WO2011086553A1 - Cyclic peptides, compositions comprising them and uses thereof as nucleases of nucleotidic macromolecules - Google Patents

Abstract

The present invention relates to metal-free cyclic peptides capable of targeting and cleaving a nucleotidic macromolecule, compositions comprising them and uses thereof in the manufacture of medicaments for the treatment of diseases and disorders associated with said targeted nucleotidic macromolecules.

Description

CYCLIC PEPTIDES, COMPOSITIONS COMPRISING THEM AND USES THEREOF AS NUCLEASES OF NUCLEOTIDIC MACROMOLECULES

FIELD OF THE INVENTION

This invention relates to cyclic peptides, compositions comprising them and uses thereof as nucleases.

BACKGROUND OF THE INVENTION

In recent years, there has been considerable interest in designing synthetic small organic molecules as catalysts that hydrolyze the phosphodiester bond (1, 2). Such compounds, when targeted to certain DNA/RNA sequences, might lead to the development of highly specific artificial restriction enzymes, as well as potential therapeutic molecules for specific gene silencing.

Although there are several reported DNA cleavage agents that are metal-free and hydrolytic in nature (1-10), nonetheless, ligand-based hydrolytic DNA cleavage remains a challenge, as it is well-known that the DNA phosphodiester bond is very stable under physiological conditions with a half-life hydrolysis rate of about 200 million years.

In addition, currently known metal-free DNA nucleases have several disadvantages, including the fact that DNA cleavage is typically observed at high peptide concentration (e.g., 10 mM) or at non-physiological conditions (e.g., 50°C). Another approach of designing metal-free DNA nucleases was based on utilizing macrocyclic compounds as synthetic scaffolds to which various ligands (e.g., guanidine) are appended. In several cases, hydrolytic DNA cleavage was achieved at physiological conditions (pH 7.5, 37°C), although ligand concentration was relatively high (100-200 μΜ). DNA cleavage was also shown to be promoted by 2,6-pyridinecarboxamide substituted with two guanidine groups. However, as in the previous cases, a relatively high concentration of ligand was required for metal-free DNA hydrolytic cleavage. Therefore, there is a growing need in the art for peptides capable of cleaving a nucleotidic macromolecule, such as DNA and/or RNA, that is metal-free and is capable of performing such cleaving at physiological conditions and relatively low peptide concentrations. The absence of a redox active metal ion has the advantage of avoiding complications such as metal dissociation and uncontrolled redox chemistry.

References

(1) Ma, Y., Chen, X., Sun, M., Wan, R., Zhu, C, Li, Y., and Zhao, Y. (2008) DNA cleavage function of seryl-histidine dipeptide and its application. Amino Acids 35,

251-256.

(2) Razkin, J., Lindgren, J., Nilsson, H., and Baltzer, L. (2008) Enhanced complexity and catalytic efficiency in the hydrolysis of phosphate diesters by rationally designed helix-loop-helix motifs. ChemBioChem 9, 1975-1984.

(3) Cheng, C. T., Lo, V., Chen, J., Chen, W. C, Lin, C. Y., Lin, H. C, Yang, C.

H., and Sheh, L. (2001) Synthesis and DNA nicking studies of a novel cyclic peptide: Cyclo[Lys-Trp-Lys-Ahx-]. Bioorg. Med. Chem. 9, 1493-1498.

(4) Feng, Y. P., Cao, S. L., Xiao, A. S., Xie, W. J., Li, Y. M., and Zhao, Y. F. (2006) Studies on cleavage of DNA by N-phosphoryl branched peptides. Peptides 27, 1554-1560.

(5) Li, Y. S., Zhao, Y. F., Hatfield, S., Wan, R., Zhu, Q., Li, X. H., McMills, M., Ma, Y., Li, J., Brown, K. L., He, C, Liu, F., and Chen, X. Z. (2000) Dipeptide seryl- histidine and related oligopeptides cleave DNA, protein, and a carboxyl ester. Bioorg. Med. Chem. 8, 2675-2680.

(6) Schmuck, C, and Dudaczek, J. (2007) Screening of a combinatorial library reveals peptide-based catalysts for phosphorester cleavage in water. Org. Lett. 9, 5389- (7) Sheng, X., Lu, X. M., Chen, Y. T., Lu, G. Y., Zhang, J. J., Shao, Y., Liu, F., and Xu, Q. (2007) Synthesis, DNA-Binding, cleavage, and cytotoxic activity of new l,7-dioxa-4,10-diazacyclododecane artificial receptors containing bisguanidinoethyl or diaminoethyl double side arms. Chem. Eur. J. 13, 9703-9712.

(8) Sheng, X., Lu, X. M., Zhang, J. J., Chen, Y. T., Lu, G. Y., Shao, Y., Liu, F., and Xu, Q. (2007) Synthesis and DNA cleavage activity of artificial receptor 1,4,7- triazacyclononane containing guanidinoethyl and hydroxyethyl side arms. J. Org. Chem. 72, 1799-1802.

(9) Wan, S. H., Liang, F., Xiong, X. Q., Yang, L., Wu, X. J., Wang, P., Zhou, X., and Wu, C. T. (2006) DNA hydrolysis promoted by 1 ,7-dimethyl- 1,4,7, 10- tetraazacyclododecane. Bioorg. Med. Chem. Lett. 16, 2804—2806.

(10) Zhao, Y. C, Zhang, J., Huang, Y., Wang, G. Q., and Yu, X. Q. (2007) DNA cleavage promoted by 2,9-dimethyl-4,7-diazadecane-2,9-dithiol (DDD) derivatives. Bioorg. Med. Chem. Lett. 17, 2745-2748.

(11) Shao, Y., Sheng, X., Li, Y., Jia, Z.-L., Zhang, J.-J., Liu, F., and Lu, G. Y.

(2008) DNA binding and cleaving activity of the new cleft molecule N,N2- bis(guanidinoethyl)-2,6-pyridinedicarboxamide in the absence or in the presence of copper(ll). Bioconjugate Chem. 19, 1840-1848.

(12) Kurreck, J. (2003) Antisense technologies. Eur. J. Biochem. 270, 1628- 1644.

(13) Fernandes, L., Fischer, F. L., Ribeiro, C. W., Silveira, G. P., Sa, M. M., Nome, F. and Terenzi, H. (2008) Metal-free artificial nucleases based on simple oxime and hydroxylamine scaffolds. Bioorg. Med. Chem. Lett. 18, 4499-4502.

SUMMARY OF THE INVENTION

The present invention is based on the development of cyclic peptide as efficient metal-free DNA and/or RNA chemical nucleases, capable of cleaving said nucleotidic macromolecules at physiological conditions (for example pH of between about 7 to 7.5, temperature of between about 35 to 37°C) and relatively low peptide concentrations (in μΜ to sub-μΜ levels). The absence of a redox active metal ion avoids complications such as for example metal dissociation and uncontrolled redox chemistry. The synthetic design of the invention allows for a relatively straightforward approach to conjugate such cyclic peptides to functional molecules (such as for example TFOs). Such conjugates provide selective and metal-free DNA cleavage.

Thus, the present invention encompasses a cyclic peptide comprising 4 to 8 acids (AA), wherein:

- at least one AA (AA^ comprising at least one positively charged group selected from -SR₂ ⁺, -NR₃ ⁺; -PR₃ ⁺, -NHCH₂C=NH₂ ⁺(NH₂), -NCH₃C=NH₂ ⁺(NH₂), -NHC=NH₂ ⁺(NH₂), imidazolium;

- wherein each R is independently selected from H or C₁-C5 alkyl and

- at least one AA (AAn) comprising at least one functional group capable of binding with a nucleotidic macromolecule;

provided that at least one AAi and at least one AAn have opposite configurations. In another aspect the invention provides a cyclic peptide having the following general formula (I):

CVC.Y1-Y2-Y3-Y4-Y5 (I)

wherein

Y₁-Y5 are each independently an AA; wherein

- at least one AA (AAj) comprises at least one positively charged group selected from -SR₂ ⁺, -NR₃ ⁺; -PR₃ ⁺, -NHCH₂C=NH₂ ⁺(NH₂), -NCH₃C-NH₂ ⁺(NH₂), -NHC=NH₂ ⁺(NH₂), imidazolium;

- wherein each R is independently selected from H or C -C alkyl; and

- at least one AA (AAn) comprises at least functional group capable of binding with said nucleotidic macromolecule;

provided that at least one AAi and at least one AAn have opposite configurations.

In the context of the present invention the term "cyclic peptide" is meant to a cyclic macromolecule that consists of 4, 5, 6, 7, or 8 amino acids (either natural or non- natural amino acids), connected to one another via a peptide bond (e.g. -C(=0)NH-), wherein the amino and carboxyl termini are linked together with a peptide bond, thereby forming the cyclic peptidic macromolecule. The terminology "eye." As used herein above and below is meant to refer to a peptidic molecule comprising Yi to Y₅ amino acids (AA), being a cyclic pentapeptide, wherein the terminal AAs, Y^'i and Y₅ are linked to one another via a peptidic bond thereby forming a cyclic peptide.

When referring to a cyclic peptide comprising "at least one AAj it should be understood to encompass one or more amino acids each independently comprising at least one positively charged group substituted on a CrC₈ alkyl chain (when said group is substituted at the terminal carbon this chain is a Ci-Cg alkylene) substituted at the C_a position of said AA backbone, said group is selected from -SR₂ ⁺, -NR₃ ⁺, -PR₃ ⁺, -NHCH₂C=NH₂ ⁺(NH₂), -NCH₃C=NH₂ ⁺(NH₂), -NHC=NH₂ ⁺(NH₂), -NRC=NR₂ ⁺(NR₂), imidazolium; wherein each R is independently selected from H or C₁-C5 alkyl. It is noted that said C_a atom of AAi is therefore a chiral carbon atom. In some embodiments, said AAj is selected from Arginine, Histidine, Lysine and

Ornithine. In other embodiments, said cyclic peptide of the invention comprises at least two AAi, that may be the same or different.

