helicity or turn conformations. Solution Structure of 23 [0266] The three dimensional structure for the hexapeptide 23 in 90% H 2 0: 10% D 2 0 at 20'C was calculated using dynamic simulated annealing and energy minimization in Xplor (3.851) from 81 ROE (26 sequential, 25 medium range, 30 intra-residue) distance restraints, and 4 phi angle 25 restraints ( 3 JNHCHa < 6Hz# = -65 ± 300 ). No explicit Hbond restraints were included in calculations. The final 16 lowest energy structures contained no dihedral angle (> 2*) or distance (> 0. 1A) violations and are displayed in Fig. 3. These lowest energy structures indicate a well defined alpha turn, with four i-+i+4 hydrogen bonds involving NH protons of the C-terminal amide, Asp6,.Ala5, Ala4 and the CO oxygens of Ala3, Lys2, Arg1 and N-terminal amide. The backbone pairwise RMSD 30 for this family of structures for Ac-(cyclo-2,6)-R[KAAAD]-NH 2 (23) in water is 0.35A, indicating a fairly tight structural convergence. When 23 was superimposed on an idealized textbook hexapeptide alpha helix (i.e. backbone dihedral angles set y = -47*, # = -57*), the backbone pairwise RMSD = 0.81A. However, for just the cyclic pentapeptide component of 23, the backbone pairwise RMSD = 0.22A over backbone carbon and nitrogen atoms. This structural evidence strongly supports the 35 conclusion that the endocyclic residues are in a highly alpha helical conformation, more so than the exocyclic Arg residue. -53 - WO 2005/090388 PCT/AU2005/000400 Effect of a Helix Stabilizing Solvent [0267] To see whether the alpha helicity exhibited by 8 in water could be increased, a CD spectrum was acquired for 8 in 50% aqueous TFE (a helix-inducing solvent which increases helicity for peptides). Figure 4 shows that the presence of 50% TFE does not increase molar elipticity at 215 5 nm, suggesting that 8 is already maximally alpha helical in water alone. This optimum alpha helicity is also supported by the ratio 0 2 15 /0 20 7 > 1 in water alone, as reported for an idealized (100%) alpha helix 20 . Although there is some controversy regarding the accuracy of this ratio"I, in this case it is at least still indicative of substantial alpha helicity. Helix Dependence On Hydrogen Bonds 10 [0268] The NMR analysis indicated the presence of 3 hydrogen bonds within the pentapeptide cycle. To investigate the importance of these hydrogen bonds for the conformational stability of 8, we synthesized a series of analogues with variable potential for forming intramolecular hydrogen bonds. Since 1,5-hydrogen bonds are characteristic of alpha helicity, inclusion or exclusion of the N-terminal acetyl group or the C-terminal amide group allowed variation between 1-3 potential 15 hydrogen bonds (Figure 1). Thus Ac-(cyclo-1,5)-[KARAD]-NH 2 (8) can form 3 potential intramolecular 1,5-hydrogen bonds (Figure 1), while Ac-(cyclo-1,5)-[KARAD]-OH (20) and H (cyclo-1,5)-[KARAD]-NH 2 (21) can form only two hydrogen bonds, and H-(cyclo-1,5)-[KARAD] OH (22) can only form 1 hydrogen bond. [0269] Of this group of four peptides, only 8 showed alpha helical structure in water at pH 20 7 (see Table 6), indicating that a minimum of 3 hydrogen bonds is required for alpha helicity in a pentapeptide sequence (see Figure 1). Since the effect of charges at the N-and C-termini might be expected to destabilize helicity, CD spectra were also recorded for 20 and 21 in 0.01M HCl (pH 2) and 0.001M NaOH (pH 11), ensuring that the uncapped termini were fully protonated and deprotonated respectively. The pH had little effect on conformation, both 20 and 21 showed slight increases in 25 helicity, but remained largely unstructured. Compound 22 was not examined at high or low pH as one of the termini will always be charged. Quantitation of helicity [0270] Assuming that mean alpha helix content (fH) is linearly related to elipticity at 222 nm, or in our case 215 nm ([0]ob,215), then the equation for calculating helix content is: 30 fH = ([ 0 obs215 - [C]0 / ([0-215 - [01C) (1) Luo and Baldwin 12 2 determined that the random coil ([0]c) and infinite alpha helix ([0]- 222 ) molar elipticities are temperature dependent based on the equations: [P]C = 2220 - 53T (2a) [0]- 2 1 5 = (-44000 + 250T)(1 - k/Np) (2b) -54- WO 2005/090388 PCT/AU2005/000400 where T is temperature in degrees Celsius, N, is the number of peptide units, and k is a finite length correction. [0271] Despite the widespread use of equation 1, a key problem is the implementation of a suitable k factor for which a range of values between 2.4-4.5 have been used" 1 2 . Baldwin has 5 suggested using k = 3.0 for carboxyamidated peptides and 4.0 for unblocked peptides 12 3 . For 8, variation of the k factor from 2.4 to 4.5 resulted in 56-135% helicity, so it was clear that the k factor can impact significantly on reported values of alpha helical content. There is no empirical method to determine a suitable k factor, and the problem of choosing one becomes particularly acute for short peptides. Given our compelling high resolution NMR data and CD studies in the presence of benign, 10 helix stabilizing, and helix destabilizing environments, we believe it is highly likely that the equilibrium between random coil, partial helix and pure alpha helix is shifted significantly towards pure alpha helix such that fH is -1 and the contribution of [0]c is ~0. Based on the assumption that 8 has 100% a-helicity, we can derive k -4, which is within the suggested range. If we apply this value to equation 2b then the percentage helicities for the alpha helical compounds in tables 6 and 7 are 100% 15 (8), 83% (23), 91% (24), 73% (25), 70% (26), 53% (27). [0272] There are however, problems associated with this derivation. Firstly, despite the significance of the Luo-Baldwin study, it does not reflect the properties of very short peptides. Secondly it is based on extrapolating TFE titration data to CD data acquired in water alone 121 . Thirdly, their study is calibrated to elipticity at 222nm, whereas the absolute minimum in these peptides occurs 20 at 215nm. Given these difficulties, we have expressed relative rather than absolute helicities for the peptides in Tables 6 and 7. [02731 The implication of these results are that appreciable alpha helicity (50-100%) can be achieved in cyclic pentapeptides using systems like for example, SEQ ID NO: 8. [0274] Twenty membered macrocycles related to that in SFQ ID NO: 8, with the same 25 ring size and same positioned amide linker but with different intervening amino acids between K and D at positions i+1, i+2, and i+3 were also examined [SEQ ID NOs: 14 to 17 and 23 to 31]. An arginine was also tacked onto the N-terminus to promote aqueous solubility but would not be expected to affect rank orders of helicity in the following compounds. The rank order for decreasing alpha-helicity in these 20-membered cyclic pentapeptides was SEQ ID NOs: 24 > 23 > 26 > 25 > 27 > 28 = 29 >30 > 30 31. Peptides having SEQ ID NOs: 30 and 31 have three of the same amino acids between linking amino acids, namely Serine or Glycine, and such amino acids are known in proteins to be the least favorable to helix formation. In fact Serine is often termed a helix breaker and Glycine is often thought of as a beta/gamma turn inducer. The peptide having SEQ ID NO: 29 suggests that even with two of these amino acids present, the cyclic pentapapetide can still have appreciable alpha helicity. 35 [02751 CD Spectra for SEQ ID NOs: 8 to 13, 18 and 19 and SEQ ID NOs: 23 to 31 can be found in Figures 1 and 2, respectively. -55- WO 2005/090388 PCT/AU2005/000400 Discussion of Example 9 [0276] Herein the present inventors have disclosed the first 5-residue peptides that display essentially complete alpha helicity in water, making them the shortest and most stable peptide alpha helices known. This result, confirmed by NMR-derived structure determination in water, was unique 5 for pentapeptides cyclized through amide formation specifically between Lysine and Aspartate at positions i and i+4 respectively. Their alpha helical nature has been convincingly established by circular dichroism and 'H-NMR spectra, neither of which were concentration dependent, thus ruling out alpha helicity due to aggregation. Failure to enhance alpha helicity (especially of 8) using 50% TFE, failure to diminish it with 8M guanidine.HCI, and lack of success in degrading it with proteolytic 10 enzymes, supports our conclusion of an exceptionally high proportion of alpha helical conformers in the structural ensemble, especially for compound 8. [0277] Conventional head to tail cyclic pentapeptides are traditionally associated with various types of P and gamma turn conformations in solution, although in water they tend to display no well defined structure at all. In the literature of peptide hormones there is evidence that lactam 15 bridges, particularly (i, i+4) linkages, can increase helicity in longer peptides and enhance bioactivity. However there is considerable disparity in the alpha helix stabilizing effects of various lactam bridges, with little agreement about which sequences, ring sizes, and ring compositions impart the highest alpha helicity 43 , and suggestions that effectiveness is case dependent". Here, the inventors have systematically examined the effects of different lactam bridges on alpha helix stability in simple 20 pentapeptides, cyclized through side chain to side chain coupling. They have shown that cyclic pentapeptides of defined size (20-membered rings) and specific composition have the capacity to adopt a single alpha turn conformation that is remarkably stable in water. Spectral data from circular dichroism and 2D 'H NMR strongly support alpha helicity for such cycles in water, even under severe peptide-denaturing conditions such as high concentrations of guanidine hydrochloride ( ;8 M). These 25 results also demonstrate that a single lactam bridge can effectively stabilize alpha helicity in short peptides, which contrasts with the best results to date where two overlapping lactam bridges were required to necessitate alpha helix stabilization in short peptides 49

