Bradykinin receptor modulators and use thereof

This application is the national stage entry under 35 U.S.C. .sctn. 371 of International Application No. PCT/IB2014/001162, filed on Jun. 24, 2014, which claims priority to Swiss Application No. 01168/13, filed Jun. 25, 2013. The contents of these applications are each incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to novel biological and purified peptides as bradykinin receptor modulators for use in the treatment and/or prevention of inflammatory diseases, pain, hyperalgesia, cardiovascular and/or cerebral ischemic diseases, in a subject in need thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 9, 2016, is named 13239.0001-00000_SL.txt and is 44,191 bytes in size.

BACKGROUND OF THE INVENTION

Angiogenesis is the development of new blood capillaries and vessels. This process occurs normally in a number of biological situations, including fetal development, menstruation, ovulation, placental development and the development of collateral blood vessels in areas of disease or ischemia, nerve regeneration, bone growth, and wound healing. All these events, especially fetal development, require the very rapid growth of endothelial cells and their migration and differentiation into a complex network of vessels.

In addition to angiogenesis, which takes place in the healthy individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessels proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.

Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated.

Vascular endothelial cell growth factor (VEGF), which is also termed VEGF-A or vascular permeability factor (VPF), has been reported as a pivotal regulator of both normal and abnormal angiogenesis (Ferrara and Davis-Smyth, 1997; Ferrara, 1999). In addition, truncated forms of VEGF-D and VEGF-C have been shown to stimulate angiogenesis through binding to VEGFR-2 (WO2012/088563).

The stimulation of angiogenesis has also been shown with agonistic substances of Bradykinin B1 and B2 receptors (WO 02/17958).

Bradykinin (BK) is a nonapeptide of sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, generated as a result of the activity of kallikreins, a group of proteolytic enzymes present in most tissues and body fluids, on kininogens. BK and related kinins, like Lys-BK (kallidin) and Met-Lys-BK are able to activate the bradykinin B2 receptor. The removal of the C-terminal Arg residue in BK or in kallidin (Lys-BK) generates desArg9-BK and desArg9-Lys-BK. These peptides are also kinins and are specific ligands of the bradykinin B1 receptor. Once released, kinins produce many physiological responses, including pain and hyperalgesia by stimulating C- and A-fibers in the periphery. There is also considerable evidence that kinins contribute to the inflammatory response.

Bradykinin, and its physiologically important related peptides described above exhibit physiological actions which qualify them as mediators of inflammatory reactions, hypotensive states, pain, hyperalgesia, cardiovascular and/or cerebral circulatory diseases, tissue repair, stem cell differentiation, angiogenesis, lymphangiogenesis, immune system cells modulation, sepsis, wasting states, diabetes, neurogenesis, cardiac function and remodeling, kidney function, neurogenesis, and tumor development (Marceau F. and Regoli D., 2004).

Although treatments exist, there remain significant unmet needs for efficient and better therapies for activating or inhibiting angiogenesis and preventing and/or treating inflammatory diseases, pain, hyperalgesia, cardiovascular and/or cerebral ischemic diseases, notably in term of improved efficacy, and diminution of side effects.

SUMMARY OF THE INVENTION

The present invention provides an isolated and purified peptide selected from the group consisting of: a) a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7; b) a biological fragment thereof having at least 8 consecutive residues in length.

The present invention also provides an isolated and purified peptide selected from the group consisting of: a) a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:49; b) a biological fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:42;

characterized in that said isolated and purified peptide is a Bradykinin receptor modulator.

Also disclosed is an isolated and purified nucleic acid molecule encoding an isolated and purified peptide, a vector comprising said nucleic acid molecule, a recombinant host cell characterized in that said cell expresses an isolated and purified peptide of the invention.

Further disclosed is a pharmaceutical composition comprising an isolated and purified peptide according to the invention, the use of said pharmaceutical compositions as analgesics, and the use of said pharmaceutical compositions in the treatment and/or prevention of inflammatory diseases, cardiovascular ischemia, pain, hyperalgesia, renal diseases, wound healing, cerebral ischemia, stroke, vascular dementia, infarct dementia, myocardial ischemia, coronary heart disease, myocardial infarction, peripheral limb disease, periphery arterial occlusive disease, sepsis, wasting states, diabetes and/or disorders associated with defective blood flow or blood vessel malformation, tissue repair, stem cell differentiation, angiogenesis, lymphangiogenesis, immune system cells modulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of 1 .mu.M Bradykinin (BK) and of different concentrations of the peptide PepD 2-10 (KPPCVNVFR; SEQ ID NO:8), corresponding to the amino acid residues number 133-141 from human VEGF-D sequence registered at Expasy (http://www.uniprot.org/uniprot/O43915), on the HUVEC angiogenesis assay. * p<0.05 compared to control (Student's "t" test).

FIG. 2 shows the effect of 1 .mu.M Bradykinin (BK) and 10 .mu.M of the peptides PepD 2-10 (KPPCVNVFR; SEQ ID NO:8) and PepD 1-10 (SEQ ID NO:1), corresponding respectively to the aminoacid residues number 133-141 and 132-141 from human VEGF-D sequence registered at Uniprot (http://www.uniprot.org/uniprot/O43915), and PepA 1-9 (FKPSCVPLM; SEQ ID NO:20) and PepA 2-9 (KPSCVPLM; SEQ ID NO:27), corresponding to the amino acid residues number 73-81 and 74-81 from the human VEGF-A sequence registered at Uniprot (http://www.uniprot.org/uniprot/P15692), on the HUVEC angiogenesis assay. * p<0.05 compared to control (Student's "t" test).

FIG. 3 shows the effect of 1 .mu.M Bradykinin (BK) and of the peptide PepD 1-9 (FKPPCVNVF; SEQ ID NO:15), corresponding to the amino acid residues number 132-140 from human VEGF-D sequence registered at Expasy (http://www.uniprot.org/uniprot/O43915), on the HUVEC angiogenesis assay. * p<0.05 compared to control (Student's "t" test).

FIG. 4 shows the result of HUVEC stimulation by PepC 2-9 (FIG. 4B) and PepC 2-10 (FIG. 4A) at 1 .mu.M and 10 .mu.M concentrations and PepC 1-10 (FIG. 4C) at 0.1, 1.0 and 10 .mu.M concentrations, 24 h after initial stimulus.