In other embodiments, said AA comprises at least one partially positively charged group substituted on a Q-Q alkylene chain substituted on the C„ position of said AA (for example imidazole, guanidine) . In further embodiments said AAi comprises at least one group substituted at the C_a position of said AA, said group is capable of being positively charged under physiological conditions (for example imidazole, guanidine). It is noted that physiological conditions are defined to include, among others, pH levels in the range of between about 7 to 7.5 and temperature in the range of between about 35 to 37°C.

When referring to a cyclic peptide comprising "at least one AA_n" it should be understood to encompass one or more amino acids each independently comprising at least one functional group capable of binding with a nucleotidic macromolecule (such as for example DNA or RNA), substituted on a Q-Q alkyl chain substituted (when substituted at the terminal carbon this chain is a Ci-Cg alkylene) at the C_a position of said AA backbone. It is noted that said C_a atom of AAn is therefore a chiral carbon atom.

In some embodiments, said functional group of AAn binds to nucleotidic macromolecule via electrostatic binding, covalent binding or intercalating to said nucleotidic macromolecule.

The term "nucleotidic macromolecule" should be understood to relate to a macromolecule comprising nucleotides. Examples of nucleotidic macromolecules encompassed by the invention include: DNA (single stranded or duplex), RNA (including mRNA or ncR A).

In some embodiments a functional group of AAn is selected such that it is capable of binding to said macromolecule via any intermolecular or intramolecular bond selected from at least one of the following:

Intramolecular bonds which include without being limited to: covalent bonds (Sigma bond, Pi bond, Delta bond, Double bond, Triple bond, Quadruple bond, Quintuple bond, Sextuple bond, 3c-2e, 3c-4e, 4c-2e, Metal to Metal bond, Agostic bond, Bent bond, Dipolar bond, Pi backbond, Conjugation, Hyperconjugation, Aromaticity, Hapticity, Antibonding), Ionic bonds (Cation-pi bond, Salt bond), Metallic bonds.

Intermolecular bonds which include without being limited to: Hydrogen bond, non-covalent bonds, van-der Waals forces, London dispersion forces, Mechanical bond, Halogen bond, Aurophilicity, Intercalating, pi-stacking, entropic forces, chemical polarity).

In some embodiments a functional group of AAn is selected such that it is capable of binding to said macromolecule via at least one bond selected from: an electrostatic bond (i.e. intermolecular bonds involving the electrostatic interaction of atoms or functional groups of two molecules, such bonds may include hydrogen bonds, van der Waals forces, London dispersion forces, pi-stacking forces and so forth), covalent bond (i.e. intramolecular bonds between two atoms wherein their electrons are shared, such bonds include sigma bond, pi bond, delta bond, double bond, triple bond, quadruple bond, quintuple bond, sextuple bond, 3c-2e, 3c-4e, 4c-2e, metal to metal bond, agostic bond, bent bond, dipolar bond, pi backbond, conjugation, hyperconjugation, aromaticity, hapticity, antibonding), intercalating bond (i.e. reversible inclusion of a molecule or a functional group thereof, between two other molecules or groups thereof, for example a group or ligand that fits in between nucleotide base pairs of a DNA molecule), or any combination thereof.

In other embodiments, said functional group of AAn binds to a specific sequence of nucleotidic macromolecule. Thus, under these embodiments, said functional group of AAn binds to a specific sequence of a nucleotidic macromolecule thereby specifically targeting and binding a peptide of the invention to a specific nucleotidic macromolecule. Such targeting and specific binding allows the other groups of a peptide of the invention (i.e. group of AAi and/or AAm) to activate a phosphodiester bond of said macromolecule (i.e. group substituted on AAi) towards nucleophilic attack (by, for example, the group substituted on AAm, or in other embodiments by another molecule capable of nucleophilically attacking said macromolecule) of said phosphodiester bond or any other phosphodiester bond of said macromolecule, thereby cleaving a specific targeted macromolecule. Such targeting provides a peptide molecule of the invention the capability of treating a condition, disorder or disease associated with such a specific nucleotidic macromolecule or a nucleotidic macromolecule comprising such a specific targeted sequence.

In yet other embodiments, said functional group of AAn is a peptide nucleic acid (PNA) or a triplex forming oligonucleotide (TFO) or any other type of oligonucleotide or oligonucleotide mimic, morpholino, DNA-LNA (LNA=locked nucleic acid) and DNA-2'-OME (O-methoxyethyl)) or any DNA analog having high binding affinity to complementary nucleotidic macromolecule target (DNA or RNA).

The term "peptide nucleic acid (PNA)" is meant to encompass any artificially synthesized polymer similar to DNA or RNA. PNA backbone comprises repeating N- (2-aminoethyl)-glycine units linked by peptide bonds, however is acyclic, achiral and neutral. Purine (A, G) and pyrimidine (C, T) bases are attached to the backbone of a PNA molecule, through methylene carbonyl linkages. Unlike DNA or DNA analogs, PNAs do not contain any (pentose) sugar moieties or phosphate groups. PNA substantially mimics the behavior of DNA, and thus can bind complementary nucleic acid strands. PNA binding modes for targeting double stranded DNA include: triplex, triplex invasion, duplex invasion, double duplex invasion. For other PNA options that may be utilized in a cyclic peptide of the invention see Nielsen, P.E. (2010) Targeted gene repair facilitated by PNA. ChemBioChem, 11, 2073-2076, which is incorporated herein by reference in its entirety.

The term "triplex forming oligonucleotide (TFO)" relates to molecules that are capable of being bound in the major groove of duplex DNA with high specificity and affinity. TFO molecules are restricted to binding DNA sites which have runs of purines on one strand and pyrimidines on the other; TFOs and their corresponding targets are typically 10-30 nt in length. Though DNA TFOs can be either polypurine or polypyrimidine or stretches of T and G nucleotides, they bind to the purine-rich strand of their target. For other TFO options that may be utilized in a cyclic peptide of the invention see Duca, M. et al., (2008) The triple helix: 50 years later, the outcome. Nucl. Acids Res. 36, 5123-5138, which is incorporated herein by reference in its entirety.

In further embodiments, said functional group of AAn is a substantially planar poly cyclic molecule. In other embodiments said functional group of AAn is an aromatic or heteroaromatic moiety. In some embodiments, said aromatic or heteroaromatic moiety is selected from anthraquinone, acridine, pyrene, ethidium bromide, daunomycin and doxorubicin. In some embodiments, said cyclic peptide of the invention comprises at least one

AA (AAm) comprising a group capable of nucleophillicly attacking said nucleotidic macromolecule, selected from -OH, -SH, -NH-OH, -SeH, -SeOH.

When referring to a cyclic peptide comprising "at least one ΑΑπ it should be understood to encompass one or more amino acids each independently comprising at least one functional group capable of nucleophillicly attacking said nucleotidic macromolecule, substituted on a C_\-C$ alkylene chain substituted on the C_a position of said AA. It is noted that said C_a atom of AAn is therefore a chiral carbon atom. The term "nucleophillicly attacking a nucleotidic macromolecule" should be understood to mean the capability of a functional group of AAm which is relatively electron rich (nucleophile) to selectively bond with or attack a positive or partially positive charged atom or group of a nucleotidic macromolecule, thereby cleaving at least one bond of a nucleotidic macromolecule.

In some embodiments, said at least one AAm is selected from the group consisting of Tyrosine (in which the nucleophile group is -OH), Serine (in which the nucleophile group is -OH), Threonine (in which the nucleophile group is -OH), Cysteine (in which the nucleophile group is -SH), Selenocysteine (in which the nucleophile group is -SeH) and Homoserine (in which the nucleophile group is -OH).

In other embodiments, said AAm is substituted with a group capable of forming an intermolecular bond with at least one molecule capable of nucleophillicly attack a nucleotidic macromolecule (in some embodiments at least one phosphodiester bond of said macromolecule). In some embodiments said molecule capable of nucleophillicly attack a nucleotidic macromolecule is a water molecule being bound by a hydrogen bond to at least one group of AAm.

In some embodiments a cyclic peptide of the invention comprises a sequence of three AA, comprising two independent AAi, as defined herein above.

In some embodiments, said sequence comprises two identical AAt, as defined herein above. In other embodiments, said sequence comprises two different AA_1; as defined herein above. In further embodiments of above noted sequence of the invention, said two AAi are directly linked to one another (i.e. said two AAiS, are linked to one another via a single peptidic bond). In other embodiments of above noted sequence of the invention, said two AAi are linked via said third AA of the sequence. In some embodiments, said third AA is a natural or non-natural AA, as defined herein above. In further embodiments above noted sequence of the invention comprises an AA (AAm) having a group capable of nucleophillicly attacking said nucleotidic macromolecule, selected from -OH, -SH, - NH-OH, -SeH, -SeOH.

Without wishing to be bound by theory, it is speculated that at least one AAi of a cyclic peptide of the invention is selected so as to activate the phosphodiester bond towards nucleophilic attack of the phosphodiester bond by AAni or an activated water molecule bound to said AAm.

In some embodiments, a cyclic peptide of the invention has the following main features:

1. -A tirad sequence of three AA (two AA] and one AA^) wherein their side chains are designed to cleave DNA, and thus are oriented towards the phosphodiester bond of the target DNA.