,

24 10278] A feature of this work is the finding that appreciable alpha helicity is dependent not only on the cyclic constraint that produces a 20-membered ring, but also on accompanying formation 30 of three intramolecular 13-membered hydrogen bonded rings. Thus three i-- i+4 hydrogen bonds together with the cyclic constraint would appear to be the minimum requirements to stabilize an alpha turn, since either removal of just one of these hydrogen bonds or minor modification to the cyclic restraint was sufficient to collapse the alpha helical structure. It is worth pointing out that, for a conventional uncapped peptide, three 1->5 hydrogen bonds would require a minimum sequence of 35 seven amino acids. In the absence of the thermodynamic stability afforded by the cyclic constraint, it is therefore now explicable as to why acyclic peptides can only be highly alpha helical if they are much longer. - 56 - WO 2005/090388 PCT/AU2005/000400 [0279] The significant difference observed in the RMSD values between the alpha helical hexapeptide structure 8 and its cyclic pentapeptide structural component is consistent with the need for the cyclization restraint, rather than just 3 (or 4) hydrogen bonds, for alpha helicity. Although NMR data at low temperature did indicate that the exocyclic Arg residue in 8 was in an alpha helical 5 environment created by 4 hydrogen bonds and the cyclization constraint, the deviation from an idealized alpha helix was larger for the hexapeptide over the pentapeptide, reflecting less alpha helicity for this residue which is outside the cycle. This does not necessarily mean that the cyclic pentapeptide cannot transmit alpha helicity to multiple attached exocyclic residues, only that it isn't very effective in inducing helicity in a single attached residue. This slight fall off in helical integrity 10 outside the cycle is not too surprising, since the termini of protein/peptide alpha helices are normally quite disordered in solution structures, and certainly more disordered than found for the ends of 8. [0280] The helix stability is dependent upon sequence, according to normal rules of protein-based structure e.g., those residues known to favor alpha helicity in proteins also favor alpha helicity in these cyclic pentapeptides. However unlike proteins, these simple systems are not 15 complicated by effects of side chain packing, folding, intra- or inter- molecular interactions other than with solvent, and thus would appear to offer excellent opportunities to investigate effects of individual natural or unnatural amino acid components on the alpha helix. Since the cycles remained intact under peptide-denaturing conditions, they may be useful as templates in longer peptides for studying unfolding/refolding and for 'seeding' structure in proteins and polypeptides. The stability of the cycles 20 under peptide-degrading conditions (trypsin, human serum) also suggests that they may have useful alpha helix-mimicking properties in biologically relevant environments. [02811 In another context the observations here complete the picture for cyclic peptides as mimetics of the key elements of protein structure. While i - i+4 side chain to side chain cyclization herein has produced a stable alpha turn with a 13-membered hydrogen bonded ring in pentapeptides, 25 i -- i+3 side chain to side chain cyclization is known to produce the beta turn with a 10-membered hydrogen bonded ring (multiples of which constitute the 310- helix) in tetrapeptides, and i -> i+2 side chain to side chain cyclization has produced the gamma turn involving a 7-membered hydrogen bonded ring as well as the beta strand, depending upon the constraints in the cycle, in tripeptides. Clearly cyclization, together with appropriate use of molecular constraints in peptide sequences, can 30 be systematically and effectively used to mimic any of the fundamental structural elements of proteins. [0282] In summary, the high conformational and proteolytic stability of these alpha helical cyclic pentapeptides suggests their use as single turn alpha helical modules, with capacity for decoration by peptidic, cyclic, or non-peptidic appendages, to mimic bioactive peptide or protein alpha helical segments. -57- WO 2005/090388 PCT/AU2005/000400 EXAMPLE 10 STABILITY Stability against denaturants [02831 Using the molar elipticity at 215 nm as an indicator of alpha helicity, Figure 5 5 clearly shows that SEQ ID NO: 8 is conformationally stable, the pentapeptide maintains full alpha helicity even in the presence of 8M guanidinium chloride. These conditions readily denature peptides and proteins, but do not affect alpha helicity in 8. Proteolytic stability of the helix. trypsin digestion [02841 A standard solution of Ac-KARAD-NH 2 and Ac-(cyclo-1,5)-KARAD-NH 2 (1 10 mg/mL) was prepared in 100mM ammonium carbonate buffer at pH-8.2. To 100 pL of each solution was added 1mg/mL of trypsin (1 pL). The digest was conducted at room temperature with 5 pL aliquots taken at 1, 4, 8, 28, 48, 55, 110, 155 minutes. Aliquots were diluted with 5 RL of 3% TFA to stop the reaction. and analyzed by LC-MS using 2.1 x 150 mm Phenomenex 300A C18 5pm column, with a 3% per minute linear gradient of 0-60% acetonitrile over 20 minutes. The amount of starting 15 material left in each sample was quantified by determination of total ion counts for the molecular ion. [0285] Compound of SEQ ID NO 8 and its acyelic version SEQ ID NO: 14 were incubated with trypsin (pH 8.2), aliquots removed at intervals between 1 and 155 minutes, and analyzed by LCMS. The cyclic compound remained intact over this period, whereas the acyclic analogue completely hydrolyzed within a few minutes to fragments Ac-KAR-OH, H-ARAD-NH1 2 , H 20 AR-OH and H-AD-NH 2 . Similarly the cyclic compound 8 was not degraded by human serum (1 h, 37 C). These results prove that the peptide sequence in 8, being held in an alpha helical conformation, is not susceptible to recognition by the proteolytic enzyme. This is consistent with other observations that only an extended or linear peptide conformation is recognized, not only by trypsin, but by all proteolytic enzymes 1 2 5 Presumably the cycle is too tightly locked into an alpha helical turn to permit 25 significant unwinding to the extended format needed for recognition by trypsin. Serum Stability [0286] Standard solutions of Ac-KARAD-NH 2 and Ac-(cyclo-1,5)-KARAD-NH2 (1 mg/mL) were prepared in water. 200 pL of each peptide was added to human serum 800 pL and incubated at 37' C. Acetonitrile/water 3:1 (300 gL)was added to aliquots (100 pL) of serum at 5, 15, 30 30, 45 and 60 minutes to precipitate serum proteins, which were removed by centrifugation. The decanted supernatant was analyzed by LC-MS MS with a 2.1 x 150mm Phenomenex 300A C18 5um column, using a 3% per minute linear gradient from 0%-60% acetonitrile over 20 minutes. The amount of starting material left in each sample was quantified by determination of total ion counts for the molecular ion. -58- WO 2005/090388 PCT/AU2005/000400 EXAMPLE 11 CD SPECTRA ON PEPTIDES HAVING ONE OR MORE CYCLIC PENTAPEPTIDE MODULES [02871 CD was performed on peptides having one [SEQ ID NO: 8] or more than one modular macrocycle [SEQ ID NOs: 46 and 47], as compared with corresponding linear peptides, 5 Ac[KARAD]f-NH 2 where n = 1 [SEQ ID NO: 14], n= 2 [SEQ ID NO: 54] and n = 3 [SEQ ID NO: 55]. The molar elipticities at 222 nm, 208 nm and 190 nm, ratios of elipticities at 222 nm/208 nm and percentage helicity are shown in Table 8. TABLE 8 Peptide [0]222 [0]208 [01192 0222/0208 % helicity* SEQ ID NO: 8 -15464 -17039 52191 0.98 79 SEQ ID NO: 46 -32340 -24957 104187 1.29 99 SEQ ID NO: 47 -31987 -23842 100811 1.34 88 SEQ ID NO: 14 -732 -5758 -14779 0.12 0.8 SEQ ID NO: 54 -1836 -8552 -12237 0.21 3 SEQ ID NO- 55 -3852 -9788 -8024 0.39. 7 * % fH X 100 , [OoH]222=- 4 4 000 degcm 2 dmol'residue 86, k = 2.685. 10 [0288] A CD Spectrum comparing the helicity of SEQ ID NOs: 46 and 47 with their acyclic linear analogues SEQ ID NOs: 54 and 55 is shown in Figure 6. EXAMPLE 12 [02891 To confirm this compelling CD evidence of high alpha helical structure, we have also examined NMR spectra for pentapeptide SEQ ID NO:46 in 90% H 2 0: 10% D 2 0. We identified 15 multiple spectral features (Figure 7) characteristic of alpha helicity including : (i) upfield CHot chemical shifts; 0 9 (ii) coupling constants 3 JNHCI 6Hz 11 0 for all amide NHs (2.2-5.2 Hz) except D 1 0 ; (iii) low temperature dependence of chemical shifts (AlT <4 ppb/deg) for 7 amide NHs," 1 consistent with all expected helix-defining H-bonds except for Ki-+Ds; and (iv) non-sequential medium range ROEs deN(i, i+3), d.N(i, i+4) and dap(i, i+3) in ROESY spectra.