FIG. 5A-E demonstrates that Calcium entry in HUVEC cells induced by PepD2-10 and detected by Fluo-3 fluorescence, is due to B1 bradykinin receptor activation.

FIG. 5A: A.F.U.--Arbitrary Fluorescence Units; BK 1 .mu.M Bradykinin; THG Thapsigargin 10 .mu.M. FIG. 5B: PepD 2-10 at 10 .mu.M; THG Thapsigargin 10 .mu.M. FIG. 5C: HOE 140 (B2 receptor antagonist) 10 .mu.M; PepD 2-10 10 .mu.M; THG Thapsigargin 10 .mu.M. FIG. 5D: DesArg9-Leu8-BK 10 .mu.M; PepD 2-10 10 .mu.M; THG Thapsigargin 10 .mu.M. FIG. 5E: PepPL 1-10 (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

Growth factors are naturally occurring substances capable of stimulating cellular growth, proliferation and cellular differentiation. Growth factors are important for regulating a variety of cellular processes. Growth factors typically act as signaling molecules between cells. For example, bone morphogenetic proteins stimulate bone cell differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate angiogenesis and blood vessel differentiation.

Individual growth factor proteins tend to occur as members of larger families of structurally and evolutionarily related proteins. For example, placental growth factor (PlGF, UNIPROT accession number P49763), Vascular endothelial growth factor (VEGF-A, UNIPROT accession number P15692; VEGF-B, UNIPROT accession number P49765; VEGF-C, UNIPROT accession number P49767 and VEGF-D, UNIPROT accession number 043915), and Platelet-derived growth factor (PDGF-A, UNIPROT accession number P04085; PDGF-B, UNIPROT accession number P01127).

In one aspect, the present invention provides an isolated and purified peptide selected from the group consisting of: a) a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:49; b) a biological fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:42;

In another aspect, the present invention provides an isolated and purified peptide selected from the group consisting of: a) a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:49; b) a biological fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:42;

wherein said isolated and purified peptide is a Bradykinin receptor modulator.

As used herein, the terms "peptide", "protein", "polypeptide", "polypeptidic", and "peptidic" are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

The present invention also includes biological variants of the isolated and purified peptide. The term "biological variants" refer to polypeptides having amino acid sequences that differ to some extent from a native sequence of the isolated and purified peptide that is amino acid sequences that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly II. Polar, positively charged residues: His, Arg, Lys III. Polar, negatively charged residues: and their amides: Asp, Asn, Glu, Gln IV. Large, aromatic residues: Phe, Tyr, Trp V. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys.

As used herein, "biological variants" have a mutation, and/or a substitution of at least one residue in an amino acid sequence selected from the group consisting of SEQ ID NO:50 to SEQ ID NO:56. Preferably, said biological variants consist of SEQ ID NO:36 to SEQ ID NO:49. More preferably said biological variants are Bradykinin receptor modulators. Even more preferably, said biological variants are anti-angiogenics.

In a further aspect, the present invention provides an isolated and purified peptide selected from the group consisting of: a) a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7; b) a biological fragment thereof having at least 8 consecutive residues in length.

The present invention also provides an isolated and purified peptide selected from the group consisting of: a) a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7; b) a biological fragment thereof having at least 8 consecutive residues in length;

wherein said isolated and purified peptide is a Bradykinin receptor modulator.

Preferably step b consists of biological fragments having an amino acid sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:28. More preferably, said biological fragments have Bradykinin receptor agonist activity.

"Biological fragments" refer to sequences sharing at least 50% amino acids in length with the respective sequence of the isolated and/or purified peptide. These sequences can be used as long as they exhibit the same properties as the native sequence from which they derive. Preferably these sequences share more than 70%, preferably more than 80%, in particular more than 90% amino acids in length with the respective sequence of the isolated and/or purified peptide. These fragments can be prepared by a variety of methods and techniques known in the art such as for example chemical synthesis.

"Homology" is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

Furthermore, since an inherent problem with native peptides (in L-form) is the degradation by natural proteases, the peptide of the invention may be prepared in order to include D-forms and/or "retro-inverso isomers" of the peptide. Preferably, retro-inverso isomers of short parts, variants or combinations of the peptide of the invention are prepared.

Protecting the peptide from natural proteolysis should therefore increase the effectiveness of the isolated and purified peptide. A higher biological activity is predicted for the retro-inverso containing peptide when compared to the non-retro-inverso containing analog owing to protection from degradation by native proteinases. Furthermore, they have been shown to exhibit an increased stability and lower immunogenicity (Sela M. and Zisman E., 1997).

Retro-inverso peptides are prepared for peptides of known sequence as described for example in Sela and Zisman, 1997.

By "retro-inverso isomer" is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.

Also encompassed by the present invention are modifications of the peptide (which do not normally alter primary sequence), including in vivo or in vitro chemical derivatization of peptides, e. g., acetylation or carboxylation. Also included are modifications of glycosylation, e. g., those made by modifying the glycosylation patterns of a peptide during its synthesis and processing or in further processing steps, e. g., by exposing the peptide to enzymes which affect glycosylation e. g., mammalian glycosylating or deglycosylating enzymes. Also included are sequences which have phosphorylated amino acid residues, e. g., phosphotyrosine, phosphoserine, or phosphothreonine.

The term "isolated and purified" refers to the state in which the peptide of the invention, or nucleic acid encoding such peptide will be, in accordance with the present invention free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e. g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo.

In one embodiment, the present invention provides an isolated and purified peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7 (Table I).

According to another embodiment of the present invention, the isolated and purified peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7 is a bradykinin receptor modulator, preferably, a B2 bradykinin receptor modulator.

More preferably, the isolated and purified peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7 is a B2 bradykinin receptor agonist.

TABLE-US-00001 TABLE I Amino acid Derived SEQ ID NO: sequence Peptide Name from RPPGFSPFR BK SEQ ID NO: 1 FKPPCVNVFR PepD 1-10 VEGF-D SEQ ID NO: 2 FKPPCVSVYR PepC 1-10 VEGF-C SEQ ID NO: 3 IWPPCVEVKR PepPDA 1-10 PDGF-A SEQ ID NO: 4 VWPPCVEVQR PepPDB 1-10 PDGF-B SEQ ID NO: 5 LVPSCVTVQR PepB 1-10 VEGF-B SEQ ID NO: 6 FKPSCVPLMR PepA 1-10 VEGF-A SEQ ID NO: 7 FSPSCVSLLR PepP1 1-10 P1GF

In another embodiment of the invention, the isolated and purified peptide is a biological fragment selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:14 (Table II). According to a further embodiment, the isolated and purified peptide consists of a biological fragment having at least 8 consecutive residues in length and selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:14. Said biological fragment is a bradykinin receptor modulator, preferably a B2 bradykinin receptor modulator.