2. An AA (AAii) having an opposite configuration as compared to the configuration of at least one AA in the triad sequence (for example D-AA when triad AA are L-AA, e.g. D-lysine), having a side chain oriented to the opposite direction of the side chains of the triad sequence, allowing conjugation of a DNA binding molecule (such as for example an organic intercalator).

It should be understood that at least one AAi and at least one AAn of a cyclic peptide of the invention have opposite configurations. The term "configuration should be understood to relate to the arrangements of atoms of a molecular entity in space that distinguishes stereoisomers (e.g. isomers that possess identical constitution, but which differ in the arrangement of their atoms in space). The different configuration may be absolute or relative (each denoted as R, S, D, L, R*or S* according to the convention used).

The configuration may be further defined by the relative relationship between two ligands attached to separate atoms that are contained in a ring. The two ligands are said to be located cis to each other if they lie on the same side of a plane. If they are on opposite sides, their relative position is described as trans. For a ring (the ring being in a conformation, real or assumed, without re-entrant angles at the two substituted atoms) it is the mean plane of the ring(s). If there are more than two entities attached to the ring the use of cis and trans requires the definition of a reference substituent.

In some embodiments, configuration of said at least one AAi is D, under this embodiment the configuration of at least one AAn of a cyclic peptide of the invention will be L. In further embodiments, configuration of said at least one AA] is L, under this embodiment the configuration of at least one AAn of a cyclic peptide of the invention will be D. Under each of the above noted embodiments, the configuration of all other AAs in the cyclic peptide of the invention may be each independently D or L. It should be understood that configuration denoted D or L, as mentioned above is the nomenclature convention used for amino acids also known as the Fischer-Rosanoff Convention (or Rosanoff Convention see 'Nomenclature and Symbolism for Amino Acids and Peptides, Recommendations 1983', Pure Appl. Chem. 56, 595-624 (1984); and 'Tentative Rules for Carbohydrate Nomenclature', Eur. J. Biochem. 21, 455-477 (1971)).

In other embodiments, at least one positively charged group of said at least one AA_\ is oriented trans to said at least one functional group capable of binding with a nucleotidic macromolecule of said at least one AAn- Thus, in some embodiments said at least one positively charged group of said at least one AAi may be oriented above the relative or mean plane of a cyclic peptide of the invention and at least one functional group capable of binding with a nucleotidic macromolecule of said at least one AAn is oriented below the relative or mean plane of a cyclic peptide of the invention. In other embodiments, said at least one positively charged group of said at least one AAi may be oriented below the relative or mean plane of a cyclic peptide of the invention and at least one functional group capable of binding with a nucleotidic macromolecule of said at least one AAn is oriented above the relative or mean plane of a cyclic peptide of the invention. In some embodiments a cyclic peptide of the invention comprises four independent AA, as defined herein above and below. In other embodiments a cyclic peptide of the invention comprises five independent AA, as defined herein above and below. It should be understood that AA comprised in a cyclic peptide of the invention, other the AAs defined herein above (i.e. AA_1; AAn and in some embodiments AAm), may be any natural and non-natural AA known in the art.

In further embodiments a cyclic peptide of the invention is a peptide having the following general formula (II):

Wherein X_1; X₂ and X₃ are each independently a straight or branched -Cs alkyl, each optionally substituted with at least one group selected from -OH, -NR₂, - NR₃ ⁺, -SR, -SR₂ ⁺, -PR₂, PR₃ ⁺, guanidine, N-methyl-guanidine, imidazolium;

wherein each R is independently selected from H or Ci-C₅ alkyl; and provided that at least one of Xi, X₂ and X₃ is positively charged;

Y is selected from straight or branched Q-C5 alkyl, optionally substituted with at least one group selected from -OH, -SH, -NH-OH, -SeH, -SeOH, phenyl, phenol, - COOH, -CONH₂;

Z is a linker which may be absent or a straight or branched Ci-C₈ alkylene, optionally substituted at the terminal end with at least one group selected from an amine, amide, ester, ether, thioester, thioether, acyl, imine, oxime, triazole, azide and maleimide; and

W is a group capable of binding to a nucleotidic macromolecule.

In other embodiments a cyclic peptide of the invention is a peptide having the following general formula (III):

~² " i (III)

Wherein X1-X3, Y, Z and W are as defined herein above, provided that the configuration of at least one carbon atom bearing groups X1-X3 is opposite to the configuration of the carbon atom bearing group Z.

In yet other embodiments a cyclic peptide of the invention is a peptide having the following general formula (IV):

(IV)

Wherein X1-X3, Y, Z and W are as defined herein above, provided that the configuration of at least one carbon atom bearing groups X X₃ is opposite to the configuration of the carbon atom bearing group Z.

In another aspect the invention provides a cyclic peptide having the following general formula (II):

wherein X_ls X₂ and X₃ are each independently a straight or branched C_\-C₅ alkyl, each optionally substituted with at least one group selected from -OH, -NR₂, - NR₃ ⁺, -SR, -SR₂ ⁺, -PR₂, PR₃ ⁺, guanidine, N-methyl -guanidine, imidazolium;

wherein each R is independently selected from H or CrC₅ alkyl; and provided that at least one of Xi, X₂ and X₃ is positively charged;

Y is selected from straight or branched C1-C5 alkyl, optionally substituted with at least one group selected from -OH, -SH, -NH-OH, -SeH, -SeOH, phenyl, phenol, - COOH, -CONH₂;

Z is a linker which may be absent or a straight or branched Q-Cs alkylene, optionally substituted at the terminal end with at least one group selected from an amine, amide, ester, ether, thioester, thioether, acyl, imine, oxime, triazole, azide, maleimide; and

W is a group capable of binding to a nucleotidic macromolecule.

In some embodiments,

and X₂ are each independently a straight or branched Q-C5 alkyl, each substituted with at least one group selected from -NH₃ ⁺, guanidine, N- methyl-guanidine and imidazolium.

In other embodiments, W is a group selected from an intercalator, a peptide nucleic acid (PNA), morpholino, DNA-LNA (or any other DNA mimic/analog that has high binding affinity to complementary DNA or RNA) and a triplex forming oligonucleotide (TFO). In further embodiments, said intercalator is selected from anthraquinone, acridine, pyrene, ethidium bromide, daunomycin and doxorubicin.

In another aspect of the invention there is provided a cyclic peptide of the invention for use in cleaving a nucleotidic macromolecule.

When referring to "cleaving of a nucleotidic macromolecule^'1'' it should be understood to encompass any breakage of a bond of a nucleotidic macromolecule, such as for example DNA or RNA where DNA can be either single stranded or duplex form and RNA is either mRNA or ncRNA. . In some embodiments, said cleavage relates to the hydrolysis of a phosphodiester bond of such a nucleotidic macromolecule.

In some embodiments said cleaving of nucleotidic macromolecule is site- selective. In other embodiments, said cleaving is obtained under physiological conditions.

Without wishing to be bound by theory, it is speculated that at least one AA] of a cyclic peptide of the invention is selected so as to activate the phosphodiester bond towards nucleophilic attack of the phosphodiester bond by AAm or an activated water molecule bound (by either an intramolecular bond or a intermolecular bond) to said AAm.

The term "Cj-Cs alkyl" as used herein represents a saturated, branched or straight hydrocarbon group having from 1, 2, 3, 4 to 5 carbon atoms. Typical C 1-5- alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl and the like.

The term "Ci-C₈ alkylene" as used herein represent a saturated, divalent, branched or straight hydrocarbon group having from 1, 2, 3, 4, 5, 6, 7 to 8 carbon atoms. Typical C 1-8-alkylene groups include, but are not limited to, methylene, ethylene, 1 ,2-propylene, 1,3-propylene, butylene, isobutylidene, pentylene, hexylene and the like. The term "imidazolium" as used herein represents an imidazole ring wherein at least one N atom is positively charged due to substitution of a group selected from H or d-Cs alkyl. The term "guanidine" as used herein represent -NHC==NH₂ ⁺(NH₂).

The term "N-methyl-guanidine" as used herein represent -N(CH₃)CHSfH₂ ⁺(NH₂).

The term "optionally substituted at the terminal end' is meant to include an optional substitution of at least one group selected from an amine (-NR-, wherein R is H or a d-C₅ alkyl), amide (-NRC(=0)-, wherein R is H or a Q-C5 alkyl), ester (-C(=0)0- ), ether (-0-), thioester (-C(=0)S-), thioether (-S-), acyl (-C(=0)-), imine (-C(=NR)- wherein R is H, alkyl, phenyl), oxime (-C(=NOH)-), triazole, azide (-N=N⁺=N-), and maleimide, at the end valancy of said C C₈ alkylene linker, either at the end connected with the cyclic peptide or at the end connected to group W.

In another aspect of the invention there is provided a cyclic peptide of the invention for use as a medicament or a pharmaceutical composition. In a further aspect the invention provides a use of a cyclic peptide of the invention for the preparation of a medicament or a pharmaceutical composition.

The present invention thus relates to pharmaceutical compositions or medicaments comprising a cyclic peptide of the subject invention in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxiliaries must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.

Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing in association compounds used in the invention or combinations thereof with any auxiliary agent. The auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavouring agents, anti-oxidants, and wetting agents.

Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets, dragees or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.

The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material, including instructions for the use of the composition for a use as hereinbefore described.

For parenteral administration, suitable compositions include aqueous and nonaqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example water, prior to use.

For transdermal administration, e.g. gels, patches or sprays can be contemplated. Compositions or formulations suitable for pulmonary administration e.g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.