100 In particular the high intensity dN(, 20 i+4) and very weak daN(i, i+2) ROEs are striking, indicating a lack of substantial contributions from beta or gamma turns to the conformational mix and establishing alpha rather than 310- helicity. [0290] Three dimensional structures were calculated for SEQ ID NO: 46 in water, initially using torsional angle dynamic simulated annealing in DYANA 1 2 , followed by dynamic simulated annealing and energy minimization in Xplor (3.851)113 from 89 ROE (24 sequential, 38 medium -59- WO 2005/090388 PCT/AU2005/000400 range, 27 intra-residue) distance restraints, 9 phi angle restraints ( 3 JNHCHa, 4 -65 ± 30*) and 2 chil angle restraints ( 3 JNHCHa, 1 -60 + 300). No explicit H-bond restraints were included in calculations. Final structures indicate 3 well-defined alpha helical turns for SEQ ID NO: 46 in water, with lactam bridges in the locations anticipated from Figure. 8. 5 [02911 The helical macrocycles were conformationally very stable even under protein denaturing conditions, as illustrated by the low dependence of their CD spectra on temperature between 5-65' C (Figure 9a) and on the concentration of guanidine.HCl (Figure 9b). [0292] In summary, 10- and 15- residue peptides were engineered to form 3 and 4 consecutive alpha helical turns via 2 and 3 macrocycles shown to maintain high conformational 10 stability in water even under strong protein-denaturing conditions. CD and 2D-NMR spectra provide compelling evidence of alpha helicity that could not be increased by adding TFE. Assembly of consecutive cyclic pentapeptide modules appears to be a suitable strategy for general mimicry of small alpha helical protein segments that bind receptors/ligands on one helical face. Their high conformational and proteolytic stability bring enormous advantages over linear peptides, and suggest 15 potential uses as biological probes and drug leads. EXAMPLE 13 TRYPSIN DIGEST [02931 Solutions of SEQ ID NO: 46 (25 pM) and linear peptide SEQ ID NO: 54 (26 LM) were incubated with trypsin (1 gg/mL) in 25 mM ammonium carbonate buffer (pH=8) at room 20 temperature. Aliquots were taken at 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour and 2 hours, and diluted with an equivalent volume of 3% trifluoroacetic acid. The resultant solutions were analyzed using a 2 x 75mm, 3 pm, Aqua C-18 column (Phenomenex) equilibrated in aqueous formic acid (0.1%). Peptide cleavage products were eluted using a linear gradient of acetonitrile from 0 to 80% in aqueous 0.01% formic acid over 20 minutes at a rate of 300 pL/min. Rate of degradation 25 of either SEQ ID NO: 32 or SEQ ID NO: 43 was quantified by determining extracted ion counts of chromatograms relative to control solutions (containing no enzyme) using a QSTAR PULSAR Electrospray QqTOF Mass Spectrometer and analyzed using BioMultiview (SCIEX Software). Retention time of SEQ ID NO: 46 = 11.64 minutes. Retention time of linear peptide SEQ ID NO: 54 = 7.43 minutes. SEQ ID NO: 46 was also found to be highly resistant to proteolytic cleavage by trypsin 30 (97% recovered intact after 2h), whereas the linear peptide Ac-KARADKARAD-NH 2 (SEQ ID NO: 54) was completely degraded within 30 seconds. - 60 - WO 2005/090388 PCT/AU2005/000400 EXAMPLE 14 HELIX MIMETICS AS ANTICANCER AGENTS BH3 Domain mimetics Background 5 [0294] Apoptosis, the process by which unwanted or damaged cells are removed during development and tissue homeostasis, has been implicated in several malignancies. The Bcl-2 family of proteins contains of several homologues that can display either pro- (Bad, Bak, Bid, Bim, Bax) or anti (Bcl-2, Bcl-xL, Bcl-W) apoptotic activity. In the case of malignancies, anti apoptotic members of the Bcl-2 family are usually up-regulated resulting in the survival of cancer cells, therefore synthetic 10 inhibitors and anti-apoptotic members are attractive chemotherapeutic agents 1 1 6 . The crystal/NMR structures of Bad-Bcl-xL (1g5j) 128 , Bak-Bcl-xL (lbxl) 127 , and Bim-Bcl-xL (lpq1) 1 29 demonstrate that pro-apoptotic members interact with Bcl-xL via and alpha helix (as indicated in Figure 10). Strategy [02951 The BH3 domains of pro-apoptotic members (Bad, Bak, Bid, Bim) interact with 15 target proteins (Bcl-2, Bcl-xL, Bcl-w) via a short region of alpha helix (indicated in Figure 10) with the general formula: Baa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Baa 5 -Xaa 6 -Xaa 7 -Baa-Xaag-Zaaio-Xaal 1 -Baan 2 [SEQ ID 56] [0296] Where subscript number indicates residue position, Xaa is any natural alpha amino acid, Baa is a hydrophobic alpha amino acid, and Zaa is a negatively charged alpha amino acid. 20 [0297] The necessity of alpha helicity for binding allows bicyclic mimetics to be constructed (around the 1-12 region of formula (VI) based on the general formula: 0 0 H ||H | 7N 7N

C

1 -K-Xaa-Xaa-Baa-D-K-Yaa-Xaa-Zaa-D-C 2 (VI) 25 [0298] Where C 1 is a hydrophobic N-terminal capping group, K is a lysine, D is an aspartate, Xaa is any natural or unnatural, Baa is any natural or unnatural hydrophobic alpha amino acid, Zaa is any natural or unnatural negatively charged alpha amino acid, C 2 is a hydrophobic C terminal capping group. [0299] Overlay of the NMR structure of the bicycle Ac-[KARAD][KARAD]-NH 2 [SEQ 30 ID NO:46] over the crystal structure of the Bad helix (Figure 10) shows it can position the side chains -61- WO 2005/090388 PCT/AU2005/000400 required for binding in the appropriate position. Examples of bicyclic molecules that can adopt this structure are given in Table 9. TABLE 9 Bicyclic Bad helix mimics cyclo(2-6,7-1 l)-Y[KRELD][KMADD]F SEQ ID NO: 57 cyclo(2-6,7-l l)-V[KRQLD][IADD]I SEQ ID NO: 58 cyclo(2-6,7-1 l)-I[KAQED][KVADD]M SEQ ID NO: 59 cyclo(2-6,7-11 )-I[KAQED][KIADD]F SEQ ID NO: 60 cyclo(2-6,7-1l)-3-(4-hydroxyphenyl)- SEQ ID NO: 61 propionyl[KRELD][KMADD]-phenethylamide cyclo(2-6,7-1 l)-iso-valeroyl[KRQLD][KIADD]2- SEQ ID NO: 62 methylbutylamide cyclo(2-6,7-11)-3-methylpentanoyl-[KAQED][KVADD]-3- SEQ ID NO: 63 methylsulfanyl-propylamide cyclo(2-6,7-1l)-3-methylpentanoyl-[KAQED][KLkDD]- SEQ ID NO: 64 phenethylamide 5 [0300] Similarly monocyclic mimetics based on the general formula (VII) 5-12 region with appropriate capping groups at either end based on the general formula: 0 H K N

C

1 -K-Xaa-Xaa-Zaa-D-C 2 (VII) 10 [0301] Where C 1 is an N-terminal capping group, Xaa is any amino acid, Zaa is any negatively charged natural or unnatural alpha amino acid, C 2 is a C-terminal capping group. An overlay of the NMR structure of the monocycle Ac-[KARAD]-NH 2 [SEQ ID NO 8] onto the crystal structure of the complexed Bad peptide places the required side chains in the appropriate positions (Figure 6). Examples of monocycles that can adopt the appropriate structure are given in Table 10. - 62 - WO 2005/090388 PCT/AU2005/000400 TABLE 10 Monocyclic Bad helix mimics with peptidic and/or non-peptidic flanking regions Cyclo(3,7)-LR[KMADD]F SEQ ID NO: 65 Cyclo(3,7)-LA[KIADD]I SEQ ID NO: 66 Cyclo(3,7)-LA[KVADD]I SEQ ID NO: 67 Cyclo(3,7)-LA[KIADD]F SEQ ID NO: 68 Cyclo(2,6)-7-methyl octanoyl-[KMADD]-Phenethylamide SEQ ID NO: 69 Cyclo(2,6)-7-methyl octanoyl-[KIADD]-2-methylbutylamide SEQ ID NO: 70 Cyclo(2,6)-7-methyl octanoyl-[KVADD]- 2-methylbutylamide SEQ ID NO: 71 Cyclo(2,6)-7-methyl octanoyl-[KMADD]-Phenethylamide SEQ ID NO: 72 Fluorescence polarization assay 5 [0302] Binding affinities can be measured relative to the native peptide by competitive fluorescence polarization assay using the fluorescein labeled Bad peptide Flu pANLWAAQRYGRELRRMSDKKFVDSFKK-NH 2 as a probe. The dissociation constant of this peptide for Bcl-xL is 0.6 nM 130 and for Bcl-2 is 3 nM 1 31 . The assay is performed according to Zhang et al.