More preferably, said isolated and purified peptide consisting of SEQ ID NO:8 to SEQ ID NO:14 is a B2 bradykinin receptor agonist.

TABLE-US-00002 TABLE II Amino acid Derived SEQ ID NO: sequence Peptide name from SEQ ID NO: 8 KPPCVNVFR PepD 2-10 VEGF-D SEQ ID NO: 9 KPPCVSVYR PepC 2-10 VEGF-C SEQ ID NO: 10 WPPCVEVKR PepPDA 2-10 PDGF-A SEQ ID NO: 11 WPPCVEVQR PepPDB 2-10 PDGF-B SEQ ID NO: 12 VPSCVTVQR PepB 2-10 VEGF-B SEQ ID NO: 13 KPSCVPLMR PepA 2-10 VEGF-A SEQ ID NO: 14 SPSCVSLLR PepP1 2-10 P1GF

In a subsequent embodiment of the invention, the isolated and purified peptide is a biological fragment selected from the group consisting of SEQ ID NO:15, to SEQ ID NO:21 (Table III). The latter is a bradykinin receptor modulator, preferably a B1 bradykinin receptor modulator. More preferably, said isolated and purified peptide is a B1 bradykinin receptor agonist.

TABLE-US-00003 TABLE 111 Amino acid Peptide Derived SEQ ID NO: sequence name from SEQ ID NO: 15 FKPPCVNVF PepD 1-9 VEGF-D SEQ ID NO: 16 FKPPCVSVY PepC 1-9 VEGF-C SEQ ID NO: 17 IWPPCVEVK PepPDA 1-9 PDGF-A SEQ ID NO: 18 VWPPCVEVQ PepPDB 1-9 PDGF-B SEQ ID NO: 19 LVPSCVTVQ PepB 1-9 VEGF-B SEQ ID NO: 20 FKPSCVPLM PepA 1-9 VEGF-A SEQ ID NO: 21 FSPSCVSLL PepP1 1-9 P1GF

In a further embodiment of the invention, the isolated and purified peptide consists in a biological fragment having at least 8 consecutive residues in length and selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:28 (Table IV). Preferably said biological fragment is a B1 bradykinin receptor modulator. More preferably, said biological fragment is a B1 bradykinin receptor agonist.

TABLE-US-00004 TABLE IV Amino acid Derived SEQ ID NO: sequence from SEQ ID NO: 22 KPPCVNVF PepD 2-9 VEGF-D SEQ ID NO: 23 KPPCVSVY PepC 2-9 VEGF-C SEQ ID NO: 24 WPPCVEVK PepPDA 2-9 PDGF-A SEQ ID NO: 25 WPPCVEVQ PepPDB 2-9 PDGF-B SEQ ID NO: 26 VPSCVTVQ PepB 2-9 VEGF-B SEQ ID NO: 27 KPSCVPLM PepA 2-9 VEGF-A SEQ ID NO: 28 SPSCVSLL PepP1 2-9 P1GF

In a further embodiment of the invention, the isolated and purified peptide consists of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:20, or SEQ ID NO:27.

The present invention also provides an isolated and purified peptide selected from the group consisting of: a) a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:49; b) a biological fragment thereof having an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:42;

wherein said isolated and purified peptide is a Bradykinin receptor modulator.

According to another embodiment of the invention, the isolated and purified peptide consists in a biological variant selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:49 (Table VI and VII).

Preferably, said biological variant is a Bradykinin receptor modulator. More preferably, said biological variant is anti-angiogenic and/or anti-lymphangiogenic.

The term "biological variants" refer to polypeptides having amino acid sequences that differ to some extent from a native sequence of the isolated and/or purified polypeptide that is amino acid sequences that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly II. Polar, positively charged residues: His, Arg, Lys III. Polar, negatively charged residues: and their amides: Asp, Asn, Glu, Gln IV. Large, aromatic residues: Phe, Tyr, Trp V. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys.

As used herein, the term "anti-angiogenics" refers to any molecule inhibiting angiogenesis and/or lymphangiogenesis.

As used herein, the term "anti-lymphangiogenic" refers to any molecule inhibiting lymphangiogenesis related to lymphatic vessels growth.

According to a further aspect, the present invention provides an isolated and purified peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:29 to SEQ ID NO:35 (Table V).

Preferably, said isolated and purified peptide gives rise, through proteolytic hydrolysis, to a Bradykinin receptor modulator. More preferably, said isolated and purified peptide generated by proteolytic hydrolysis is a Bradykinin receptor agonist.

TABLE-US-00005 TABLE V SEQ ID NO: Amino acid sequence SEQ ID NO: 29 KSTNTF FKPPCVNVFR CGGC SEQ ID NO: 30 VATNTF FKPPCVSVYR CGGC SEQ ID NO: 31 TSANFL IWPPCVEVKR CTGC SEQ ID NO: 32 TNANFL VWPPCVEVQR CSGC SEQ ID NO: 33 GTVAKQ LVPSCVTVQR CGGC SEQ ID NO: 34 DEIEYI FKPSCVPLMR CGGC SEQ ID NO: 35 SEVEHM FSPSCVSLLR CTGC

According to a further aspect, the present invention provides an isolated and purified peptide consisting of a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:42 (Table VI).

According to a preferred embodiment, said isolated and purified peptide is a Bradykinin receptor modulator.

According to a further aspect, it is provided an isolated and purified peptide consisting of SEQ ID NO:36 to SEQ ID NO:42 wherein said peptides are anti-angiogenics.

TABLE-US-00006 TABLE VI SEQ ID NO: Amino acid sequence SEQ ID NO: 36 KSTNTF FKPPCVNVFP PGGC SEQ ID NO: 37 VATNTF FKPPCVSVYP PGGC SEQ ID NO: 38 TSANFL IWPPCVEVKP PTGC SEQ ID NO: 39 TNANFL VWPPCVEVQP PSGC SEQ ID NO: 40 GTVAKQ LVPSCVTVQP PGGC SEQ ID NO: 41 DEIEYI FKPSCVPLMP PGGC SEQ ID NO: 42 SEVEHM FSPSCVSLLP PTGC

According to a further aspect, the present invention provides an isolated and purified peptide consisting of a biological variant having an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:49 wherein said peptide is a VEGF, PDGF or PlGF receptor modulator (Table VII).