The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered. In yet a further aspect the invention provides a use of a cyclic peptide of the invention for the preparation of a medicament for nucleotidic-macromolecule targeted therapy. The term "nucleotidic-macromolecule targeted therapy" should be understood to include therapy of a condition, disease or disorder associated with a specific nucleotidic macromolecule (such as for example DNA or RNA) which is achieved by targeting (i.e. binding to a nucleotidic macromolecule or a sequence thereof) and neutralizing of said nucleotidic macromolecule (thereby reducing or substantially eliminating its activity, transcription and/or translation). In some embodiments, such nucleotidic- macromolecule targeted therapy includes antigene and/or antisense therapies.

In some embodiments, said medicament is for the treatment of a disease or disorder associated with a targeted (specific) nucleotidic macromolecule. Under these embodiments the treatment of the condition, disease or disorder is achieved upon cleaving said targeted (specific) nucleotidic macromolecule.

In other embodiments said medicament is for the treatment of a disease or disorder associated with a target specific gene.

In some embodiments a gene cleaved by a cyclic peptide of the invention may be any gene capable of being associated with a P A, other DNA mimics or TFO ligated to a cyclic peptide of the invention (see for example Beane, R. L., Ram, R., Gabillet, S., Arar, K., onia, B. P., and Corey, D. R. Inhibiting gene expression with locked nucleic acids (LNAs) that target chromosomal DNA. Biochemistry, 46: 7572-7580, 2007; and Hu, J. X. and Corey, D. R. Inhibiting gene expression with peptide nucleic acid (PNA)- peptide conjugates that target chromosomal DNA. Biochemistry, 46: 7581-7589, 2007).

Non-limiting examples of genes that may be cleaved by a cyclic peptide of the invention include:

1. Target gene: HER-2/neu (potential target in breast cancer) (Ebbinghaus, S.W., Fortinberry, H., and Gamper, H.B. (1999). Inhibition of transcription elongation in the HER-2/neu coding sequence by triplex-directed covalent modification of the template strand. Biochemistry 38, 619-628).

2. Target gene: rhodopsin (autosomal dominant retinitis pigmentosa (blindness));

((a) Perkins, B.D., Wensel, T.G., Vasquez, .M., and Wilson, J.H. (1999). Psoralen photo-cross-linking by triplex forming oligonucleotides at multiple sites in the human rhodopsin gene. Biochemistry 38, 12850-12859. (b) Oh, D.H., and Hanawalt, P.C. (2000). Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides. Photochem. Photobiol. 72, 298-307).

3. Target gene: interleukin receptor IL-2Ra (anticancer); (Grigoriev, M, Praseuth, D., Guieysse, A.L., Robin, P., Thuong, N.T., Helene, C, and Harelbellan, A. (1993). Inhibition of gene-expression by triple helix-directed D A cross-linking at specific sites. Proc. Natl. Acad. Sci. USA. 90, 3501-3505).

4. Target gene: Type I Collagen (liver fibrosis); (S. Koilan, D. Hamilton, N.

Baburyan, M. K. Padala, K. T. Weber, and R. V. Guntaka. (2010). Prevention of Liver Fibrosis by Triple Helix-Forming Oligodeoxyribonucleotides Targeted to the Promoter Region of Type I Collagen Gene. Oligonucleotides 20, 231-237).

In some embodiments, said medicament is for the treatment of a disease or disorder selected from cancer, autoimmune diseases, genetically hereditary diseases, inflammation and conditions or states associated therewith.

In a further aspect of the invention there is provided a method of cleaving a nucleotidic macromolecule comprising contacting said macromolecule with an effective amount of a cyclic peptide of the invention.

In another one of its aspects the invention provides a method of treating a disease or disorder associated with a specific gene, comprising administering to a subject in need thereof an effective amount of a cyclic peptide of the invention.

In some embodiments of a method of the invention said cleaving is achieved under physiological conditions. Such conditions include pH in the range of between about 7 to 7.5 and temperature in the range of between about 35 to 37°C. In further embodiments, an effective amount of a cyclic peptide of the invention is in the micromolar range (i.e. at least about 1 μΜ). In other embodiments, an effective amount of a cyclic peptide of the invention is in the range of between about ΙμΜ to about 50μΜ (i.e. between about ΙμΜ to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50μΜ).

In other embodiments of a method of the invention said cleaving of said macromolecule is site-specific. In yet further embodiments of a method of the invention, said nucleotidic macromolecule is associated with a disease or disorder. In some embodiments, said disease or disorder is selected from cancer, autoimmune diseases, genetically hereditary diseases and inflammation. As used herein, the term "effective amount" means that amount of a cyclic peptide of the invention or a pharmaceutical composition/medicament of the invention that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. The term "treatment" as used herein refers to the administering of a therapeutic amount of a cyclic peptide or a composition of the present invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context 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 integer or step or group of integers and steps.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figs. 1A-1B. Fig. 1A shows an agarose gel (1%) electrophoresis of supercoiled DNA (pSP73 plasmid, superhelical density = -0.063) incubated for 4 and 16 h with 1- AQ at 37 °C. Lanes: 0, 4, 16 h = DNA incubated with 1-AQ for 0, 4, and 16 h, respectively; M = 1 kb DNA ladder. Fig. IB shows the DNA nuclease activity of 1-AQ on a 32P-labeled 50-mer DNA duplex as corroborated on a 20% polyacrylamide/8 M urea gel. Lanes: C+T and G+A = Maxam-Gilbert sequencing reactions; no peptide = control, no peptide added; 0, 4, and 16 = DNA incubated with 1-AQ for 0, 4, and 16 h, respectively. For both gels: Incubation temperature= 37 °C, [peptide]: [DNA in bp] = 1 :1, final peptide concentration =20 μΜ. All samples were kept in the dark. Incubation buffer: [Tris] =50 mM, [CaCl₂] = 10 mM, pH = 7.4.

Fig. 2 shows the fluorescence decay of EtBr bound to CT-DNA after the addition of Glu-AQ. The estimated binding affinity (Xapp = 5.4* 10⁵ M^"1) of Glu-AQ to dsDNA was determined according to the amount of Glu-AQ required for a 50% decrease in fluorescence. Fig. 3 shows the DNA nuclease activity of compounds 1-AQ, 1, and Lin-1 in [Tris] = 50 mM, [CaCl₂] = 10 mM, pH - 7.4 buffer at 37 °C as corroborated on an agarose gel. In all lanes: 95 ng PCR product (591 bp). Lane 1, control (without peptide); lane 2, 4 μΜ of 1-AQ; lane 3, 16 μΜ of 1-AQ; lane 4, 16 μΜ of 1; lane 5, 48 μΜ of 1; lane 6, 144 μΜ of 1; lane 7, 16 μΜ of Lin-1; lane 8, 48 μΜ of Lin-1; lane 9, 144 μΜ of Lin-1. DNA incubated with all peptides for 4 h. All samples were kept in the dark.

Fig. 4 shows that DNA nuclease activity of compounds 1-AQ, Gly-AQ, 1-Ac, and 1-L-AQ in [Tris] = 50 mM, [CaCl₂] = 10 mM, pH = 7.4 buffer at 37°C as corroborated on an agarose gel. In lanes 2-12: 95 ng PCR product (591 b.p.). Lane 1, DNA ladder; lane 2, DNA control (without peptides); lanes 3-5, 1-AQ at 4, 16, and 64 μΜ, respectively; lanes 6-8, Gly-AQ at 4, 16, and 64 μΜ, respectively; lanes 9-1 1, 1-Ac at 4, 16, and 64 μΜ, respectively; lane 12, 16 μΜ of 1-L-AQ. DNA incubated with all cyclic peptides for 4 h. All samples were kept in the dark.

Fig. 5 shows the DNA nuclease activity of 1-AQ in the presence of ROS scavengers. 95 ng PCR product (591 bp) was incubated with 1-AQ (20 μΜ) for 4 h at 37 °C. Lane 1, DNA ladder; lane 2, only DNA; lane 3, no inhibitor added; lane 4, 10 mM NaN3; lanes 5-6, 1 mM of DMSO and tBuOH, respectively; and lane 7, 10 mM KI. All samples were kept in the dark during incubation. Incubation buffer: [Tris] = 50 mM, [CaCl₂] = 10 mM, pH = 7.4.

Figs. 6A-6B. Fig. 6 A shows the hyperchromicity of 15-mer dsD A substrate after the addition of peptide conjugates 1-AQ, Gly-AQ, and 1-Ac at 2.5 molar excess concentrations. Fig. 6B shows the estimated kobs values of 0.70 h^"1, 0.85 h^'1, and 0.65h^"' for peptide conjugates 1-AQ, Gly-AQ, and 1-Ac, respectively. k_0bS values were extracted form the linear fit (slope = Aobs) for all three peptides conjugates.

Figs. 7A-7B shows the Agarose gel (1%) electrophoresis of supercoiled DNA (pSP73 plasmid) incubated for 4 (Fig. 7A) and 16 hours (Fig. 7B) with compounds 1- AQ and Glu-AQ at 37°C and 50°C. [Peptide]: [DNA in base pairs] = 1 :1. Cont = control (no peptide). Final peptide concentration = 20 OM. All samples were kept in the dark during incubation. Incubation buffer: [NaPi] = 50 mM, [CaCl₂] = 10 mM, pH = 7.4. Fig. 8 shows the polyacrylamide gel electrophoresis (20% polyacryamide/8M urea) of 50-mer duplex DNA (³²P-labeled) incubated for 1, 2 and 4 hours with compounds 1-AQ and Glu-AQ at 50°C and 16 hours at 37°C. [peptide]: [DNA in base 5 pairs] = 1 : 1. C = control (no peptide), Ser = compound 1-AQ, GIu = compound Glu- AQ. C+T and G+A = Maxam-Gilbert sequencing reactions. Final peptide concentration = 20 μΜ. All samples were kept in the dark during incubation. Incubation buffer: [NaPi] = 50 mM, [CaCl₂] = 10 mM, pH = 7.4.