131 Briefly, a stock solution of 5 nM of the fluorescein labeled Bad peptide, 25nm of Bcl-2, 1 mM 10 EDTA, and 0.05% PEG-8000 in 2Q mM phosphate buffer pH 7.4 is prepared. 120 pL of this solution is placed into 8 wells of a 96 well plate, and 25, 5, 1, 0.5, 0.25, 0.1, 0.05, 0.01 pM compound from a DMSO stock. The total DMSO concentration is adjusted to 5% by the addition of neat DMSO. A negative (5nm Flu-Bad, 5% DMSO in buffer), and positive control (5 nm Bad peptide, 25 nm Bcl-2, 5% DMSO in buffer) were used to determine the free and bound polarization values for the assay. The 15 plate is incubated for 2hr at room temperature, and then polarization was measured at room temperature using a BMG FLUOstar fluorescence polarization spectrometer with excitation at 485nm and emission at 530nm. IC 50 values were obtained by non-linear least squares fitting of the data to the equation: 20 Observed polarization =(bound polarization - free polarization)1+1 0 (Log ICso-Concentration) p53 Tumor suppressor mimetics Background [03031 The protein p53 acts as a potent tumor suppressor that prevents the proliferation of 25 malignant cells causing cell cycle arrest or apoptosis, however it is the most frequently inactivated -63 - WO 2005/090388 PCT/AU2005/000400 protein in human cancers 132 . p53 is tightly controlled by the protein MDM2, however its up-regulation results in the proliferation of cancer cells. It has been demonstrated that inhibitors of MDM2 can restore the cell cycle arrest of apoptotic mechanism resulting in the destruction of cancer cells 3 a indicating that p53 binds to MDM2 via and alpha helical conformation. Furthermore is has been 5 demonstrated that synthetic peptides corresponding to the p53 sequence, that contain helix inducing residues such as Aib (ac-amino isobutyric acid) possess a higher affinity for MDM2 than the native peptide 1 3 s Strategy [0304] The interaction of p53 with MDM2 depends on 3 critical hydrophobic contacts in a 10 12 residue sequence indicated by the general formula indicated below (FB) Xaa-Xaa-Xaa-Baa-Xaa-Xaa-Xaa-Baa-Xaa-Xaa-Baa-Xaa (FB) Where Xaa is any natural amino acid and Baa is any natural hydrophobica-amino acid. 15 [0305] Modification of this sequence by others incorporating helix inducing amino acid residues has resulted in high affinity ligands for MDM2. Acetyl-Phe-Met-Aib-Pmp-6CW-Glu-Ac 3 c-Leu-NH 2

IC

50 =5 nm 20 [0306] Where Aib is a-aminoisobutyric acid, Pmp is 4-(Phosphonomethyl)-L phenylalanine, 6ClW is 6-Chloro-L-Tryptophan, and Ac 3 c is 1 -aminocyclopropane carboxylic acid. [0307] Application of the monocycle prototype would constrain the p53 sequence into the required conformation for binding to MDM2, resulting in high affinity, metabolically stable anticancer agents of the general formula (VIII). 25 0 H 7 N

C

1 -K-Zaa-Baa-Xaa-D-C 2 (VIII) [0308] Where C 1 is a hydrophobic N-terminal capping group (natural or unnatural a amino acid or dipeptide, aliphatic or aromatic carboxylic acid), K is a lysine, Zaa is a negatively, 30 natural or unnatural a-amino acid, Baa is a hydrophobic, natural or unnatural a-amino acid preferably an L-tryptophan derivative with halogen or alkyl substitution in the 5 or 6 position, Xaa is any natural or unnatural a-amino acid , D is aspartate , and C 2 is a hydrophobic C-terminal capping - 64 - WO 2005/090388 PCT/AU2005/000400 group (natural or unnatural c-amino acid or dipeptide, aliphatic or aromatic amine). Examples of molecules of this sort could include: Cyclo(3,7)-FM[K(Pmp)(6ClW)ED]L SEQID: 73 Cyclo(3,7)-3-Phenylpropanoyl-M[K(Pmp)(6ClW)ED] SEQID: 74 isopentylamide Cyclo(2,6)-6-Phenylheptanoyl-[K(Pmp)(6CIW)ED]-isopentylamide SEQID: 75 5 [0309] Overlay of the NMR structure of monocycle on the crystal structure of p53 shows that the monocycle places the binding residues in the required positions (Figure 11). Fluorescence polarization assay [0310] Binding affinities can be measured relative to the native peptide by competitive fluorescence polarization assay using the fluorescein labeled p53 peptide Ac-FR(Dpr-Flu)(Ac 6 c)(6-Br 10 W)EEL-NH 2 as a probe. The dissociation constant of this peptide for MDM2 is 2 nM (5). The assay is performed according to Zhang et. al."'. Briefly, a stock solution of lOnM of the fluorescein labeled p53 peptide, 30 nM of MDM2, 1 mM EDTA, and 0.05% PEG-8000 in 20 mM phosphate buffer pH 7.4 is prepared. 120 tL of this solution is placed into 8 wells of a 96 well plate, and 25, 5, 1, 0.5, 0.25, 0.1, 0.05, 0.01 ptM compound from a DMSO stock. The total DMSO concentration is adjusted to 5% 15 by the addition of neat DMSO. A negative (10 nM Flu-p53, 5% DMSO in buffer), and positive control (10 nM Flu-p53 peptide, 30 nM MDM2, 5% DMSO in buffer) were used to determine the free and bound polarization values for the assay. The plate is incubated for 2 hr at room temperature, and then polarization was measured at room temperature using a BMG FLUOstar fluorescence polarization spectrometer with excitation at 485 nm and emission at 530nm. IC 50 values were obtained by non 20 linear least squares fitting of the data to the equation: Observed polarization =(bound polarization - free polarization)1+10(Log IC50-Concentration) EXAMPLE 15 25 GPCR AGONISTS/ANTAGONISTS Pain/Addiction therapies: ORL-i receptor agonists antagonists Background [0311] The function of the ORL-1 receptor includes pain transmission, stress and anxiety, learning and memory, locomotor activity, food intake, motivational properties of drugs of abuse 3 - 65 - WO 2005/090388 PCT/AU2005/000400 Agonists of the ORL-1 receptor have likely uses for anti-anxiety therapy, appetite suppressants, alcohol and opiate withdrawal therapies, anti-epilepsy drugs. Antagonists of the ORL-1 receptor have likely uses for analgesia/pain therapy, alleviation of memory disorders". Strategy 5 [0312] The receptor is activated by the endogenous ligand nociceptin which has the sequence:

F

1

-G

2

-G

3

-F

4

-T

5

-G

6

-A

7 -R-K-Sio-All-R 12

-K

13

-L

14 -Ai 5 10 [0313] Portions of this sequence serve different roles, the'four N-terminal residues (FGGF) serves as a 'message sequence', actually activating the receptor, whilst the remaining sequence serves as an 'address' and is responsible for receptor binding and specificity, SAR data indicated that the dibasic repeat (residues 8,9 and 12,13) is especially important for binding 1 38 . On the basis of NMR evidence and SAR, it has been proposed that the address sequence binds to the ORL-1 15 receptor in an ac-helical conformation 1 39 . Further support for this hypothesis has been evidenced in the synthesis of nociceptin analogues containing Aib residues (which are known helix promoters) resulting in 10-fold increases in affinity of these peptides for the ORL-1 receptor. Based on previous data 1 40 it appears constraining the address sequence into an ca-helical conformation would improve activity. Therefore mimetics based on constraining the address sequence could provide highly active 20 and selective agonists for treating anxiety, obesity, epilepsy and drug/alcohol addiction. Replacing the N terminal residue from a Phenylalanine (F) and N-benzylglycine (Nphe) switches the activity from agonism to antagonism, it has also been shown that attaching non-selective, non-peptidic opioid antagonists to the nociceptin address sequence can target the antagonists selectively to the ORL-1 receptor 141 which could be useful therapeutics for chronic pain. Thus the proposed agonists/antagonists 25 would bear the general formula (IX): 0 0 HH N N

