WO2013035107A1 - Therapeutic peptides - Google Patents

Abstract

The present invention provides peptides which specifically react with B-subunit of Plasmodium falciparum vacuolar-H⁺ATPase (V-H⁺ ATPase). The peptides were identified by panning phage display peptide libraries with recombinant B subunit of P.falciparum V-H⁺ ATPase. The selected phages specifically react with P.falciparum infected cells and corresponding free peptides inhibit parasite growth in vitro. The identified peptides can be used for therapy of malaria and other diseases caused by pathogens expressing B subunit of V-H⁺ ATPase.

Description

THERAPEUTIC PEPTIDES

The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF THE INVENTION

The present invention relates to cysteine constrained cyclic peptide against recombinant subunit B of P falciparum V-H⁺ ATPase. These peptides inhibit P falciparum growth in vitro and can be exploited for their therapeutic values in malaria and also in other diseases such as osteoporosis and cancer where V-H⁺ ATPase over expression is a characteristics of diseased cells. In conclusion, the invention relates to a peptide mediated therapy of diseases caused by pathogens having similar B subunit of V-H⁺ ATPase.

BACKGROUND OF THE INVENTION

Malaria remains one of the most devastating diseases of mankind, inflicting serious health and economic burdens on many countries throughout the world. Out of 109 countries which were declared endemic for malaria, 45 are only in Africa {WHO report, 2009). Identification and functional characterization of infected cell associated molecules in malaria is central not only to understand the basic mechanism involved in parasite survival and growth but also in selection of targets for controlling the disease. Malaria-infected red blood cells (IRBCs) undergo drastic membrane changes (Smith et al., 2001) resulting in modifications in erythrocyte cytoskeleton as well as the extra cellular face of the membrane (Smith and Craig, 2005). A plethora of target antigens have been identified from Plasmodium genome using advanced proteomics technologies in combination with bioinformatics tools (Florens et al., 2004; Hiller et al., 2004). An array of parasite-derived molecules, including PfEMP-1, stevor, rifins, are present on infected cell membrane (Craig and Scherf, 2001 ; Maier et al., 2008; Deitsch and Hviid, 2004; yes et al., 2001 ; Petter et al., 2007). Unfortunately, number of these molecules suffer from allelic heterogeneity between strains, antigenic variation within a single strain and high sequence polymorphism at critical target epitopes (Blythe et al., 2004; Craig and Scherf, 2001 ; Florens et al, 2004). Besides, out of the 5300 proteins expressed during the life cycle of Plasmodium, the functionality of majority of these molecules is unknown. The role of V-rf^1" ATPase has been implicated in the maintenance of the intracellular pH (pHi) of infected erythrocyte (Hayashi et al., 2000), and also in energizing the secondary transport of diverse solutes (Moriyama et al., 2003). Functional malaria parasite-encoded vacuolar V-H⁺ ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte (Marchesini et al., 2005). So, it is a challenge to look for the inhibitors based on the functional properties of the target molecule, which ultimately can arrest the growth of intracellular parasite.

Phage display technology has been used in cancer not only for the identification of targets but also in the development of peptide based drugs (Ladner et al., 2004) and vaccines (Wang et al., 2004). Earlier reports have demonstrated that high-affinity ligands for protein targets could be selected from phage display peptide libraries (Cwirla et al., 1990). In tuberculosis, screening of peptides from phage display technique was done to inhibit the function of Hspl 6.3 (Gupta et al 2009). Thus, targeting with a peptide inhibitor may provide a useful reagent to assist small- molecule drug discovery (Arkin and Wells, 2004; DeLano et al., 2000). In malaria, phage display system has been used to investigate host-pathogen interactions (Ghosh et al., 2001). 15-mer peptides specific to P. falciparum AMA 1 were selected by panning phage display library. These peptides were found effective in blocking erythrocyte invasion thereby leading to inhibition of P .falciparum growth in vitro (Li et al., 2002). In another study, the peptides that bound specifically to recombinant serine repeat antigen (SERA5) were studied for their effect on parasite growth. SBP1, a 14 residue cyclized peptide resulted in the retention of late-stage parasites at levels similar to those observed using protease inhibitors, E64 and antipain-leupeptin, thus impeding the parasite development (Fairlie et al 2008).