10 Fig. 9 shows the estimated kobs values for 1-AQ based on hyperchromicity studies using a 15-mer dsDNA substrate. Based on linear fit for each 1-AQ concentration, kobs values were 0.26 h^"1, 0.70 h^"1, 1.13 h^"1, 1.62 h^"1 and 0.53 h^"1 for 1, 2.5, 5, 7.5 and 10 equivalents of 1-AQ, respectively.

15 DETAILED EXPERIMENTAL DESCRIPTION

MATERIALS AND METHODS

All commercially available chemical reagents were used without further purification. The solvents DMF, DCM, and MeOH were purchased from Acros (dry under molecular sieves) and used for peptide synthesis without further purification.

20 Phosphoramidites and reagents for DNA synthesis were purchased from Biolab (Jerusalem, Israel) and used as received. Fmoc-6-aminocaproic acid (Fmoc-Ahx) (27), anthraquinone-2-cabonyl chloride (AQ-C1), and acridine-9-carbonyl chloride (28) were synthesized according to the literature. Thin-layer chromatography (TLC) was performed on aluminum sheets Merck silica gel 60 F254. Compounds were visualized

25 by UV absorption at 254 run and stained with 3% ninhydrin solution in 97% EtOH to monitor the presence of free amines.

Semipreparative RP-HPLC separations were performed on a Shimadzu instrument (LC-2010C) by using a Phenomenex CI 8 column (300A0, 5 μτη, 250 mm * 30 20 mm) with a flow rate of 4 mL/min-1. The eluent was a linear gradient from 95% water (with 0.1% TFA) to 30% acetonitrile over 5 min followed by a gradient of 30% to 80% acetonitrile over 25 min. The detection was performed at 255 nni. ESI-MS spectra were carried out either on an Orbi-trap mass spectrometer (Thermo Finnigan) using a nanospray attachment or on a ThermoQuest Finnigan LCQ Duo MS. Preparative normal-phase CombiFlash separations were performed on a Mercury instrument. The eluent was a linear gradient from 0% MeOH to 20% MeOH (in DCM).

SOLID-PHASE PEPTIDE SYNTHESIS

All cyclic peptides were prepared on the solid support using standard Fmoc peptide chemistry. D-Fmoc-Lys(X)-OH, where X=Anhraquinone or Acridine was prepared according to Scheme 1. This modified amino acid was then introduced during solid phase synthesis (Scheme 2). The trityl chloride resin was utilized as the peptide and was readily cleaved form the solid support in mild acidic conditions (3% TFA), allowing all protection group on side chains to stay intact. Thus, the linear peptide was then readily cyclized in a dilute solution of DMF (with the appropriate coupling reagent) from C- to N-terminus. Subsequently, all the protection groups were removed in 88% TFA to afford the desired cyclic peptide.

Schemes 1 and 2 below provide an exemplary embodiment of a synthesis procedure of the invention:

D-Fmoc-Lys(BOC)-OH

1. TFA / DCM

2. 9-carboxy-AQ, HATU, DIEA ,

Scheme 1: Synthesis of D-Fmoc-Lys(AQ)-OH monomer for solid phase synthesis.

1. 20% piperidine

2. Fmoc-NH-aa-OH / HATU / HOBT / DIEA

Gly-NHFmoc

Scheme 2: Solid and solution phase syntheses of a cyclic pentapeptide with Arg- 5 Ser-Arg (1-AQ) as the active triad and Anthraquinone as DNA intercalator.

In some embodiments, group Y in a compound of formula (II), may be hydroxylamine. In such cases the following unnatural AA may be synthesized possessing an hydroxylamine functionality on its side chain (Scheme 3):

10

u

(Fmoc-L-homoserine)

, MeOH

Scheme 3: Synthesis of a modified AA with a THP-protected hydroxylamine.

In order to achieve site-selective, metal free hydrolytic cleavage of DNA, the cyclic peptide can be conjugated to a TFO (triplex forming oligonucleotide) through the D-lysine AA. A Non-limiting example of DNA/RNA analogs suitable for this purpose includes metabolically stable RNA analogs with improved triplex propensity. For example, a 2'-OMe RNA or DNA sequence containing several LNAs (locked nucleic acid) that have been shown to improve triplex hybridization. Another possible conjugate may include a PNA (peptide nucleic acid) sequence with 3-4 L-lysines at its C-terminus for improved solubility and cellular uptake. Homopurine or homopyrimidine PNAs have excellent affinities to duplex DNA leading to duplex strand invasion. For example, down-regulation of the mdm2 oncogene can result in anticancer activity as the mdm2 protein has an important role in the degradation of p53. In this case the TFO of choice may be, for example, a 14-mer homopurine sequence (AAAGGGAAAGGAAA) that is complementary to a coding region in the mdm2 oncogene.

Exemplary synthesis of PNA conjugates are presented in Schemes 4 and 5. The synthetic strategy may be such that a pNZ protecting group is introduced at the ε-amine of D-Lysine. After cyclization in solution, this pNZ group is selectively deprotected from D-Lys allowing coupling to the PNA of choice with high selectivity. Finally, after conjugation, the other protection groups (e.g. Pbf on L-Arg) are removed by standard acidic conditions.

Fmoc-D-Lys(BOC)-OH

1. TFA/DCM

1 2. p-nitrophenyl-chloroformate + NaN₃

Fmoc-D-Lys(pNZ)-OH

Solid phase synthesis

-NH, -Arg(Pbf)-Ser(OtBu)-Arg(Pbf)-Gly-D-Lys(pNZ)-NH₂

of cyclic peptide

using Fmoc-D-Lys(pNZ)-OH 1. 3% TFA

2. Cyclization in solution (HATU / DIEA) i 3. pNZ removal (2 SnCI₂ / AcOH / Phenol)

cyc-1

Scheme 4: Solution followed by solid phase synthesis of the amine-free cyclic peptide. (K₃)-PNA-NH₂

Scheme 5: Synthesis of PNA conjugated to cyclic pentapeptide via a diacid linker.

GENERAL PROCEDURES FOR SOLID-PHASE PEPTIDE SYNTHESIS la. Coupling with HBTU/HOBt (used for Ser/Gly/Glu/Lys Amino Acids and Ahx). Fmoc-amino acid (4 equiv), 2-(lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate (HBTU, 4 equiv), 1-hydroxybenzotriazole (HOBt, 4 equiv), and N,N-diisopropylethylamine (DIEA, 7 equiv) were dissolved in DMF (5 mL) to give a reaction mixture which was added to the 2-chlorotrityl-Gly resin (0.5 g, theoretical loading 0.55 mmol g-1, 0.275 mmol). The reaction mixture was shaken at room temperature (2*90 min) and washed with DMF (3*5 mL) and DCM (3*5 mL). 90% average yield/amino acid based on monitoring Fmoc removal by UV-vis spectroscopy (ε³⁰⁰ =7800 cm^"1 M^'1). Ib. Coupling with DIC/HOBt (used for Arg Amino Acid). Fmoc-amino acid (4 equiv), N. N-diisopropylcarbodiimide (DIC, 4 equiv), HOBt (4 equiv), DIEA (7 equiv), and 4-(dimethylamino)-pyridine (DMAP, 0.1 equiv) were dissolved in DMF to give a clear solution which was added to the resin. The reaction mixture was shaken at room temperature for 90 min, and then a fresh solution (of the above mixture) was added to the resin and shaken overnight. The resin was washed with DMF (3*5mL), DCM (3^*5mL). Typically 85% yield.

//. Fmoc Deprotection. The Fmoc-protected resin was suspended in a solution of 20% piperidine in DMF (5 mL) for 2*10 min. The resin was washed with DMF (3*5 mL) and DCM (3^*5 mL).

III. Peptide Cleavage from Resin by TFA Treatment. The resin was washed with DCM (3^*5 mL) and treated with a solution of 3% TFA/DCM (5 mL) for 2*10 min. After removal of the resin by filtration, the filtrates were combined and the product was precipitated by the addition of cold Et20 (25 mL). After filtration, the product was obtained as a white powder.

IV. Cyclization of the Linear Pentapeptide. The linear pentapeptide was dissolved in DMF (high dilution, C=2*10^"3M). After addition of 2-(lH-7- azabenzotriazol-l-yl)-l,3,3-tetramethyluranium hexafluorophosphate methanaminium (HATU,2 equiv) and DIEA (7 equiv), the suspension was stirred for 12 h at room temperature. After the solution was concentrated under vacuum to a minimum volume of DMF, the cyclic peptide was precipitated by the addition of water.

V. Pbf and OtBu Deprotection with TFA. The protected peptide was dissolved in TFA/H₂0/Et₃SiH/phenoi (88:5:2:5). After 3 h, Et₂0 was added to the reaction mixture and a white precipitate was collected by filtration. The final product was HPLC purified using conditions stated above. Compounds of the invention which were synthesized and examined here are summarized in Table 1 (in accordance with general formula (V)):

Intercalates

Table 1: Compounds of the invention (general formula (V))

Compound Intercalator

Glu-AQ (CH₂)₂COOH Anthraquinone (AQ)

Anthraquinone (AQ)

1-Ac -CH₂OH Acridine (Ac) * D-Lys is replaced with L-Lys as Intercalator connecting linker.