C

1 -K-Xaa-R-K-D-K-R-K-Xaa-D-C 2 (IX) [0314] Where C 1 is a suitable N-terminal capping group (1-5 natural or unnatural a-amino 30 acids, aliphatic/aromatic carboxylic acid or non-peptidic opioid antagonist), K is lysine, Xaa is any amino acid, D is aspartate, C 2 is a suitable C-terminal capping group (primary or an aliphatic/aromatic secondary amide). Compounds of this type could include: - 66 - WO 2005/090388 PCT/AU2005/000400 Cyclo(6-10,11-15)-FGGFT[KARKD][KRKLD]-NH 2 (agonist) SEQ ID NO: 76 Cyclo(6-10,11-15)-NpheGGFT[KARKD][KRKLD]-NH 2 (antagonist) SEQ ID NO: 77 Cyclo(2-6,7-1 1)-Ac-T[KARKD][KRKLD]-NH 2 (antagonist) SEQ ID NO: 78 Cyclo(2-6,7-1l)-(8-napthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza- SEQ ID NO: 79 spiro[4,5]dec-3-yl)-acetoyl-[KARKD][KRKLD]-NH 2 (antagonist) [03151 Furthermore the endogenous ligand is known to be cleaved by endopeptidases at the several positions 137 (see below). Given that the monocyclic and bicyclic strategy prevent cleavage by proteases within the cycle, the mimetics described above would be expected to be metabolically 5 stable. endopeptidase cleavage sites FGGFTGARKSARKLANQ Assay: Binding Affinity 10 [03161 Membranes from recombinant HEK-293 cells expression hORL-l are prepared as described in (Zhang et al.) 140 . The above peptides are incubated with 0.1 nM [ 3 H]-nociceptin, 20 pg membrane protein/well in a final volume of 500 pL of binding buffer in 96-well plates for 2 hr at room temperature. Binding reactions are terminated by rapid filtration onto 96well Unifilter GF/C filter plates pre-soaked in 0.5% polyethylenamine using 96-well tissue harvester, followed by three 15 washes with ice cold binding buffer. Filter plates are dried at 500 C for 3 hrs, and then scintillation cocktail is added (50 pg/well) and plates counted in a scintillation counter for 1min/well. Data is analyzed using the one-site competition curve fitting function in PRISM. [0317] Incorporation of the bicyclic strategy as indicated above.has been shown by CD spectroscopy to increase helicity in 76 and 77 compared to the reported native agonist and antagonist 20 sequences (F/Nphe-GGFTGARKSARK) as indicated in Figure 12. EXPERIMENTAL MATERIALS AND METHODS GENERAL [03181 Fmoc-Asp(OAllyl)-OH and tetrakis(triphenylphosphino)palladium were obtained 25 from Sigma-Aldrich (Sydney, Australia). Boc-Lys(Fmoc)-OH, Rink Amide MBHA resin and other L amino acids were obtained from Novabiochem (Melbourne, Australia). Benzotriazol-1-yl-1,1,3,3 tetramethyluronium (HBTU) and benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium (BOP) were - 67 - WO 2005/090388 PCT/AU2005/000400 obtained from Richelieu Biotechnologies (Quebec Canada). All other reagents were of peptide synthesis grade and obtained from Auspep (Melbourne, Australia). NMR SPECTROSCOPY [03191 Samples for NMR analysis of peptides were prepared by dissolving the peptide 5 3mg in 450ul H20 and 50ul D 2 0 (5mmol) and adjusting the pH of the solution to 4.5 by adding HCl or NaOH and stirring for 30 min. 1D and 2D 11 NMR spectra were recorded on both Bruker ARX-500 and Bruker Avance DMX-750 spectrometers at 278K. All spectra were recorded in the phase sensitive mode using time proportional phasing incrementation 96 . 2D experiments included TOCSY using MLEV-17 spin lock sequence with a mixing time of 100ms, NOESY with a mixing time of 300ms. 10 Water suppression was achieved using watergate W5 pulse sequences with gradients using double echo 97 . 2D TQCSY and NOESY experiments were recorded over 7936.5 Hz with 4096 complex data points in F2 and 512 increments in F1 with 16 and 48 scans per increment respectively. Spectra were processed using XWINNMR (Bruker, Germany). The t1 dimensions of all 2D spectra were zero filled with 2048 real data points, and 90* phase-shifted sine bell window functions applied in both. 15 dimensions followed by fourier transformation and fifth order polynomial baseline correction. Chemical shifts were referenced to TSP an internal standard at 0.00ppm. Processed spectra were analyzed using the program SparkyNMR 98 and assigned using the sequential assignment technique". STRUCTURE CALCULATIONS [0320] Cross peaks in NOESY spectra were integrated and calibrated in SparkyNMR 98 , 20 and distance constraints derived using the standard CALIBA function in DYANA"'. Corrections for pseudo atoms were added to distance constraints where needed. Backbone dihedral angle restraints were inferred from 3 JNHCHa coupling constants in 1D spectra at 278K and 288K, # was restrained to 65 300 for 3 JNHCHa 6Hz. Peptide bond o angles were all set to trans, and structures were calculation without explicit hydrogen bond restraints. Stereospecific assignments of p-methylene protons and X, 25 dihedral angles were derived from 1D 'H spectra 3 Jap and set to -60 ± 30' for both aspartic acid residues. Initial structures were generated using a torsion angle simulated annealing protocol in DYANA until no violations were obtained. Final structures were calculated using XPLOR 3.851. Starting structures with randomized * and y angles and extended side chains were generated using an ab initio simulated annealing protocol' 0 '. The calculations were performed using the standard 30 forcefield parameter set (PARALLHDG.PRO) and topology file (TOPALLHDG.PRO) in XPLOR with in house modifications to generated lactam bridges between lysing and aspartic acid residues. Refinement of structures was achieved using the conjugate gradient Powell alogrithm with 1000 cycles of energy minimization and a refined forcefield based on the program CHARMmi 0 2 . Structures were visualized with MOLMOL 0 3 and InsightII" 4 . -68- WO 2005/090388 PCT/AU2005/000400 CD SPECTROSCOPY [0321] CD experiments were performed on a Jasco Model J-710 spectropolarimeter which was routinely calibrated with (1S)-(+)-10-camphorsulfonic acid. Temperature control was achieved using a Neslab RTE-1 11 circulating water bath. Spectra were recorded in a 0.1 cm Jasco cell between 5 310-185 nm at 50 nm/min wit a band width of 1.Onm, response time of 2 sec, resolution step width of 0. lnm and sensitivity of 20, 50 or 100 mdeg. Each spectrum represents the average of 5 scans with smoothing to reduce noise. Peptide samples for CD spectroscopy were dissolved in distilled water (~1 mg/mL). Each stock solution was diluted to a final concentration of 50 pM in 10 mM sodium phosphate buffer (pH7.4), with or without additives (2,2,2-trifluoroethanol (TFE) or guanidine.HCl). 10 Guanidine.HCI denaturation experiments were performed according to Creighton' 05 . [0322] Accurate concentration determination of stock solutions were obtained by ID 'H NMR using the method of Larive et. al.' 06 Briefly 475 gL of the initial peptide stock solution was mixed with 50 pL of D 2 0, and spiked with an internal standard 25 pL of 10.077mM DSS (the concentration of which had carefully been back-calibrated by NMR from a standard solution of L 15 tryptophan - the concentration of which was determined by UV 827=5579). 1D 'H NMR spectra were then recorded with presaturation and a relaxation delay (dl) of 30 seconds to allow for full relaxation of peptide and DSS 11 signals to facilitate accurate integration of proton signals with S/N >250:1. This method provides reproducible concentrations within 2%. CIRCULAR DICHROISM DATA ANALYSIS 20 [0323] CD data in ellipticity was converted to mean peptide ellipticity using the equation: [0] = 0 /(10 x C x N, x 1) where 0 is the ellipticity in millidegrees, C is the peptide molar concentration (M), I is the cell path length (cm), and N, is the number of peptide units (ie. pentapeptides N,=6, hexapeptides N,=7, 10 & 11 Np=5, 12 N, = 4.) 25 [0324] Relative helicities were employed to allow scaling of compounds by dividing the [6]obs215( 8 )/ [O]ob,215(X) for pentapeptides, and [0]obs21s( 2 4 )/ [6]obs21s(X) for hexapeptides. [0325] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [0326] The citation of any reference herein should not be construed as an admission that 30 such reference is available as "Prior Art" to the instant application. [0327] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified - 69 - WO 2005/090388 PCT/AU2005/000400 without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims. - 70 - WO 2005/090388 PCT/AU2005/000400 BIBLIOGRAPHY (1) Barlow, D. J.; Thornton, J. M. J. Mol. Biol. 1988, 201, 601. (2) Fairlie, D.; West, M.; Wong, A. Curr. Med. Chem. 1998, 5, 29. (3) Andrews, M. J. I.; Tabor, A. B. Tetrahedron 1999, 55, 11711. 5 (4) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P. Science 1996, 274, 948-953. (5) Burley, S. K.; Roeder, R. G. 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Claims (39)