Preferably, said isolated and purified peptide is anti-angiogenic.

TABLE-US-00007 TABLE VII SEQ ID NO: Growth factors Variants SEQ ID NO: 43 Variant VEGF-D R141P; C142P SEQ ID NO: 44 Variant VEGF-C R161P; C162P SEQ ID NO: 45 Variant PDGF-A R128P; C129P SEQ ID NO: 46 Variant PDGF-B R129P; C130P SEQ ID NO: 47 Variant VEGF-B R77P; C78P SEQ ID NO: 48 Variant VEGF-A R82P; C83P SEQ ID NO: 49 Variant PIGF R82P; C83P

TABLE-US-00008 TABLE VIII SEQ ID NO: Human Growth factors SEQ ID NO: 50 VEGF-D SEQ ID NO: 51 VEGF-C SEQ ID NO: 52 PDGF-A SEQ ID NO: 53 PDGF-B SEQ ID NO: 54 VEGF-B SEQ ID NO: 55 VEGF-A SEQ ID NO: 56 PIGF

In a further aspect, the present invention provides an isolated and purified peptide selected from the group consisting of: a) a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7; b) a biological fragment thereof having at least 8 consecutive residues in length;

wherein said isolated and purified peptide is a Bradykinin receptor modulator inducing calcium entry in endothelial cells expressing B1 or B2 bradykinin receptors.

Preferably step b is a biological fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:28 wherein said isolated and purified peptide is a Bradykinin receptor modulator inducing calcium entry in endothelial cells expressing B1 or B2 bradykinin receptors.

In a further aspect, the present invention provides an isolated and purified peptide selected from the group consisting of: a) a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:7; b) a biological fragment thereof having at least 8 consecutive residues in length;

wherein said isolated and purified peptide is a Bradykinin receptor modulator inducing angiogenesis.

Other expected actions of B1 and B2 agonists are inflammatory reactions, hypotensive states, pain, hyperalgesia, cardiovascular and/or cerebral circulatory diseases, tissue repair, stem cell differentiation, angiogenesis, lymphangiogenesis, immune system cells modulation, sepsis, wasting states, diabetes, diabetic neuropathy, allergies, brain edema neurogenesis, cardiac function and remodeling, kidney salts balance function, neurogenesis, tumor development (Marceau F. and Regoli D., 2004; Trujillo C. A. et al., 2012).

Preferably step b is a biological fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:28 wherein said isolated and purified peptide is a Bradykinin receptor modulator inducing angiogenesis.

The present invention also relates to peptides having Cys residues that can form dimers, homo or heterodimers, through S-S bridges, and these dimers can be active as homodimers or heterodimers (e.g., Seq ID No 8 dimer with itself or Seq ID No 8 dimer with Seq ID No 23, for example). The present invention considers also dimers obtained by other methods of homo or heterodimerization.

In another aspect, the present invention relates to any cyclic peptides of SEQ ID NO: 1 to SEQ ID NO:56.

The isolated and purified peptide of the present invention may be provided by chemical synthesis, obtained from a biotechnological method and/or extracted from a natural source.

Preferably, the isolated and purified peptide is provided by chemical synthesis. In the context of peptides, the term "chemical synthesis" may refer to SPPS, liquid phase peptide synthesis or a combination of both. Here, the synthesis typically bases on the stepwise coupling of amino acids bearing protected side chains (orthogonal protecting groups). Typically, during synthesis, the peptide strand grows from the C-terminus to the N-terminus. However, there are alternative methods wherein the peptide strand grows from the N-terminus to the C-terminus. Nowadays, the most common methods base on at least two different types of protecting groups that are cleavable under at least two different conditions, such as, e.g., the fluorenyl-9-methoxycarbonyl/tert-butanyl- (Fmoc/tBu) protecting group scheme (Sheppard Tactics) or the tert-butoxycarbonyl/benzyl- (Boc/Bzl) protecting group scheme (Merrifield Tactics). Alternatively or additionally, the peptide may be also provided by conjugating two or more peptide strand(s) with another by any conjugation method known in the art such as, e.g., Native Chemical Ligation (NCL), Click Chemistry, Maleimide-Thiol Conjugation, enzymatic conjugation, biochemical protein ligation and/or soluble handling conjugation.

Alternatively, the isolated and purified peptide may be obtained from a biotechnological method. Today, numerous biotechnological methods are well-known in the art such as, e.g., overexpression and/or heterologous expression, in particular heterologous expression based on cloning of one or more gene(s) in bacteria, insect cells, mammalian cells, plant cells or yeast cells. The isolated and purified peptide may further be extracted by any means known in the art.

Alternatively, the isolated and purified peptide may be extracted from a natural source by any means known in the art. Additionally, the isolated and purified peptide may be purified by any means known in the art, such as, e.g., one or more chromatographic method(s), one or more filtration method(s), one or more electrophoretic method(s), one or more precipitation-based method(s), due or more dialysis method(s) or a combination of two or more thereof. The natural source may be any biological material such as, e.g., bacterial material, plant material, animal material or fungal material, such as e.g. tissue, liquids or secretion(s). The isolated and purified peptide obtained from a natural source may also be digested or partly digested by one or more enzymes, such as protease(s).

It will be understood by a person skilled in the art, that the aforementioned methods for providing an isolated and purified peptide may also be combined with another. In particular, a bradykinin receptor agonist obtained from a biotechnological method or a natural source may further be purified and/or modified by chemical means known in the art.

According to a further aspect, the present invention provides an isolated and purified nucleic acid molecule encoding the isolated and purified peptide.

DNA which can be used herein is any polydeoxynucleotide, including, e.g. double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently-closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically-synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNA and fluorochrome-labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid.

DNA sequences that encode the isolated and purified peptide, or a part thereof, can be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods. The purified and isolated DNA sequence encoding the isolated and purified peptide according to the invention may also be produced by enzymatic techniques. Thus, restriction enzymes, which cleave nucleic acid molecules at predefined recognition sequences can be used to isolate nucleic acid sequences from larger nucleic acid molecules containing the nucleic acid sequence, such as DNA (or RNA) that codes for the isolated and purified peptide or a part thereof.