Synthesis of Compound 1-AQ

Linear-Gly-Arg(Pbfl-Ser(tBu)-Arg(PbJ)-D-Lys(AQ). The linear peptide GRSRK was initially synthesized on the solid support as described in the general procedures (sections I-III). 235 mg of crude linear peptide was subsequently cyclized without purification. Cyclic-Gly-Arg(Pbj)-Ser(tBu)-Arg(Pbf)-D-Lys(AQ). The linear peptide was cyclized according to general procedure (section IV). The cyclic peptide was purified by column chromatography (CombiFlash) using conditions described in materials and equipment. Yield: 164 mg (0.13 mmol, 70%) of cyclic peptide.

Deprotection of Cyclic-Gly-Arg-Ser-Arg-D-Lys(AQ). Final deprotection of Pbf and tBu protecting groups was carried out as described in general procedures (section V). MS:[M+1] (Observed) ) 820.01 ; [M+l] (expected) ) 819.88.

Synthesis of Compound 1-L-AQ.

Cyclic-Gly-Arg-Ser-Arg-Lys(AQ) was synthesized as described for compound 1-AQ. 290 mg of crude linear peptide was subsequently cyclized without purification. Yield: 223 mg (0.179 mmol, 78%) of cyclic peptide. Final deprotection in TFA afforded the pure cyclic peptide after it's precipitation in cold di-ethyl ether. MS: [M/2] (observed) ) 410.3; [M+l] (expected) ) 819.88. Synthesis of Compound 1-Ac.

Cyclic-Gly-Arg-Ser-Arg-Lys(Ac) was synthesized as described for compound 1- AQ. 210 mg of crude linear peptide were subsequently cyclized without purification. Yield: 170 mg (0.136 mmol, 82%) of cyclic peptide. Final deprotection in TFA afforded the pure cyclic peptide after it's precipitation in cold di-ethyl ether. MS: [M/2] (observed) ) 395.93; [M+l] (expected)) 789.88.

Synthesis of compound Gly-AQ.

Linear-Gly-Arg(Pbf)-Gly-Arg(Pbj)-D-Lys(AQ). The linear peptide GRGRK was initially synthesized on the solid support as described in the general procedures (sections I-III). 230 mg of crude linear peptide was subsequently cyclized without purification.

Cyclic-Gly-Arg(Pbfi-Gly-Arg(Pbf)-D-Lys(AQ). The linear peptide was cyclized according to general procedure (section IV). The cyclic peptide was purified by column chromatography (CombiFlash) using conditions described in materials and equipment. Yield: 190 mg (0.164 mmol, 84%) of cyclic peptide. Deprotection of Cyclic-Gly-Arg-Gly-Arg-D-Lys(AQ). Final deprotection of Pbf and tBu protecting groups was carried out as described in general procedures (section V). MS:[M/2] (observed) ) 395.3; [M+l] (expected) ) 788.85. Synthesis of Compound 1 (No Intercalator, see scheme 6).

Cyclic-Gly-Arg-Ser-Arg-Gly. The cyclic control peptide GRSRG was synthesized on the solid support as described in the general procedures (sections I-IV). 205 mg of linear peptide was subsequently cyclized without purification. Cyclic peptide was purified by column chromatography (CombiFlash) using the conditions described in materials and equipment. Total yield of cyclization was 170 mg (0.158 mmol, 84%).

Deprotection of Cyclic Peptide. Final deprotection of Pbf and tBu protecting groups was carried out as described in general procedures (section V). MS: [M/2] (observed) ) 257.64; [M+l] (expected) ) 513.55.

Synthesis of Compound Lin-1 (see scheme 6).

Linear-Gly-Arg-Ser-Arg-Ahx-(AQ). The linear peptide GRSR-Ahx was initially synthesized on the solid support (150 mg Rink Amide resin, L) 0.5 mmol/g) as described in general procedures (sections I-II).

Coupling of AQ-Cl to Resin-Bound Linear Peptide. AQ-C1 (4 equiv) and DIEA (7 equiv) dissolved in dry DCM (3 mL) were added to the linear peptide GRSRAhx (on the solid support) and shaken for 90 min. Cleavage from the resin and removal of the protecting groups were carried out as described in general procedures (section V). 6.5 mg of compound Lin-1 were obtained (7.9 μταοΐ, 10% yield). MS: [M/2] (observed) ) 411.2; [M] (expected) ) 820.89.

DNA Cleavage Monitored by Agarose Gel Electrophoresis.

DNA cleavage experiments were performed using 95 ng DNA (PCR product or plasmid) per reaction. DNA (dissolved in 50 mM Tris-HCl/10 mM CaC12 buffer, pH ) 7.4) was incubated (in the dark) at 37 °C with the various peptides. After incubation, 1.5 //L of the loading buffer (30 mM EDTA, 0.05% (w/v) glycerol, 36% (v/v) bromophenol blue) was added to each sample followed by loading samples (total volume ) 15 μ on al% agarose gel containing 1.0 /g/mL ethidium bromide. Electrophoresis was carried out at 90 V for 1.5 h in 0.5 M TAE buffer. Bands were visualized by UV light and photographed. DNA Cleavage Monitored by Polyacrylamide Gel Electrophoresis.

Double-stranded oligonucleotide (50 bp), 52-end labeled at the top strand, was incubated at 37 °C with 1-AQ in a 50 mM Tris, in 10 mM CaC12 buffer (pH 7.4), and in the dark. The aliquots were withdrawn at various time intervals, and the reaction was stopped by placing samples on dry ice. DNA cleavage products were resolved by polyacrylamide gel electrophoresis under denaturing conditions (20% PAA/8 M urea). The autoradiograms were visualized by using the bioimaging analyzer BAS-2500 (Fuji Photo Film Co. Ltd., Tokyo, Japan) and Aida image analyzer software (Raytest GmbH, Strauben, Germany). DNA Synthesis.

Oligonucleotides (15-mer 52-CGCGATGACTGTACT and its complementary sequence) were synthesized on an Applied Biosystems 3400 DNA/RNA synthesizer, purified by reverse-phase HPLC (Phenomenex, Clarity 5 μ oligo RP), and characterized by MALDI-TOF MS.

DNA Cleavage as Monitored by Hyperchromicity.

A 60 piL quartz cuvette was loaded with 1.8 μΜ dsDNA (15 mer-52- CGCGATGACTGTACT and its complementary sequence) dissolved in Tris buffer (50 mM Tris, 10 mM CaC12, pH 7.4). The peptide conjugate (at a given concentration between 1.8 μΜ to 18 μΜ) was added, and the adsorption of the sample at 260 nm was monitored by UV-vis spectroscopy every 5 min.

Binding Affinity of Inactive Peptide Conjugate (Glu-AQ) to CT-DNA as Determined by EtBr Displacement.

A 1 mL four-sided quartz cuvette was loaded with ethidium bromide (1.3 μΜ) dissolved in Tris buffer (50 mM Tris, 10 mM NaCl, pH 7.4). The fluorescence was measured (ex. 545 nm, em. 595 nm, RT). Next, CT-DNA was added (254 μΜ) in aliquots (¾tiL), and the increase in fluorescence was measured after the continuous addition of CT-DNA. This procedure was continued until no change in fluorescence was noticed (saturation). Finally, a solution of Glu-AQ (4 μΜ) was added in aliquots of 2.6 μ\,, and fluorescence quenching was measured. The binding affinity of Glu-AQ was estimated according to:

K_app = (K_EBX[EB])/[Glu - AQ]₅₀

where K_EB = l O'M^"1, [EB]=1.3*10^"6M and [Glu-AQ]₅₀ is the concentration of concentration of Glu-AQ where 50% reduction in fluorescence is achieved (29). Structural Considerations

On the basis of the active site found in Staphylococcal nuclease which consists of two L-Arg (Arg35 and Arg87) that electrostatically bind and activate the phosphodiester bonds toward hydrolysis by an activated water as nucleophile, several cyclic pentapeptides were synthesized (Scheme 1) with the following variations: Triads of the type Arg-X-Arg (where X = L-Ser, Gly, or L-Glu) positioned on the y-turn (25, 26) as mediators of DNA cleavage were studied. The choice of X = L-serine (compounds 1-AQ and 1-Ac) stems from the potential of the hydroxyl group to act as a nucleophile; attacking the phosphodiester bond that is more susceptible to nucleophilic attack due to it is electrostatic interactions with two L-Arg. In the case of L-Glu (compound Glu-AQ), the carboxyl side chain of this amino acid is designed to activate a water molecule (as elucidated in the enzyme's active site) that, in turn, would then attack the susceptible phosphodiester bond. Positioning Gly (compound Gly-AQ) between two L-Arg should determine whether it is necessary to have a nucleophile at position X, as H₂0 may provide this role. In addition, a cyclic pentapeptide conjugated to AQ (1-L-AQ, Wide post) was synthesized, where L-lysine replaces D-Lys. This change in stereochemistry should dramatically change the cyclic backbone conformation, thus allowing assessment of the importance of the designed cyclic peptides (with the βΙΓ/7-turn arrangement 25, 26) to DNA nuclease activity. Chemical Synthesis

The synthesis of the cyclic pentapeptides (Scheme 1) was carried out by synthesizing the linear peptide (GRSRK(X), GRSRK(X), GRGRK(X), and GRERK(X), where = D-lysine and X = AQ or Acridine) on the solid support (2-chlorotrityl Gly resin) followed by C- to N-terminus peptide cyclization in a diluted DMF solution using HATU as a coupling reagent. After cyclization, peptides were deprotected in TFA (88:2:5:5 TFA/triethylsilane/water/phenol) for 3 h and precipitated in cold di-ethyl ether. Purity of cyclic compounds was determined by HPLC and Maldi-TOF MS,

DNA Nuclease Activity of Cyclic Peptides

Chemical nuclease activity was initially evaluated in a phosphate buffer after incubating both 1-AQ and Glu-AQ (20 μΜ) with plasmid DNA (pSP73) in the dark to avoid UV -induced DNA photocleavage by AQ (27, 28). At 37°C, no activity of either cyclic pentapeptide was seen. However, at 50°C, 1-AQ was extremely active (Figs. 7 and 8). Taking into account that a high concentration of phosphate ions (phosphate buffer) might interfere with the nuclease activity of the cyclic pentapeptides, it was decided instead to incubate the cyclic pentapeptides in a TRIS buffer. In this buffer (50 mM TRIS, 10 mM CaCl₂, pH=7.4), incubation of 1-AQ with plasmid DNA at 37 °C leads to complete disappearance of the band after overnight incubation (Fig. 1A). To verify that this observation is due to DNA cleavage, a 50-mer ³²P-radiolabeled duplex was incubated with 1-AQ. As shown in Fig. IB, small DNA fragments are observed already after 4 h of incubation. Glu-AQ did not show any substantial activity.