1. A compound having a plurality of alpha helical cyclic pentapeptide sequences, which is represented by formula (IV): (IV) (CR'R)m- L (CR R)r H H R 1 N-C(R)-C-Xaa-Xaa-Xaa-N- C(R')-C 0 0 5 -P wherein the Xaa are L-alpha amino acid residues 2 or 3 of which are favorable to alpha helix formation; or wherein the Xaa are D-alpha amino acid residues 2 or 3 of which are favorable to alpha helix 10 formation; wherein respective alpha helical cyclic pentapeptide sequences have at least three (1,5) intramolecular hydrogen bonds; R, is selected from H, an N-terminal capping group, a peptide of I to 5 amino acid residues optionally capped by an N-terminal capping group, a non-peptidic group or a 15 group that mimics an amino acid side chain; R 2 is selected from H, a C-terminal capping group, a peptide of I to 5 amino acids optionally capped by a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; 20 each R'and R" are independently selected from H, Ci-Cjoalkyl, C 2 -Coalkenyl, C 2 Cloalkynyl, C 3 -Cocylcoalkyl, C-Clocycloalkenyl, -OH, -OCI-Cloalkyl, -NH 2 , NH(CI-Cloalkyl), -N(CI-Cioalkyl) 2 , C 6 -Cloaryl, C 3 -Coheterocyclyl, C 5 -Cloheteroaryl and halo; L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH 2 -, CH 2 CH(OH)-, 25 CH=CH-, -CH 2 -CH 2 -, -NH-CH 2 - -CHr-NH-, -CH 2 -S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 C(O)-, -S(O),-NH-, -NH-S(O),-, CH 2 -P(=0)(OH)- and -P(=O)(OH)-CH 2 -; m is 4, n is 1, -79- t is 0, 1 or 2, and p is an integer from 2 to 4.

2. A compound according to claim 1, wherein the amino acid residues that are favorable to 5 alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid. . 3. A compound according to claim 1, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine. 10 4. A compound according to any one of claims I to 3, wherein an individual pentapeptide sequence is a macrocycle formed by consecutively linking at least 18 to 22 atoms, wherein the first and last atoms are bonded to one another to form a ring.

5. A compound according to claim 4, wherein the macrocycle is formed from 19 to 21 atoms

6. A compound according to claim 4, wherein the macrocycle is formed from 20 atoms. 15 7. A compound according to any one of claims I to 3, wherein the amino-terminal and carboxy-terminal residues of an individual pentapeptide sequence are Lys and Asp, respectively.

8. A compound according to any one of claims I to 3, wherein the amino acid side chains of the amino-terminal and carboxy-terminal residues of an individual pentapeptide sequence are covalently linked to one another by a lactam bridge between a side chain amino group and a side chain 20 carboxylic acid group.

9. A compound according to any one of claims I to 3, wherein individual pentapeptide sequences are different.

10. A compound according to any one of claims I to 3, wherein individual pentapeptide sequences in the peptide are the same. 25 11. A compound according to any one of claims I to 3, selected from: cyclo(I-5, 6-10)-Ac-[KARADKARAD]-NH 2 [SEQ ID NO: 46); and cyclo(I-5, 6-10, 1 1-15)-Ac-[KARADKARADKARAD]-NH 2 [SEQ ID NO: 47].

12. A compound having the formula (1): - (I) (CR'R)m- L-(CR'R)n H H R-N-C(R)-C-Xaa-Xaa-Xaa-NC(R) 30 0 -80- wherein the Xaa are L-alpha amino acid residues 2 or 3 of which are favorable to alpha helix formation; or wherein 2 or 3 of the Xaa. are D-alpha amino acid residues 2 or 3 of which are favorable to alpha helix formation; 5 wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tryptophan, histidine and aspartic acid; wherein the compound has at least three (1,5) intramolecular hydrogen bonds; R is selected from H, an N-terminal capping group, a non-peptidic group or a group that 10 mimics an amino acid side chain, an amino acid residue optionally capped by an N terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain; R 2 is selected from H, a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to 15 nucleophilic substitution, an amino acid residue optionally capped by a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; each R'and R" are independently selected from H, C 1 -Cioalkyl, C 2 -Cloalkenyl, C 2 Cioalkynyl, C 3 -Clocylcoalkyl, C 5 -Ciocycloalkenyl, -OH, -OCI-Cloalkyl, -NH 2 , -NH(C 20 Cloalkyl), -N(CI-Cloalkyl) 2 , C 6 -Cl 2 aryl, C 3 -Cloheterocyclyl, C-Cioheteroaryl and halo; L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH 2 -, CH 2 CH(OH)-, -CH=CH-, -CH 2 -CH 2 -, -NH-CH 2 - -CH 2 -NH-, -CH 2 -S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 -C(O)-, -S(O), NH-, -NH-S(O),-, CH 2 -P(=0)(OH)- and -P(=O)(OH)-CH-; m is 4, 25 n is 1, and t is 0, 1 or 2.

13. A compound according to claim 12, wherein Ri is selected from H; an N-terminal capping group that stabilizes the terminus of a helix; a non-peptidic group; or a mimic of an amino acid side chain. 30 14. A compound according to claim 13, wherein the N-terminal capping group is selected from acyl and N-succinate.

15. A compound according to claim 13, wherein the mimic of the amino acid side chain is selected from any natural or unnatural amino acid side chain that is attached to the N-terminal amino group of the peptide through a carbonyl group derived from a carboxylic acid by formation of an 35 amide bond. -81 -

16. A compound according to claim 13, wherein the mimic of the amino acid side chain is selected from: CH 3 CH2C(O)(Cl 2 )uC(O)-, NH 2 (NH=)CNHC(O)(CH 2 )uC(O)-, 'H2NC(O)(CH2)2C(O)(C H2)uC(0)-, HOC(O)(CH2)2C(0)(CH2)uC(O)-, HS(CH2)2C(O)(CH2)uC(0)-, H2NC(O)(CH2)3C(O)(CH2)uC(0)-, HOC(0)(CH2)2C(0)(CH2)uC(O)-, (4 5 imidazolyl)(CH 2 )C(O)(CH 2 ),C(O)-, CH 3 CH 2 CH(CH 3 )CH 2 C(O)(CH 2 )uC(O)-, (CH 3 ) 2 CH(CH 2 ) 2 C(O)(CH 2 )uC(O)-, H 2 N(CH 2 ) 5 C(O)(CH 2 )uC(O)-, CH 3 S(CH 2 ) 3 C(O)(CH 2 ).C(O)-, Ph(CH 2 ) 2 C(O)(CH 2 )uC(O)-, Ph(CH 2 ) 4 C(O)(CH 2 )uC(O)-, HO(CH 2 ) 2 C(O)(CH 2 )uC(O)-, HOCH(CH 3 )CH 2 C(O)(CH 2 )uC(O)-, (3-indolyl)(CH 2 ) 2 (CH 2 )uC(O)-, (4 hydroxyphenyl)(CH 2 ) 2 C(O)(CH 2 )uC(O)-, (4-hydroxyphenyl)(CH 2 ) 3 C(O)(CH 2 ) 1 C(O)-, 10 (CH 3 ) 2 CHCH 2 C(O)(CH 2 )uC(O)-, CH 3 CH 2 CH 2 C(O)(CH 2 )uC(O)-, C 6 H 1 oCH 2 C(O)(CH 2 ).C(O)-, CsH 8 CH 2 C(O)(CH 2 )uC(O)-, CH 3 C(O)(CH 2 )uC(O)-, CH 3 (CH 2 ) 4 C(O)(CH 2 )uC(O)-, CH 3 (CH 2 ) 5 C(O)(CH 2 ) 1 C(O)-, HOC(O)CH 2 C(O)(CH 2 ), 1 C(O)-, HS(CH 2 )C(O)(CH 2 )uC(O)-, H 2 N(CH 2 ) 4 C(O)(CH 2 )uC(O)- and HOCH 2 C(O)(CH 2 )uC(O)- wherein u is 0 or an integer from I to 10.

17. A compound according to claim 13, wherein the non-peptidic group enhances the stability, 15 bioavailability or activity of the peptides.