The criteria for selecting a target protein in this study were: a) presence on infected red blood cell membrane, b) conserved nature in different species of Plasmodium and, c) its function. Presence of V-H+ ATPase protein in different species of Plasmodium with closer E-values and high identity revealed its significance in the parasite. Besides, it is the first functional enzyme reported on infected cell membrane (Marchesini et al, 2005).

V-H⁺ ATPase has been targeted by different class of inhibitors. A benzolactone based class of inhibitors e.g. Bafilomycin and concanamycin, are effective in blocking the proton extrusion from V-H⁺ ATPase pump. The structural mimics of these compound have been screened which effectively inhibit parasite growth specifically blocking subunit C of enzyme (Saliba et al., 2010). Further attempts to screen inhibitors against other subunits will provide a momentum for drug discovery. There has been a growing evidence for the existence of B subunit diversity among V-ATPases and it has been reported that these are expressed differently in organelles and tissues (Forgac et al, 2002; Sun-Wada et al., 2003; Ueda et al., 2003), thus making such molecules as a possible target for therapeutic intervention of the disease.

In this study, 7-mer cysteine constrained phage display library was screened against recombinant B subunit of P. falciparum V-H⁺ ATPase. Some of these peptides react ' with the P .falciparum infected cell and inhibit the parasite growth in vitro. These ^; identified peptides can be used for therapeutic intervention of malaria and other pathogenic diseases which share the same target molecules.

OBJECTS OF THE INVENTION

Main object of the present invention is to provide 7-mer cyclic peptides useful in peptide mediated therapy of diseases caused by pathogens having similar B subunit of V-H⁺ ATPase. Another object of the present invention is to Identify conserved parasite protein expressed on infected erythrocytes as a drug target.

Yet another object of the present invention is cloning, expression and purification of recombinant subunit B of V-H⁺ ATPase of P. falciparum.

Still another object of the present invention is recognition of P.falciparum infected cells by peptide phages reactive to recombinant subunit B.

Yet another object of the present invention is inhibition of P. falciparum growth in vitro by peptides corresponding to phages. SUMMARY OF THE INVENTION

Accordingly, the present invention provides 7-mer cyclic peptides useful in peptide mediated therapy of diseases caused by pathogens having similar B subunit V-H⁺ ATPase

In an embodiment of the present invention, the peptide is a 7-mer cysteine constrained cyclic peptide.

In another embodiment of the present invention, the peptide targets the highly conserved B subunit of V-^H+ ATPase

In still another embodiment of the present invention, the peptide inhibit P. falciparum growth in vitro.

In yet another embodiment of the present invention, the peptide is useful for setting of screens to identify other potent therapeutic agents.

In still another embodiment of the present invention, the peptide is selected for setting of screens to identify chemical analogues.

In yet another embodiment of the present invention, a pharmaceutical composition comprising a peptide for use as a therapeutic including pharmaceutically acceptable carrier.

In still another embodiment of the present invention, the peptide is used for the treatment of Malaria caused by pathogen having similar B subunit of V-H⁺ ATPase. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : SDS-PAGE of recombinant subunit B of P. falciparum V-H⁺ ATPase, purified by Ni-NTA affinity chromatography, followed by commossie staining. Molecular weight of purified protein is indicated with arrow.