The present invention also includes variants of the aforementioned sequences, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with same characteristics.

According to a preferred embodiment, the inventions concerns an isolated and purified cDNA molecule encoding the isolated and purified peptides of the invention.

As used herein, the term "cDNA" refers to complementary DNA that is a single stranded DNA complementary to messenger RNA, a DNA synthesized from messenger RNA by reverse transcriptase and/or double stranded complementary DNA synthesized from messenger RNA suitable for subsequent ligation into an expression vector.

Another subject matter of the present invention is a vector comprising the nucleic acid molecule of the invention. The vector being capable of replication in eukaryotes, prokaryotes or in eukaryotes and prokaryotes. It can be a vector capable of integration into the genome of the host cell (e.g. bacteriophage lambda) or a vector which is present extrachromosomally (e.g. a plasmid). Preferably, the vector is a plasmid.

Further, the vector can be an expression vector, i.e. a vector in which the isolated and purified DNA sequence is operably linked to a promoter. This means that the linked isolated and purified DNA sequence encoding the isolated and purified peptide of the present invention is under control of a suitable regulatory sequence which allows expression, i.e. transcription and translation of the inserted isolated and purified DNA sequence. Suitable expression vectors for prokaryotic host cells are described in Sambrook et al., Molecular Cloning; a Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, especially in Chapters 1-4 and 17. Suitable vectors for expression of cloned genes in mammalian cells are described in Sambrook et al., Chapter 16. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, marker genes and other sequences as appropriate.

According to a preferred embodiment of the invention, the expression vector comprises at least one copy of the isolated and purified cDNA molecule encoding the isolated and purified peptide of the invention.

The terms "introducing a purified DNA into a eukaryotic or prokaryotic host cell" or "transfection" denote any process wherein an extracellular DNA, with or without accompanying material, enters a host cell. The term "cell transfected" or "transfected cell" means the cell into which the extracellular DNA has been introduced and thus harbors the extracellular DNA. The DNA might be introduced into the cell so that the nucleic acid is replicable either as a chromosomal integrant or as an extra chromosomal element.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e. g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2u plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences (i.e. sequences that control the expression of a DNA sequence operatively linked to it) may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e. g., Pho5), the promoters of the yeast-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

Another subject matter of the present invention is a recombinant host cell characterized in that said cell expresses a peptide or a host cell capable of expressing the isolated and purified DNA molecule as defined above, preferably a cDNA molecule, particularly a host cell which is stably transformed with an expression vector containing said DNA molecule.

The terms "host cell" and "recombinant host cell" are used interchangeably herein to indicate a eukaryotic or prokaryotic cell into which one or more DNA or vectors of the invention have been introduced. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations clue to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"Eukaryotic cell" refers to any mammalian or non-mammalian cell from a eukaryotic organism. By way of non-limiting example, any eukaryotic cell that is capable of being maintained under cell culture conditions and subsequently transfected would be included in this invention. Especially preferable cell types include, e. g., stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, cancer cells, and primary differentiated or undifferentiated cells. Other suitable host cells are known to those skilled in the art.

A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1. 1, B-W and L-M cells, African Green Monkey kidney cells (e. g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e. g., Sf9), and human cells and plant cells in tissue culture. Preferably, the host cell is a bacterial cell, more preferably an E. coli cell.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large-scale animal culture.

The host cells used to produce the isolated and purified peptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media, described in Ham et al., 1979; Barnes et al., 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430 or WO 87/00195 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Another subject matter of the present invention is a pharmaceutical composition comprising the isolated and purified peptide of the invention.

A further subject matter of the present invention is a pharmaceutical composition comprising as an active substance a pharmaceutically effective amount of the isolated and purified peptide as defined above, optionally in combination with pharmaceutically acceptable carriers, diluents and adjuvants. Preferably, the pharmaceutical composition of the present invention comprises pharmaceutically acceptable carriers, diluents and adjuvants: Such acceptable carriers, diluents and adjuvants should be non-toxic and should not interfere with the efficacy of the active ingredient. This pharmaceutical composition is preferably used as an agent for the preparation of a medicament for the treatment and/or prevention of inflammatory diseases, cardiovascular ischemia, pain, hyperalgesia, renal diseases, wound healing, cardiovascular ischemia, cerebral ischemia, stroke, vascular dementia, infarct dementia, myocardial ischemia, coronary heart disease, myocardial infarction, peripheral limb disease, periphery arterial occlusive disease, sepsis, wasting states, diabetes and/or disorders associated with defective blood flow or blood vessel malformation, tissue repair, stem cell differentiation, angiogenesis, lymphangiogenesis, immune system cells modulation.

The pharmaceutical composition can be in any suitable form, e.g. in the form of a solution, suspension, powder, lyophilisate, ointment or tincture. The composition can be administered by any suitable method, e.g. per injection (systemically or locally) or topically. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, topical, or by injection, e.g. intravenous or intradermal.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.

Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

The respective pharmaceutically effect amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific polypeptide used, especially if the polypeptide additionally contains a cytotoxic component or not. The treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks. The pharmaceutically effective amount of a dosage unit of the polypeptide usually is in the range of 0.001 ng to 100 .mu.g per kg of body weight of the patient to be treated.

Pharmaceutical compositions used in accordance with the present invention are prepared for storage by mixing a pharmaceutically effective amount of the isolated and purified peptide as defined above having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. 1980), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).

A further subject matter of the present invention concerns a pharmaceutical composition for use in medicine.

Another subject matter of the present invention is a pharmaceutical composition comprising the isolated and purified peptide of the invention for use as analgesics.

Preferably the pharmaceutical composition of the invention is used in the treatment and/or prevention of inflammatory diseases, cardiovascular ischemia, pain, hyperalgesia, renal diseases, wound healing, cerebral ischemia, stroke, vascular dementia, infarct dementia, myocardial ischemia, coronary heart disease, myocardial infarction, peripheral limb disease, periphery arterial occlusive disease, sepsis, wasting states, diabetes and/or disorders associated with defective blood flow or blood vessel malformation, tissue repair, stem cell differentiation, angiogenesis, lymphangiogenesis, immune system cells modulation.

In particular, the pharmaceutical composition of the invention is used in the treatment and/or prevention of inflammatory diseases, pain, hyperalgesia, cardiovascular and/or cerebral ischemic diseases, and/or disorders associated with angiogenesis, and lymphangiogenesis in a subject in need thereof.