It is speculated that the inactivity of Glu-AQ might be a consequence of an electrostatic interaction between the guanidine group(s) of L-Arg (positively charged at pH = 7.4) and the carboxylate group of L-Glu. Thus, the designed activity for both L- Arg and L-Glu might be hampered by such an electrostatic interaction.

As Glu-AQ showed no DNA nuclease activity, it was exploited in order to estimate the apparent binding affinity of this class of peptide conjugates to dsD A. An EtBr displacement experiment was conducted (see experimental section for details). By following the reduction in EtBr fluorescence (when excluded from CT-DNA by Glu- AQ), a app value of 5.4^*10⁵ M^"1 was determined (Fig. 2). This value is in the range of the intrinsic binding constants reported for peptidyl anthraquinones. Control peptides Lin-1 and Compound 1 (Scheme 6), were synthesized, as indicated hereinabove.

Scheme 6: (A) compound Lin-1: linear version of 1-AQ (cyclic pentapeptide with AQ as DNA intercalator) with 6-aminohexanicacid (Ahx) as a substitute for D- lysine. (B) Compound 1, cyclic version (with Arg-Ser-Arg as triad) without DNA intercalator.

Compound Lin-1 is a linear version of the cyclic peptide where a 6- aminocaproic acid (Ahx) linker is used to separate AQ from the triad (Arg-Ser-Arg). Compound 1 (Scheme 2) is a cyclic pentapeptide that lacks AQ. Both compounds were incubated with a PCR product (591 bp's) and compared to the DNA nuclease activity of the parent compound 1-AQ (Fig. 3). Both compounds Lin-1 and 1 were considerably less active. Thus, it is apparent that both cyclic scaffold and DNA intercalator are required for the superior DNA nuclease activity exerted by compound 1-AQ. In order to gain some insight into the importance of the triad (Arg-Ser-Arg), a cyclic peptide was synthesized where glycine replaces L-Serine. The title compound (cRGRGK conjugated to AQ, Gly-AQ) has two L-arginine amino acids for phosphodiester binding and activation; however, it lacks a nucleophile. DNA nuclease activity of such a conjugate would suggest that a water molecule acts as a nucleophile. Indeed, it was observe that Gly-AQ exerts similar DNA nuclease activity to that of 1-AQ (Fig. 4, lanes 6-8). The high DNA nuclease activity observed for these conjugates also coincides with water as a nucleophile. In such a scenario, phosphodiester cleavage would regenerate the chemical nuclease providing catalytic activity to such conjugates. DNA nuclease activity of both cyclic peptides with the Arg- Ser-Arg triad but with different DNA intercalators (AQ and Ac) were compared (Fig. 4, compounds 1-AQ and 1-Ac, lanes 9-1 1). No significant difference in activity was observed, highlighting the versatility of such cyclic peptide- DNA intercalator conjugates as metal-free chemical nucleases. L-Lysine was introduced into the cyclic pentapeptide (cRSRGK, 1-L-AQ) replacing D-lysine. This is expected to bring about a dramatic change in the conformation of the cyclic peptide's backbone. As shown in Fig. 4 (lane 12), a concentration of 16 μΜ of 1-L-AQ results only in modest DNA nuclease activity (as corroborated by the weakened DNA band) in comparison to complete disappearance of the DNA band obtained with 1-AQ, 1-Ac, and Gly-AQ at the same concentration. Mechanism and Potency of DNA Cleavage

To gain evidence that supports a hydrolytic mechanism of cleavage by the active cyclic pentapeptides, ROS scavengers were added to incubated samples (1-AQ + DNA) in order to exclude an oxidative mechanism for DNA cleavage. As shown in Fig. 5, the addition of a singlet oxygen quencher (NaN₃), hydroxyl radical scavengers (DMSO or tBuOH), and a superoxide scavenger (KI) had no effect on 1-AQ's DNA nuclease activity. In all cases, the plasmid is cleaved as observed for the control sample (lane 3) without inhibitor. The fact that DNA cleavage of plasmid DNA or duplex DNA (591 bp) by 1-AQ (and other active cyclic peptides) yielded either DNA smears or complete disappearance of the parent band precluded the straightforward analysis of cleavage kinetics (i.e., nicking (form Il-coiled) and double strand breaks (form Ill-linear)). Thus, DNA cleavage of the active peptide conjugates was monitored by following DNA degradation by monitoring the change in absorbance at 260 nm (hyperchromicity). A synthetic 15-mer DNA duplex was chosen as a DNA substrate. The various peptide conjugates were added to this 15-mer duplex DNA substrate, and the change in adsorption was followed at 260 nm. Degradation of dsDNA to ssDNA and to further single bases is expected to lead to an increase in absorption (hyperchromicity) (31, 32). As complete degradation of the radiolabeled 50-mer duplex (Fig. IB), was observed it was assumed that the change in adsorption on a longer time scale in which the change in O.D. is negligible (e.g., 2 h) represents a good approximation for complete degradation of the DNA substrate. Accordingly, it was estimated the amount of intact DNA as follows:

% intact DNA = 100 - (O.D, - O.D₀/O.D_∞)

Where O.D, is the measured adsorption, O.D₀ is the measured adsorption at t 0, and O.D.oo is the measured adsorption at the end of the reaction (plateau region). By plotting the logarithm of this value as a function of time (within the first hour of kinetics), a linear fit was obtained where the slope is an estimation for kobs. Fig. 6 presents the hyperchromicity data (change in O.D.) related to the initial DNA nuclease activity (within 2 h) and the linear plots of the three active peptide conjugates, namely, 1-AQ, 1-Ac, and Gly-AQ at 2.5 equiv peptide conjugate to dsDNA substrate. On the basis of the fobs values that range 0.65-0.85 h^"1, all three peptide conjugates seem to be similarly active. The peptide conjugate (Gly-AQ) that has no bulky group situated between the two L-Arg in its triad (Gly instead of L-Ser) seems to be the most active of all three compounds. Without being bound by theory it was speculated that the introduction of L-Ser at this position diminishes DNA nuclease activity perhaps due to steric hindrance. However, further peptide analogues are required in order to verify this argument. The small differences in fobs between 1-AQ and 1-Ac are attributed to the DNA intercalator (anthraquinone vs. acridine), as this is the only difference in structure between these peptide conjugates. These experiments were also repeated with one of the active compounds (1-AQ) at various peptide-conjugate concentrations. Table 2 lists the fobs values determined for this peptide conjugate (see Fig. 9 for kinetic profiles of 1- AQ at various concentrations). Interestingly, it was found that at a high peptide conjugate concentration (10 equiv) the fobs value is reduced. Thus, an optimal value is obtained at 7.5 equiv of 1-AQ. It is possible that, at higher concentrations, compound 1- AQ forms aggregates in buffer by 7r-stacking of the appended anthraquinone moieties on each D-Lys of the cyclic peptide. At 7.5 equiv of 1-AQ, a fobs of 1.62 h^"1 is found based on the hyperchromicity measurements. It is noted that this value is by an order of magnitude higher than the most efficient metal-free DNA nucleases reported to date (at physiological conditions).

Table 2: Kinetics of DNA cleavage by 1-AQ as determined by hyperchromicity at 260nm

1)

Claims

CLAIMS:

1. A cyclic peptide comprising 4 to 8 amino acids (AA), wherein:

- at least one AA (AA^ comprising at least one positively charged group selected from -SR₂ ⁺, -NR₃ ⁺; -PR₃ ⁺, -NHCH₂C=NH₂ ⁺(NH₂), -NCH₃C-NH₂ ⁺(NH₂), -NHC=NH₂ ⁺(NH₂), imidazolium;

- wherein each R is independently selected from H or Ci-C₅ alkyl; and

- at least one AA (AAn) comprising a functional group capable of binding with a nucleotidic macromolecule;

provided that at least one AAj and at least one AAn have opposite configurations.

2. A cyclic peptide according to claims 1 , comprising at least one AA (AA_ni) comprising a group capable of nucleophillicly attacking said nucleotidic macromolecule, selected from -OH, -SH, -NH-OH, -SeH, -SeOH.

3. A cyclic peptide according to claim 2, wherein said at least one AAni is selected from the group consisting of Tyrosine, Serine, Threonine, Cysteine, Selenocysteine, and

Homoserine.

4. A cyclic peptide according to any one of claims 1 to 3, comprising at least two AAi, that may be the same or different.

5. A cyclic peptide according to claim 4, wherein said AAj is selected from Arginine, Histidine, Lysine and Ornithine.

6. A cyclic peptide according to any one of claims 1 to 5, wherein said functional group of AAn binds to nucleotidic macromolecule via electrostatic binding, covalent binding or intercalating to said nucleotidic macromolecule.