Figure 2: Localization of recombinant B subunit specific phage peptide binding sites in P.falciparum infected erythrocytes (IRBC) as revealed by immunofluorescence assay. IRBC, preincubated with selected phages (ATPase 10; row 1, ATPase 13; row 2 ), were coated on to the slide, treated with mouse anti- M13 antibody followed by goat anti-mouse IgG Alexa594 conjugate at 1 :600 dilution (Panel B). The parasite was counterstained with DAPI (Panel A).

Figure 3: In vitro parasite growth inhibition by recombinant B subunit specific peptides. EGTA was taken as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to cysteine constrained cyclic peptide against recombinant subunit B of P falciparum V-H⁺ ATPase. These peptides inhibit P falciparum growth in vitro and can be exploited for their therapeutic values in malaria and also in other diseases such as osteoporosis and cancer where V-H⁺ ATPase over expression is a characteristics of diseased cells. In conclusion, the invention relates to a peptide mediated therapy of diseases caused by pathogens having similar B subunit of V-H⁺ ATPase.

EXAMPLES

The following examples are given by way of illustrations and therefore, should not * be construed to limit the scope of the present invention.

EXAMPLE 1

Identifying conserved parasite protein expressed on infected erythrocytes as drug target

The criteria for selecting a target protein were: 1) presence on infected erythrocyte membrane, 2) conserved nature in different species of Plasmodium and, 3) its function. Due to high complexity, only few infected cell surface molecules have been characterized. Most of these molecules such as PfEMP-1, rifins, stevors etc. are present on infected cell membrane with well known function but suffer from antigenic diversity/polymorphism or clonal antigenic variations (Hisaeda et al., 2004; Hisaeda et al., 2005) which cause great hinderance to target them. Accordingly, other proteins e.g. Exp-2 (Export 2 protein), PIESP-1 , PIESP-2, V-H⁺ ATPase present on infected cell membrane were selected for their sequence analysis by Blast homology available at www.ncbi.nlm.nih.gov. No conservation of Exp-2 and PIESP-2 was found in different species of plasmodium as indicated by differences in E-value, while PIESP-1 was relatively conserved but its function is still not known. On the other hand, presence of V-H+ ATPase protein in different species of Plasmodium {P. falciparum. P.berghei, P.yoelii, P.chabaudii and P.vivax) with closer E-values and >97% identity revealed its functional significance in the parasite. Besides, it is the first functional enzyme reported on infected cell membrane (Marchesini et al., 2005). The role of V-H⁺ ATPase has been implicated in the maintenance of the intracellular pH of infected erythrocyte. This proton pump is also responsible for increased uptake of metabolites into infected erythrocytes. Localization studies using antiserum to B subunit of V-H⁺ ATPase has revealed its presence on surface of infected cell membrane (Marchesini et al, 2005). No such V- H⁺ ATPase is present on NRBC membrane. Recently, there has been a growing evidence for the existence of B subunit diversity among V-H⁺ ATPase and it has been reported that these are expressed differently in organelles and tissues (Nishi and Forgac, 2002; Sun-Wada et al, 2003; Toyomura et al., 2003; Ueda et al., 2003). Moreover, functional properties vary among various V-H⁺ ATPase. Different cells and tissue specific isoforms of B subunit of V-H⁺ ATPase make them a suitable drug target. Sequence comparison of V-H⁺ ATPase from different species of Plasmodium by using clustal X showed that this protein is conserved in human malaria parasite e.g. P. vivax, P.falciparum as well as in rodent parasite e.g. P. berghei, P. yoelii. Thus, B subunit of V-H⁺ ATPase (Seq Id no. 3) was selected as a target for peptide mediated therapy because of its accessibility on infected erythrocyte surface, conserved nature in different species and the functional importance of enzyme.