More preferably, the pharmaceutical composition of the invention is used in the treatment and/or prevention of disorders associated with angiogenesis, and lymphangiogenesis in a subject in need thereof.

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.

EXAMPLES

Example 1

In Vitro Angiogenesis Assay on Matrigel

The angiogenesis assay was performed according to the protocol described in Paschoalin T, et al (2007).

Human umbilical vein endothelial cells (HUVEC) were maintained in complete RPMI 1640 medium, pH 7.2, supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 24 mM sodium bicarbonate, 10% heat-inactivated fetal calf serum (FCS) from Gibco (Minneapolis, Minn., USA) and 40 .mu.g/mL gentamicin sulfate (Hipolabor Farmaceutica, Sabara, MG, Brazil).

In vitro angiogenesis assay on Matrigel was performed as described previously (Paschoalin et al, 2007). Briefly, BD Matrigel Matrix (B&D Biosciences, Bedford, Mass., USA) was thawed on ice, distributed in 96-well plates (50 .mu.L per well) and allowed to polymerize for 1 h at 37.degree. C. HUVECs (15.times.103 cells/well) were suspended in 100 .mu.L of RPMI medium supplemented with 2% of FCS and added to each well in the presence of peptides with possible angiogenic activity. Angiogenic stimulators and inhibitors can be incubated with the cells before adding on the Matrigel if necessary. The plates were incubated at 37.degree. C. for 18 h and then images were captured at 8.times. magnification with a Sony Cyber-shot camera coupled to a light inverted microscope. The number of pro-angiogenic structures (closed rings arising from dendritic extensions of endothelial cells) was counted from 4 different wells, and the average value was determined for each sample.

When stimulated by angiogenic factors, like BK, HUVEC cells undergo the development of pro-angiogenic structures and it allows studying angiogenic or anti-angiogenic molecules. The number of pro-angiogenic structures is counted and compared to a control and to BK. Generally, peptides of the invention derived from VEGF-A were named PepA, peptides from VEGF-C were named PepC, peptides from VEGF-D were named PepD and peptides from Placental Growth Factor were named PepPl.

FIG. 1 shows the result of HUVEC stimulation by PepD 2-10 (SEQ ID NO:8) at 1 .mu.M, 10 .mu.M and 50 .mu.M concentrations. Results show clearly a pro-angiogenic effect at 1 and 10 .mu.M PepD 2-10 concentrations. FIG. 2 shows the results for the same assay, now using PepD 1-10 (SEQ ID NO:1), PepD 2-10 (SEQ ID NO:8), PepA 1-9 (SEQ ID NO:20) and PepA 2-9 (SEQ ID No 27), at 10 .mu.M concentration, compared to BK 1 .mu.M and the control without any peptide added. Clearly PepD 1-10, PepD 2-10, PepA 1-9 and PepA 2-9 are able to induce angiogenesis.

FIG. 3 shows the result of HUVEC stimulation by PepD 1-9 (SEQ ID NO: 15; FKPPCVVF) 10 .mu.M compared to BK 1 .mu.M and to the control. It shows that PepD 1-9 is able to induce angiogenesis.

FIG. 4 shows the result of HUVEC stimulation by PepC 2-9 (FIG. 4B) and PepC 2-10 (FIG. 4A) at 1 .mu.M and 10 .mu.M concentrations and PepC 1-10 (FIG. 4C) at 0.1, 1.0 and 10 .mu.M concentrations, 24 h after initial stimulus. It shows that PepC 2-9, PepC 2-10 and PepC 1-10 are able to induce angiogenesis.

* p<0.05, Student's "t" test, when comparing the mean number of pro-angiogenic structures from three different wells of HUVEC cultures to the control.

Example 2

Intracellular Calcium Measurement in HUVEC Cells Stimulated with PepD 2-10 Peptides

The intracellular calcium measurement was performed according to the method described in Bagnaresi P et al, 2012 and Lungato L, et al., 2012.

Human umbilical vein endothelial cells were cultured in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal bovine serum (FBS), 24 mM sodium bicarbonate, 40 mg/mL gentamicin, 10 mM HEPES, pH 7.4 at 37.degree. C. in a 5% CO2 humidified atmosphere. 5.times.10.sup.4 cells were plated on 60 mm dishes (Costar 3260), and incubated in culture medium containing 5 .mu.M Fluo-3 AM (Invitrogen Corporation, CA) for 40 min at 37.degree. C. in a buffer containing the channel blocker Probenecid (40 .mu.M), followed by 3 washes with culture medium to discard remaining extracellular probe. The cells were then placed into a culture chamber at 37.degree. C. on the stage of a fluorescence microscope (Zeiss LSM 510 META) with excitation at 488 nm and emission at 505-530 nm. After calcium probe incubation, assays were performed with 1 uM bradykinin (BK), 10 uM PepD 2-10. To demonstrate which B receptor is activated by PepD 2-10 (KPPCVNVFR; SEQ ID NO:8), B1 receptor antagonist Des-Arg9-Leu8-BK (10 .mu.M) and B2 receptor antagonist HOE-140 (10 .mu.M) where added to HUVEC cell culture for 10 min before the addition of: PepD 2-10 (10 uM) or 5 .mu.M Thapsigargin (THG).

Fluorescence data were normalized as F/Fmax, where Fmax represents maximal intracellular fluorescence obtained with Ca2+ released from ER with addition of THG, a Ca2+ATPase inhibitor (Bagnaresi et al. 2012). THG blocks calcium pumps that recover cytosolic calcium from the cytoplasm. Under the action of THG the cytosolic calcium reaches its maximal concentration.

The software-based analysis allowed fluorescence imaging in the whole field or in a selected cell as a function of time (Image J 1.47h, NIH, USA). This was accomplished by defining areas of interest (e.g., an entire cell) on a given image frame and directing the software to construct a graphical representation of intensity over time (GraphPad Software 6 Prisma). Individual traces for typical 15 cells responses represent the intracellular calcium fluorescence changes in the analyzed cell population (Lungato et al. 2012).

FIG. 5A-E demonstrates that calcium entry in HUVEC cells induced by PepD2-10 and detected by Fluo-3 fluorescence, is due to B1 bradykinin receptor activation.

FIG. 5A is a control to show the effect of adding BK to HUVEC in culture (at .about.1:50 min) induces calcium increase in the cytoplasm as shown by the fluorescence increase around 2:20 min. Thapsigargin added at the end allows the maximum increase of intracellular calcium to be used in ratio calculations.