18. A compound according to claim 13, wherein the non-peptidic group is selected from: hydrophobic groups; groups which stabilize or mimic alpha-helices, groups which mimic the secondary structure of peptides; groups which improve bioavailability; and groups which are recognized by transport receptors to allow or improve transport of the peptides to the site of activity. 20 19. A compound according to claim 12, wherein R 2 is selected from: H; a C-terminal capping group that stabilizes the terminus of an alpha helix; a peptide of 1, 2, 3, 4 or 5 amino acid residues optionally capped with a C-terminal capping group that stabilizes the terminus of an alpha helix; a mimic of an amino acid side chain; or a group which activates the terminal carboxylic acid carbonyl group to nucleophilic substitution. 25 20. A compound according to claim 19, wherein the C-terminal capping group is NH 2 .

21. A compound according to claim 19, wherein the mimic of the amino acid side chain is any common or unnatural amino acid side chain that is attached to the C-terminal carbonyl group of the peptide through an amine group by formation of an amide bond.

22. A compound according to claim 19, wherein the mimics of the amino acid side chain is 30 selected from: -NH(CH 2 )uNHCH 2 CH 3 , -NH(CH 2 )uNH(CH 2 ) 4 NHC(=NH)NH 2 , NH(CH 2 )uNH(CH 2 ) 2 C(O)N H 2 , -NH(CH 2 )uN H(CH 2 ) 2 CO 2 H, -NH(CH 2 ) 1 NH(CH 2 ) 2 SH, NH(CH 2 )uNH(CH 2 ) 3 C(O)NH 2 , -NH(CH 2 ).NH(CH 2 ) 3 CO 2 H, -NH(CH 2 )uNH(CH 2 ) 2 (4-imidazolyl), NH(CH 2 )uNHCH 2 CH(CH 3 )CH 2 CH 3 , -NH(CH 2 )uNH(CH 2 ) 2 CH(CH 3 ) 2 , -NH(CH 2 )uNH(CH 2 )sNH 2 , NH(CH 2 )uN H(CH 3 ) 3 SCH 3 , -N H(CH 2 ),,NH(CH 2 ) 2 (3-indolyl), -NH(CH 2 )uNH(CH 2 ) 2 (4-hydroxyphenyl), 35 -NH(CH 2 ) 1 NH(CH 2 ) 3 (4-hydroxyphenyl), -NH(CH 2 )uNH-CH 2 CH(CH 3 ) 2 , -NH(CH 2 )uNHCH 2 CH 2 CH 3 , NH(CH 2 )uNHCH 2 C 6 H o, -NH(CH 2 )uNHCH 2 CsH 8 , -NH(CH 2 )uNHCH 3 , -NH(CH 2 )uNH(CH 2 ) 4 CH 3 , NH(CH 2 )uNH(CH 2 )sCH 3 , -NH(CH 2 )uNHCH 2 CO 2 H, -NH(CH 2 ),NHCH 2 SH, -NH(CH 2 )uNH(CH 2 ) 2 OH, -NH(CH 2 )uNH(CH 2 )sNH 2 and -NH(CH 2 )uNHCH 2 OH; wherein u is 0 or an integer from I to 10. -82-

23. A compound according to claim 19, wherein the group, which activates the C-terminal carboxylic carbonyl group to nucleophilic substitution, converts the C-terminal carboxylic acid to a group selected from an acid chloride, an acid anhydride, an acyl azide, an 0-acylisourea, a phosphonium derivative or an activated ester. 5 24. A compound according to claim 19, wherein the non-peptidic group enhances the stability and circulating time, or decrease immunogenicity, or increase solubility, bioavailability or activity of the peptides,

25. A compound according to claim 13, wherein the non-peptidic group is selected from: hydrophobic groups; groups which stabilize or mimic alpha-helices; groups which mimic the 10 secondary structure of peptides; groups which improve bioavailability; groups that are recognized by transport receptors to allow or improve transport of the peptide(s) to the site of activity.

26. A compound according to claim 12, wherein each R9 is selected from H, C13, CH2CH 3 , vinyl, OH, OCH 3 , NH 2 , Ni(CH 3 ), N(CH 3 )2, phenyl, F or Cl.

27. A compound according to claim 12, wherein each RO is selected from H, CHa, CH 2 CH 3 or 15 vinyl.

28. A compound according to claim 12, wherein each Xaa is selected to mimic amino acid residues in a known alpha helical peptide of interest.

29. A compound according to claim 12, wherein an individual Xaa is the same or different as another Xaa , 20 30. A compound according to claim 12, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine.

31. A compound according to claim 12, wherein L is -NH-C(O)- or -C(O)-NH-,

32. A compound according to claim 12, wherein the amino-terminal and carboxy-terminal 25 residues of the compound are Lys and Asp, respectively.

33. A compound according to claim 12, selected from: Ac.(cyclo-l,5)-[KARAD]-NH2, [SEQ ID NO, 81; AcR-cyclo-2,6-[KLLLD]-NH 2 , [SEQ ID NO. 15]; AcR-cyclo-2,6-[KLALD]-NHz, (SEQ ID NO. 16]; 30 AcR-cyclo-2,6-[KLFAD-NH2, [SEQ ID NO. 17]; Ac-(cyclo-l,5)-[KARAD]-OH, [SEQ ID NO. 20]; H-(cyclo-1,5)-[KARAD]-NH 2 , (SEQ ID NO. 21]; H-(cyclo-l,5)-(KARAD]-OH, [SEQ ID NO. 221; Ac-(cyclo-2,6)-R[KAAAD]-NH2, [SEQ ID NO. 23]; 35 Ac-(cyclo-2,6)-R[KALAD]-NH2, [SEQ ID NO. 24]; Ac-(cyclo-2,6)-R[KAMAD]-NH 2 , [SEQ ID NO. 25]; Ac-(cyclo-2,6)-R[KAQAD]-NHz, [SEQ ID NO. 26]; Ac-(cyclo-2,6)-R[KAFAD)-NH 2 , [SEQ ID NO, 27]; - 83 - Ac-(cyclo-2,6)-R{KAGAD]-NH 2 , [SEQ ID NO, 28]; Ac-(cyclo-2,6)-R[KGSAD]-NH2, [SEQ ID NO. 29]; Ac-(cyclo-2,6)-R[KSSSD)-NH 2 , [SEQ ID NO. 30]; and Ac-(cyclo-2,6)-R[KGGGD]-NHt [SEQ ID NO, 31] 5 34. A method for constructing a constrained helical peptide, the method comprising: (1) synthesizing a peptide that comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the individual side chains of the first and second terminal residues are linkable to each other; and (2) cyclizing the peptide by linking the side chain of the first terminal 10 residue with the side chain of the second terminal residue, thereby yielding an alpha helical cyclic peptide, wherein 2 or 3 of the three amino acid residues inthe intervening sequence are 0- or L- alpha amino acid residues and are selected on the basis that they are favorable to alpha helix formation and wherein the alpha helical peptide so constructed has at least three (1,5) intramolecular hydrogen bonds. 15 35. A method according to claim 34, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid.

36. A method according to claim 34, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid 20 and glutamine.

37. A method according to any one of claims 34 to 36, wherein the first terminal residue has a side chain containing an amide bond-forming substituent and the second terminal residue has a side chain containing a functional group capable of forming an amide linkage with the side chain aide bond-forming substituent of the first terminal residue and the peptide is cyclized by reacting the side 25 chain aide bond-forming substituent of the first terminal residue with the functional group of the second terminal residue to form an aide bond linkage, thereby yielding an alpha helical cyclic peptide. 3$. A method according to any one of claims 37 to 40, wherein in step (1) the reactive groups on the side chains, including the amide forming substituents, are protected by a protecting group. 30 39. A method according to any one of claims 37 to 40, wherein the reactive groups on the side chains, including the amide forming substituents, are deprotected prior to cyclization.

40. A method according to any one of claims 37 to 40, wherein step (2) comprises activating the carboxylic acid to nucleophilic attack by forming an acyloxyphosphonium or uronium derivative of the carboxylic acid. 35 41. A method of producing a mimic of an alpha helical binding determinant, comprising: providing a protein of interest that comprises an alpha helical domain that interacts with a ligand; identifying a candidate binding determinant situated within a sequence of 3 or more contiguous amino acid residues in the alpha helical binding domain; selecting a first residue and a second residue in the -84- sequence (designated i and i+4), which are separated by an intervening sequence of 3 amino acid residues, and which do not do not interact substantially with the ligand, for linkage to each other, wherein 2 or 3 of the amino acid residues in the intervening sequence are selected on the basis that they are favorable to alpha helix formation; and synthesizing a peptide that comprises the first and 5 second residues and the intervening sequence and linking the side chains of the first and second residues, wherein the peptide so synthesized has at least three (1,5) intramolecular hydrogen bonds,

42. A method according to claIm 41, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid, 10 43. A method according to claim 41, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine.