EXAMPLE 2

Cloning, expression and purification of recombinant P.falciparum V-H⁺ ATPase

Characterized as a single copy gene on chromosome 4 of P. falciparum genome which is expressed as a 56 kDa protein (Cowman et al,. 1994). Gene fragment of 1.48 Kb encoding for B subunit of V-H⁺ ATPase was amplified from P. falciparum genomic DNA by overlap extension PCR strategy and removing an intron of 474bp from the whole fragment. Primers were designed for first fragment (1 -109 base pairs) and second fragment (585-1960 base pairs) with the overlap in reverse primer ElRl of first fragment and forward primer E2F2 of 2" fragment (Table 1). First and second PCR amplification was carried out using -200 ng template DNA in a final reaction volume of 50μ1 containing 200μΜ of each dNTP, 200 nM of each primer ElFl (forward),ElRl (reverse) for 1^st fragment and E2Fl(forward),E2R2(reverse) for 2^nd fragment, IX PCR buffer and 2 units of enzyme (Barnes, 1994). PCR amplifications were carried out for total of 35 cycles (denaturation: 95°C for 30 sec; annealing: 54°C for 30 sec in 1^st PCR and 47°C for 30 sec in 2^nd PCR; elongation: 72°C for 1.5 min depending on insert size to be amplified) followed by final extension cycle at 72°C for 10 min in an eppendorf thermal cycler. Products of both PCR reactions were mixed and 50 ng of DNA was used as the template with ElFlas a forward primer and E2R2 as a reverse primer in third PCR amplification at the annealing temperature of 54° C.

Table 1 Primers for amplification of subunit B of V-H ATPase

Ligation of insert into pET 28 a plasmid was done by overnight incubation in ice and then transformed into DH5a competent cells. Further, the plasmid was isolated and transformed into expression host BL21 codon plus. The protein was over expressed in LB medium supplemented with kanamycin (30μg/ml) and allowed to grow at 37°C until A₆oo reached~0.4-0.5. For bringing the protein in soluble form, heat shock was given at 42°C for 45 minutes followed by cooling of culture at room temperature for 15-20 minutes. The culture was then induced with 0.25 mM IPTG and further incubated at 25°C for 4 hrs. The protein was purified from soluble fraction by Ni-NTA affinity chromatography. Dialysis of eluted fractions was done for 48 hrs, followed by concentration and purity analysis by SDS-PAGE (Fig-1). EXAMPLE 3

Phage Display Peptide Library

The phage library used in the present invention, Ph.D. ™-C7C Phage Display Peptide Library Kit, was purchased from New England BioLabs (Beverly, Mass.). This kits is based on a combinatorial library of random peptide 7 fused to a minor coat protein (pill) of Ml 3 phage. The displayed peptide is expressed at the N- terminus of pill, such that after the signal peptide is cleaved, the first residue of the coat protein is the first residue of the displayed peptide. The Ph.D.™-C7C library consist of approximately 2.8.times. l0.sup.9 and 2.7.times. l(hsup.9 sequences, respectively. A volume of 10 .mu.L contains about 55 copies of each peptide sequence. Each initial round of experiments was carried out using the original library provided by the manufacturer in order to avoid introducing any bias into the results.

EXAMPLE 4

Panning of phage display peptide library onto recombinant V-H⁺ ATPase

7-mer cysteine constrained phage display peptide library was used to first select the short cyclic peptides that bind to the recombinant B subunit (represented by SEQ ID NO: 3) of V-H⁺ ATPase of P. falciparum and are able to recognize the infected cells. Briefly, the recombinant protein was coated onto the 96 well ELISA plate at pH 7.4, and incubated with lx lO^{1 1} pfu of phage peptides for 2 hours at room temperature. The bound phage peptides were eluted under acidic conditions. The plaques corresponding to protein bound phage peptides were randomly picked from 3rd pan titer plate, amplified in E.Coli ER2738 followed by their DNA isolation and then subjected to automated sequencing. The selected phage peptides (Table 2) were checked for their binding with recombinant protein and infected RBC membrane by ELISA and immunofluorescence assay respectively. Table 2: Panning of random 7 mer cysteine constrained phage display peptide library onto rec.B subunit of P.falciparum V-H^1" ATPase