7. A cyclic peptide according to any one of claims 1 to 6, wherein said functional group of AAn binds to a specific sequence of nucleotidic macromolecule.

8. A cyclic peptide according to any one of the preceding claims, wherein said functional group of AAn is an aromatic or heteroaromatic moiety.

9. A cyclic peptide according to claim 8, wherein said aromatic or heteroaromatic moiety is selected from anthraquinone, acridine, pyrene, ethidium bromide, daunomycin and doxorubicin.

10. A cyclic peptide according to claims 1 to 7, wherein said functional group of AAn is a peptide nucleic acid (PNA), morpholino, DNA-LNA, triplex forming oligonucleotide (TFO) or any DNA analog having high binding affinity to a complementary DNA or RNA.

11. A cyclic peptide according to claim 1, comprising a sequence of three AA, comprising two independent AAi.

12. A cyclic peptide according to claim 11, wherein said two AA] are directly linked to one another.

13. A cyclic peptide according to claim 11, wherein said two AAi are linked via said third AA of the sequence.

14. A cyclic peptide according to any one of claims 11 to 13, comprising an AA (AA_ffl) having a group capable of nucleophillicly attacking said nucleotidic macromolecule, selected from -OH, -SH, -NH-OH, -SeH, -SeOH.

15. A cyclic peptide according to any one of claims 1 to 14, wherein the configuration of said at least one AAi is D.

16. A cyclic peptide according to any one of claims 1 to 14, wherein the configuration of said at least one AAj is L.

17. A cyclic peptide according to any one of the preceding claims comprising independent four AA.

18. A cyclic peptide according to any one of the preceding claims comprising independent five AA.

19. A cyclic peptide according to any one of the preceding claims, having the following general formula (II):

wherein wherein Xj, X₂ and X₃ are each independently a straight or branched C1-C5 alkyl, each optionally substituted with at least one group selected from -OH, -NR₂, - NR₃ ⁺, -SR, -SR₂ ⁺, -PR₂, PR-3⁺, guanidine, N-methyl-guanidine, imidazolium;

wherein each R is independently selected from H or CrC₅ alkyl; and provided 5 that at least one of Xi, X₂ and X3 is positively charged;

Y is selected from straight or branched CpC₅ alkyl, optionally substituted with at least one group selected from -OH, -SH, -NH-OH, -SeH, -SeOH, phenyl, phenol, - COOH, -CONH2;

Z is a linker which may be absent or a straight or branched Cj-C₈ alkylene, 10 optionally substituted at the terminal end with at least one group selected from an amine, amide, ester, ether, thioester, thioether, acyl, imine, oxime, triazole, azide, and maleimide; and

W is a group capable of binding to a nucleotidic macromolecule.

20. A cyclic peptide having the following general formula (I):

15 cyc-Y_i-Y₂-y₃-Y4-Y5 (I)

wherein

Y1-Y5 are each independently an AA; wherein

- at least one AA (AAj) comprises at least one positively charged group selected from -SR₂ ⁺, -NR₃ ⁺; -PR₃ ⁺, -NHCH₂C=NH₂ ⁺(NH₂), 20 -NCH₃C=NH₂ ⁺(NH₂), -NHC-NH₂ ⁺(NH₂), imidazolium;

- wherein each R is independently selected from H or Q-C5 alkyl; and

at least one AA (AAn) comprises at least functional group capable of binding with said nucleotidic macromolecule;

provided that at least one AA_t and at least one AAn have opposite 25 configurations.

21. A cyclic peptide according to claims 20, wherein AAi is selected from Arginine, Histidine, Lysine and Ornithine.

22. A cyclic peptide according to claims 20 or 21, comprising at least two AAj, which are the same or different.

30 23. A cyclic peptide according to claim 20, wherein said functional group of AAn binds to nucleotidic macromolecule via electrostatic binding, covalent binding or intercalating to said nucleotidic macromolecule.

24. A cyclic peptide according to claim 20, wherein said functional group of AAn binds to a specific sequence of nucleotidic macromolecule.

25. A cyclic peptide according to claims 20 or 24, wherein said functional group is an aromatic or heteroaromatic moiety.

5 26. A cyclic peptide according to claim 25, wherein said aromatic or heteroaromatic moiety is selected from anthraquinone, acridine, pyrene, ethidium bromide, daunomycin and doxorubicin.

27. A cyclic peptide according to claim 20, wherein said functional group is a peptide nucleic acid (PNA), morpholino, DNA-LNA, triplex forming oligonucleotide

10 (TFO) or any DNA analog having high binding affinity to a complementary DNA or RNA.

28. A cyclic peptide according to claims 20 to 27, comprising at least one AA (AAni) comprising a group capable of nucleophillicly attacking said nucleotidic macromolecule, selected from -OH, -SH, -NH-OH, -SeH, -SeOH.

15 29. A cyclic peptide according to claim 28, wherein said at least one AAm is selected from the group consisting of Tyrosine, Serine, Threonine, Cysteine, Selenocysteine, and Homoserine.

30. A cyclic peptide according to any one of claims 20 to 29, wherein the configuration of said at least one AA_! is D.

20 31. A cyclic peptide according to any one of claims 20 to 30, wherein the configuration of said at least one AAi is L.

32. A cyclic peptide according to any one of claims 20 to 31, having the following general formula (II):

25 wherein wherein Xj, X₂ and X₃ are each independently a straight or branched Q- alkyl, each optionally substituted with at least one group selected from -OH, -NR₂, - NR₃ ⁺, -SR., -SR₂ ⁺, -PR₂, PR₃ ⁺, guanidine, N-methyl-guanidine, imidazolium;

wherein each R is independently selected from H or d-Cs alkyl; and provided that at least one of Xi, X₂ and X₃ is positively charged;

Y is selected from straight or branched CpC₅ alkyl, optionally substituted with at least one group selected from -OH, -SH, -NH-OH, -SeH, -SeOH, phenyl, phenol, - COOH, -CONH₂;

Z is a linker which may be absent or a straight or branched C C₈ alkylene, optionally substituted at the terminal end with at least one group selected from an amine, amide, ester, ether, thioester, thioether, acyl, imine, oxime, triazole, azide, and maleimide; and

W is a group capable of binding to a nucleotidic macromolecule.

33. A cyclic peptide having the following general formula (II):

wherein

wherein X_l5 X₂ and X₃ are each independently a straight or branched C₁-C5 alkyl, each optionally substituted with at least one group selected from -OH, -NR₂, - NR₃ ⁺, -SR, -SR₂ ⁺, -PR₂, PR₃ ⁺, guanidine, N-methyl-guanidine, imidazolium;

wherein each R is independently selected from H or Ci-C₅ alkyl; and provided that at least one of X_1; X₂ and X₃ is positively charged;

Y is selected from straight or branched Q-C5 alkyl, optionally substituted with at least one group selected from -OH, -SH, -NH-OH, -SeH, -SeOH, phenyl, phenol, - COOH, -CONH₂; Z is a linker which may be absent or a straight or branched C]-Cg alkylene, optionally substituted at the terminal end with at least one group selected from an amine, amide, ester, ether, thioester, thioether, acyl, imine, oxime, triazole, azide, and maleimide; and

5 W is a group capable of binding to a nucleotidic macromolecule.

34. A cyclic peptide according to claim 33, wherein X] and X₂ are each independently a straight or branched C1-C5 alkyl, each substituted with at least one group selected from -NH₃ ⁺, guanidine, N-methyl-guanidine and imidazolium.

35. A cyclic peptide according to claims 33 or 34, wherein W is a group selected 10 from an intercalator, a peptide nucleic acid (PNA), morpholino, DNA-LNA, triplex forming oligonucleotide (TFO) or any DNA analog having high binding affinity to a complementary DNA or RNA.

36. A cyclic peptide according to claim 35, wherein said intercalator is selected from anthraquinone, acridine, pyrene, ethidium bromide, daunomycin and doxorubicin.

15 37. A cyclic peptide according to any one of the preceding claims, for use in cleaving a nucleotidic macromolecule.

38. A cyclic peptide according to claim 37, wherein said cleaving of nucleotidic macromolecule is site-selective.

39. A cyclic peptide according to claims 37 or 38, wherein said cleaving is obtained 20 under physiological conditions.

40. A cyclic peptide according to any one of the preceding claims for use as a medicament.

41. Use of a cyclic peptide according to any one of the preceding claims for the preparation of a medicament.

25 42. Use of a cyclic peptide according to any one of the preceding claims, in the preparation of a medicament for nucleotidic-macromolecule targeted therapy.

43. Use according to claim 42, wherein said medicament is for the treatment of a disease or disorder selected from cancer, autoimmune diseases, genetically hereditary diseases and inflammation.

30 44. A method of cleaving a nucleotidic macromolecule comprising contacting said macromolecule with an effective amount of a cyclic peptide according to any one of claims 1 to 40.

45. A method according to claim 44, wherein said cleaving is achieved under physiological conditions.

46. A method according to claims 44 or 45, wherein the effective amount of said cyclic peptide is in the range of between about 1 μΜ to about 50μΜ.

47. A method according to any one of claims 44 to 46, wherein said cleaving of said macromolecule is site-specific.

48. A method according to any one of claims 44 to 47, wherein said nucleotidic macromolecule is associated with a disease or disorder.

49. A method according to claim 48, wherein said disease or disorder is selected from cancer, autoimmune diseases, genetically hereditary diseases and inflammation.

50. A method of treating a disease or disorder associated with a specific gene, comprising administering to a subject in need thereof an effective amount of a cyclic peptide according to claims 1 to 40.