44. A method according to any one of claims 41 to 43, wherein the binding determinant is identified using mutagenesis, 15 45, A method according to claim 41 to 44, fWther comprising detecting binding of the peptide to the ligand. 46, A composition comprising a compound according to any one of claims I to 33 and a pharmaceutically acceptable carrier, diluent or adjuvant,

47. Use of a compound having at least one alpha helical cyclic pentapeptide sequence in the 20 manufacture of a medicament for treating or preventing a disease or condition associated with a ligand-receptor interaction that Is mediated at least in part by an alpha helical domain present in the ligand or the receptor, wherein each pentapeptide sequence comprises a sequence of five amino acid residues having a first terminal residue and a second terminal residue that are separated by an intervening sequence of three amino acid residues, and wherein the side chains of the first and second 25 terminal residues are linked to each other and wherein the side chains of at least some of the amino acid residues of the or each pentapeptide sequence are in a (three-dimensional) configuration that is analogous to the configuration of amino acid side chains of at least a portion of the alpha helical domain of the ligand or the receptor, and wherein an individual -pentapeptide sequence has at least three (1,5) intramolecular hydrogen bonds, wherein the compound is represented by formula (IV) or 30 formula (1), as follows: (IV) (CR'R)m-l-L (CRR)n H R 1 -- N- C(R")- -- Xaa- Xaa-Xaa----C(R- C--R2 p -85- wherein the Xaa are L-alpha amino acid residues 2 or 3 of which are favorable to alpha helix formation; or wherein the Xaa are D-alpha amino acid residues 2 or 3 of which are favorable to alpha helix 5 formation; R, is selected from H, an N-terminal capping group, a peptide of I to 5 amino acid residues optionally capped by an N-terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain; R2 is selected from H, a C-terminal capping group, a peptide of I to 5 amino acids 10 optionally capped by a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; each R' and R" are independently selected from H, C 1 -Cioalkyl, C-Cloalkenyl, Cr Cloalkynyl, CrCi ocylcoalkyl, CrCi 0 cycloalkenyl, -OH, -OCI-Cloalkyl, -NH 2 , 15 NH(C-Cioalkyl), -N(Cr-Cioalkyl) 2 , C 6 -Cioaryl, C 3 -Cioheterocyclyl, C-Cloheteroaryl and halo; L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)CH-, CH 2 CH(OH)- CH=CH-, -CH-CH 2 -, -NH-CHr -CH 2 -NH, CHrS, -S-CH, -C(O)CH 2 -, -CHr C(O)-, -S(O),-NH-, -NH-S(O),-, CH 2 -P(=O)(OH)- and -P(O)(OH)-CH 2 -; 20 m is 4, n is 1, t is 0, 1 or 2, and p is an integer from 2 to 4; 25 (I) - (CRR)m-L-(CR'R)n H H wherein the Xaa are L-alpha amino acid residues 2 or 3 of which are favorable to alpha helix 30 formation; or -86- wherein the Xaa are D-alpha amino acid residues 2 or 3 of which are favorable to alpha helix formation; R, is selected from H, an N-terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain, an amino acid residue optionally capped by an N 5 terminal capping group, a non-peptidic group or a group that mimics an amino acid side chain; R 2 is selected from H, a C-terminal capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution, an amino acid residue optionally capped by a C-terminal 10 capping group, a group that mimics an amino acid side chain or a group that activates the terminal carboxylic acid carbonyl group to nucleophilic substitution; each R'and R" are independently selected from H, C 1 -Ctoalkyl, C 2 -Cloalkenyl, C 2 Cloalkynyl, C-Cacylcoalkyl, Cs-Ciocycloalkenyl, -OH, -OCr-Cloalkyl, -NH 2 , -NH(Cr Coalkyl), -N(CI-Cloalkyl) 2 , C 6 -C 12 aryl, C 3 -C 1 heterocyclyl, C-Cjoheteroaryl and halo; 15 L is selected from -NH-C(O)-, -C(O)-NH-, -S-S-, -CH(OH)Cr, CH 2 CH(OH), -CH=CH-, -CH 2 -CH 2 -, -NH-CH 2 - -CW-NH-, -CH-S-, -S-CH 2 -, -C(O)-CH 2 -, -CH 2 -C(O), -S(O) NHA-, -NH-S(O),-, CH 2 -P(=O)(OH)- and -P(=0)(OH)-CH-; m is 4, n is 1, and 20 t is 0, 1 or 2.

48. A use according to claim 47, wherein the amino acid residues that are favorable to alpha helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid.

49. A use according to claim 47, wherein the amino acid residues that are favorable to alpha 25 helix formation are selected from alanine, arginine, lysine, methionine, leucine, glutarnic acid and glutamine.

50. A use according to claim 47, wherein the disease or condition is related to an aberration in DNA transcription, RNA reverse transcription, transcriptional antitermination, apoptosis regulation, tumor suppression, calcium homeostasis, pain transmission, memory, lipid metabolism, cholesterol 30 homeostasis or stress response or to anxiety, appetite, alcohol withdrawal, opiate withdrawal or epilepsy.

51. A use according to claim 47, wherein the disease or condition is related to aberrant apoptosis regulation or tumor suppression.

52. A use according to claim 51, wherein the compound is selected from a BH3 domain 35 mimetic or a p53 tumor suppressor mimetic. -87 -

53. A use according to claim 51, wherein the the compound is a BH3 domain mimetic selected from cyclo(2-6,7-l 1)-Y[KRELD][KMADD]F [SEQ ID NO; 57], cyclo(2-6,7-1 I) V[KRQLD][KIADD]I [SEQ ID NO: 58], cyclo(2-6,7-1 1)-I[KAQED][KVADDIM [SEQ ID NO: 59], cyclo(2-6,7-1 1)-I[KAQED][KIADD]F [SEQ ID NO: 60], cyclo(2-6,7-11)-3-(4-hydroxyphenyt) 5 propionyl[KRELD][KMADD]-phenethylamide [SEQ ID NO: 61], cyclo(2-6,7-1 1)-iso valeroyl[KRQLD]IKIADD]2-methylbtylamide [SEQ ID NO: 62], cyclo(2-6,7-1l)-3 methylpentanoyl-[KAQED][KVADD]-3-methylsulfanyl-propylamide [SEQ 1D NO: 63], cyclo(2-67 11)-3-inethylpentanoyl-[KAQED][KIADD]-phenethylamide [SEQ ID NO: 64], Cyclo(3,7) LR[KMADDIF [SEQ ID NO: 65], Cyclo(3,7)-LA[KIADD]I [SEQ ID NO: 661, Cyclo(3,7) 10 LA[KVADD]I [SEQ ID NO: 67], Cyclo(3,7)-LA[KIADD]F [SEQ ID NO: 68], Cyclo(2,6)-7-methyl octanoyl-[KMADD]-Phenethylamide [SEQ ID NO: 69], Cyclo(2,6)-7-methyl octanoyl-[KIADD]-2 methylbutylamide [SEQ ID NO: 70], Cyclo(2,6)-7-methyl octanoyl-[KVADD]- 2-methylbutylamide [SEQ ID NO: 71] and Cyclo(2,6)-7-inethyl octanoyl-[KMADD]-Phenethylamide [SEQ ID NO: 72],

54. A use according to claim 52, wherein the p53 tumor suppressor mimetic is selected from 15 Cyclo(3,7)-FM[K(Pmp)(6ClW)ED]L [SEQ ID NO: 73], Cyclo(3,7)-3-Phenylpropanoyl M[K(Pmp)(6C1W)ED]isopentylamide [SEQ ID NO: 74] and.Cyclo(2,6)-6-Phenylheptanoyl [K(Pmp)(6ClW)ED]isopentylamide [SEQ ID NO:75].

55. A use according to claim 50, wherein the disease or condition is related to pain transmission, anxiety, appetite, alcohol withdrawal, opiate withdrawal, epilepsy or memory. 20 56. A use according to claim 55, wherein the compound is an agonist or antagonist of ORL-l receptor.

57. A use according to claim 55, wherein the compound is selected from Cyclo(6-10,11-15) FGGFT[KARKD][KRKLD]-NH2 (agonist) [SEQ ID NO: 76], Cyclo(6-10,11-15) NpheGGFT[KARKD][KRKLD]-NH 2 (antagonist) [SEQ ID NO: 77], Cyclo(2-6,7-1 1)-Ac 25 T[KARKD][KRKLD]-NH 2 (antagonist) [SEQ ID NO: 78] and Cyclo(2-6,7-1 I)-(8-napthalen-1-yl methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-acetoyl-[KARKD][KRKLD]-NH (antagonist) [SEQ ID NO: 79]. .88-