FIG. 5B shows that 10 .mu.M PepD 2-10 also induces an increase of cytoplasmatic calcium in HUVEC cells. Previous addition of HOE140 a Bradykinin B2 receptor antagonist does not block calcium entry but clearly shows a disruption in the pattern of calcium increase, indicating the involvement of B2 receptors in the observed effect, as shown in FIG. 5C. Previous addition of DesArg9-Leu8-BK, a Bradykinin B1 receptor antagonist, completely blocks the effect of PepD 2-10, by blocking calcium entry induced by PepD 2-10 demonstrating the involvement of B1 receptor on the observed effect as shown in FIG. 5D.

Data on calcium entry indicate a B2 receptor activity (FIG. 5B) that after B2 blockage by the B2 receptor antagonist HOE140, it shows a B1 receptor activity (FIGS. 5C and 5D), that can be blocked by the B1 receptor antagonist DesArg9-Leu8-BK. It is likely that PepD 2-10 is converted to PepD 2-9. From a B2 agonist it may become a B1 agonist: PepD 2-10 is a B2 agonist (FIG. 5B), but calcium entry results shown on FIG. 5C is due to a B1 agonist activity.

TABLE-US-00009 B2 agonist B1 agonist BK RPPGFSPFR DesArg9-BK RPPGFSPF PepD 2-10 KPPCVNVFR PepD 2-9 KPPCVNVF

FIG. 5E shows the effect of PepPL 1-10 (SEQ ID NO:7) on cultured HUVEC cells, in the presence of the B1 receptor antagonist DesArg9-Leu8-BK, at 100 s PepPL 1-10, 10 .mu.M is added. When applied to HUVEC cells previously treated with the fluorescent calcium indicator Fluo-3 in culture, PepPl 1-10 at 10 .mu.M concentration induces an increase in cell fluorescence due to calcium entry in the cytoplasm. A transient fluorescence increase is observed that lasts around 5 minutes and corresponds to cytoplasmic calcium increase. Thapsigargin is then added to release all the calcium content to the cytoplasm and give a measure of the maximum fluorescence that could be expected under these conditions.

FIG. 5A: A.F.U.--Arbitrary Fluorescence Units; BK 1 .mu.M Bradykinin; THG Thapsigargin 10 .mu.M.

FIG. 5B: PepD 2-10 at 10 .mu.M; THG Thapsigargin 10 .mu.M.

FIG. 5C: HOE 140 (B2 receptor antagonist) 10 .mu.M; PepD 2-10 10 .mu.M; THG Thapsigargin 10 .mu.M.

FIG. 5D: DesArg9-Leu8-BK 10 .mu.M; PepD 2-10 10 .mu.M; THG Thapsigargin 10 .mu.M

FIG. 5E: AxN is the mean signal of 10 selected cells. PepPl 1-10 is the sequence derived from Placental Growth Factor (SEQ ID NO: 7; FSPSCVSLLR).

REFERENCE LIST

Bagnaresi, P., Barros, N. M. T., Assis, D. M., Melo, P. M. S., Fonseca, R. G., Juliano, M. A., Pesquero, J. B., Juliano, L., Rosenthal, P. J., Cannona, A. K., and Gazarini, M. L. (2012) Intracellular proteolysis of kininogen by malaria parasites promotes release of active kinins. Malaria Journal 11: 156. Barnes, D., and Sato, G. (1980) Methods for growth of cultured cells in serum-free medium, Analytical Biochemistry 102, 255-270. Cleber A. Trujillo et al., Journal of Biological Chemistry, vol. 287, No. 53, pp. 44046-44061, 2012. Ferrara, N. (1999) Molecular and biological properties of vascular endothelial growth factor. Journal of Molecular Medicine-Jmm 77, 527-543 Ferrara, N., and DavisSmyth, T. (1997) The biology of vascular endothelial growth factor. Endocrine Reviews 18, 4-25. Ham et al., Media and growth requirements, Meth. Enz. 58:44 (1979). Lungato L, et al., Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response. Biochim Biophys Acta 1820:1997-2006 (2012). Marceau, F., and Regoli, D. (2004) Bradykinin receptor ligands: Therapeutic perspectives. Nature Reviews Drug Discovery 3, 845-852 Paschoalin, T., Carmona, A. K.; Rodrigues, E. G., Oliveira, V., Monteiro, H. P., Juliano, M. A., Juliano, L., and Travassos, L. R. (2007) Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth. Molecular Cancer 6: 25-34 Sela, M., and Zisman, E. (1997) Different roles of D-amino acids in immune phenomena. Faseb Journal 11, 449-456 Trujillo, C. A., Negraes, P. D., Schwindt, T. T., Lameu, C., Carromeu, C., Muotri, A. R., Pesquero, J. B., Cerqueira, D. M., Pillat, M. M., de Souza, H. D. N., Turaca, L. T., Abreu, J. G., and Ulrich, H. (2012) Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells. Journal of Biological Chemistry 287, 44046-44061.

SEQUENCE LISTINGS

1

56110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1Phe Lys Pro Pro Cys Val Asn Val Phe Arg 1 5 10 210PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 2Phe Lys Pro Pro Cys Val Ser Val Tyr Arg 1 5 10 310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Ile Trp Pro Pro Cys Val Glu Val Lys Arg 1 5 10 410PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Val Trp Pro Pro Cys Val Glu Val Gln Arg 1 5 10 510PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Leu Val Pro Ser Cys Val Thr Val Gln Arg 1 5 10 610PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Phe Lys Pro Ser Cys Val Pro Leu Met Arg 1 5 10 710PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Phe Ser Pro Ser Cys Val Ser Leu Leu Arg 1 5 10 89PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Lys Pro Pro Cys Val Asn Val Phe Arg 1 5 99PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 9Lys Pro Pro Cys Val Ser Val Tyr Arg 1 5 109PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 10Trp Pro Pro Cys Val Glu Val Lys Arg 1 5 119PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 11Trp Pro Pro Cys Val Glu Val Gln Arg 1 5 129PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Val Pro Ser Cys Val Thr Val Gln Arg 1 5 139PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Lys Pro Ser Cys Val Pro Leu Met Arg 1 5 149PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Ser Pro Ser Cys Val Ser Leu Leu Arg 1 5 159PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Phe Lys Pro Pro Cys Val Asn Val Phe 1 5 169PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Phe Lys Pro Pro Cys Val Ser Val Tyr 1 5 179PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 17Ile Trp Pro Pro Cys Val Glu Val Lys 1 5 189PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Val Trp Pro Pro Cys Val Glu Val Gln 1 5 199PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Leu Val Pro Ser Cys Val Thr Val Gln 1 5 209PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 20Phe Lys Pro Ser Cys Val Pro Leu Met 1 5 219PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 21Phe Ser Pro Ser Cys Val Ser Leu Leu 1 5 228PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 22Lys Pro Pro Cys Val Asn Val Phe 1 5 238PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 23Lys Pro Pro Cys Val Ser Val Tyr 1 5 248PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 24Trp Pro Pro Cys Val Glu Val Lys 1 5 258PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 25Trp Pro Pro Cys Val Glu Val Gln 1 5 268PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 26Val Pro Ser Cys Val Thr Val Gln 1 5 278PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 27Lys Pro Ser Cys Val Pro Leu Met 1 5 288PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 28Ser Pro Ser Cys Val Ser Leu Leu 1 5 2920PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 29Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg 1 5 10 15 Cys Gly Gly Cys 20 3020PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 30Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg 1 5 10 15 Cys Gly Gly Cys 20 3120PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 31Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg 1 5 10 15 Cys Thr Gly Cys 20 3220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 32Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg 1 5 10 15 Cys Ser Gly Cys 20 3320PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 33Gly Thr Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg 1 5 10 15 Cys Gly Gly Cys 20 3420PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 34Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg 1 5 10 15 Cys Gly Gly Cys 20 3520PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 35Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu Leu Arg 1 5 10 15 Cys Thr Gly Cys 20 3620PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 36Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Pro 1 5 10 15 Pro Gly Gly Cys 20 3720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 37Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Pro 1 5 10 15 Pro Gly Gly Cys 20 3820PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Pro 1 5 10 15 Pro Thr Gly Cys 20 3920PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 39Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Pro 1 5 10 15 Pro Ser Gly Cys 20 4020PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 40Gly Thr Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Pro 1 5 10 15 Pro Gly Gly Cys 20 4120PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 41Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Pro 1 5 10 15 Pro Gly Gly Cys 20 4220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 42Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu Leu Pro 1 5 10 15 Pro Thr Gly Cys 20 43354PRTHomo sapiens 43Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val 1 5 10 15 Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser 20 25 30 Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser 35 40 45 Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu 50 55 60 Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg 65 70 75 80 Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95 Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110 Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr 115 120 125 Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Pro Pro Gly Gly 130 135 140 Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155 160 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro 165 170 175 Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu 180 185 190 Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln 195 200 205 Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile 210 215 220 Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu 225 230 235 240 Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 245 250 255 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val 260 265 270 Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys 275 280 285 Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His 290 295 300 Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 305 310 315 320 His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys 325 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys 340 345 350 Asn Pro 44419PRTHomo sapiens 44Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 40 45 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 65 70 75 80 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90 95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 145 150 155 160 Pro Pro Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220 Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 245 250 255 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys 290 295 300 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys 305 310 315 320 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340 345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro 405 410 415 Gln Met Ser 45211PRTHomo sapiens 45Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Pro 115 120 125 Pro Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu 195 200 205 Lys Pro Thr 210 46241PRTHomo sapiens 46Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Pro Pro Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala 47207PRTHomo sapiens 47Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val Val

Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Pro Pro Gly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Asp Ser Ala Val Lys Pro Asp Arg Ala Ala Thr Pro His His Arg 130 135 140 Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 145 150 155 160 Ser Pro Ala Asp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 165 170 175 His Ala Ala Pro Ser Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala 180 185 190 Ala Ala Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 195 200 205 48232PRTHomo sapiens 48Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Pro Pro Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 49221PRTHomo sapiens 49Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Pro Pro Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg His Ser Pro Gly Arg Gln Ser Pro Asp Met Pro Gly Asp 130 135 140 Phe Arg Ala Asp Ala Pro Ser Phe Leu Pro Pro Arg Arg Ser Leu Pro 145 150 155 160 Met Leu Phe Arg Met Glu Trp Gly Cys Ala Leu Thr Gly Ser Gln Ser 165 170 175 Ala Val Trp Pro Ser Ser Pro Val Pro Glu Glu Ile Pro Arg Met His 180 185 190 Pro Gly Arg Asn Gly Lys Lys Gln Gln Arg Lys Pro Leu Arg Glu Lys 195 200 205 Met Lys Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg 210 215 220 50354PRTHomo sapiens 50Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val 1 5 10 15 Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser 20 25 30 Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser 35 40 45 Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu 50 55 60 Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg 65 70 75 80 Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95 Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110 Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr 115 120 125 Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly 130 135 140 Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155 160 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro 165 170 175 Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu 180 185 190 Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln 195 200 205 Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile 210 215 220 Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu 225 230 235 240 Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 245 250 255 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val 260 265 270 Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys 275 280 285 Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His 290 295 300 Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 305 310 315 320 His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys 325 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys 340 345 350 Asn Pro 51419PRTHomo sapiens 51Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 40 45 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 65 70 75 80 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90 95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 145 150 155 160 Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220 Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 245 250 255 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys 290 295 300 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys 305 310 315 320 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340 345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro 405 410 415 Gln Met Ser 52211PRTHomo sapiens 52Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu 195 200 205 Lys Pro Thr 210 53241PRTHomo sapiens 53Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala 54207PRTHomo sapiens 54Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Asp Ser Ala Val Lys Pro Asp Arg Ala Ala Thr Pro His His Arg 130 135 140 Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 145 150 155 160 Ser Pro Ala Asp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 165 170 175 His Ala Ala Pro Ser Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala 180 185 190 Ala Ala Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 195 200 205 55232PRTHomo sapiens 55Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 56221PRTHomo sapiens 56Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5

10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg His Ser Pro Gly Arg Gln Ser Pro Asp Met Pro Gly Asp 130 135 140 Phe Arg Ala Asp Ala Pro Ser Phe Leu Pro Pro Arg Arg Ser Leu Pro 145 150 155 160 Met Leu Phe Arg Met Glu Trp Gly Cys Ala Leu Thr Gly Ser Gln Ser 165 170 175 Ala Val Trp Pro Ser Ser Pro Val Pro Glu Glu Ile Pro Arg Met His 180 185 190 Pro Gly Arg Asn Gly Lys Lys Gln Gln Arg Lys Pro Leu Arg Glu Lys 195 200 205 Met Lys Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg 210 215 220