EXAMPLE 5

Binding of phage peptides with P .falciparum infected erythrocyte

For checking the binding of selected phage peptides on intact P.falciparum infected erythrocytes, fluorescence microscopy was performed, taking phage peptides ATPasel, ATPase3, ATPase5, ATPase6, ATPase 10 and ATPase 13 based on- differences in their reactivity with recombinant B subunit and P.falciparum extract. Distinct fluorescence was observed in P. falciparum infected RBC treated with phage peptides ATPasel , ATPase3, ATPase 10 and ATPase 13 while no fluorescence could be seen in other phage peptides ATPase5 and ATPase6 and also with control phage peptide (data not shown). Fig.2 presents a representative picture of immunofluorescence pattern as observed with ATPase 10 (Seq Id No. 1 ) and ATPase 13 (Seq Id No. 2) phages. Thus among phages reactive with rec.B subunit of P.falciparum V-H⁺ ATPase, few also reacted with the infected cell.

EXAMPLE 6

Effect of phage peptides on the survival of parasite

Since peptides screened for recombinant B subunit; ATPasel , ATPase3, ATPase 10 and ATPase 13 were found to localize on infected erythrocytes and therefore these peptides were assessed for their effect on parasite growth in culture. For this, hypoxanthine incorporation assa was done. The P.falciparum (MRA102) culture was doubly synchronized by 5% sorbitol and suspended at 5% hematocrit and two different parasitemia of 2% and 5%. Then, the culture was incubated with different custom synthesized peptides at concentrations varying from 100 μg/ml to 1 mg/ml. After 4 hrs of incubation with the peptide 10 μθϊ/πιΐ of [³H]-hypoxanthine (GE Healthcare) was added followed by incubation for 40 hrs at 37°C. The cells were then freeze/thawed to lyse the erythrocytes, and content of each well was collected on filtermat and washed using a cell harvester. Filters were dried and counts were taken using scintillation counter after addition of scintillation fluid to dried filtermat. ATPase l3 resulted in 40% and 77% inhibition of parasite growth at 100 μg/ml and 1000 μg/ml peptide concentrations. While ATPaselO peptide showed 60% inhibition at 1000 μg/ml concentration (Fig.3). In contrast, peptides corresponding to ATPasel, ATPase3 and ATPase 5 did not have any effect.

ADVANTAGES OF THE INVENTION

1. The target protein, B subunit of V-H⁺ ATPase, is highly conserved functional molecule in different species of Plasmodium. Thus, malaria caused by different species can be treated by the set of drugs developed against this target.

2. Different species of malaria parasite have highly conserved B subunit as a target, so the chances of drug resistance due to mutations are unlikely.

3. The claimed peptides can be used as a potent inhibitor of B subunit restricting the malaria parasite growth in a dose dependent manner.

4. These claimed peptides are conformational, showing better specificity to the target protein in its native form and inhibiting the parasite growth in vitro.

5. These claimed peptides can further be modified using peptidomimetics for better efficacy as a drug candidate to treat malaria.

6. As the target molecule is localized on infected erythrocyte surface, parasitophorous vacuolar membrane and parasite surface. So the peptide inhibitors can be targeted at multiple sites during malaria infection. 7. The claimed peptides can be used in other pathogen associated diseases where the selected peptides can be used against similar target as in malaria.

REFERENCES

Arkin, M.R., and Wells, J.A. (2004) Small□ molecule inhibitors of protein□ protein interactions: progressing towards the dream. Nat Rev Drug Discov 3 : 301□ 317.

Blythe, J.E., Surentheran, T., and Preiser, P.R. (2004) STEVORD Da multifunctional protein? Mol Biochem Parasitol 134: 1 1□ 15.

Craig, A., and Scherf, A. (2001) Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion. Mol Biochem Parasitol 115: 129D 143.

Cwirla, S.E., Peters, E.A., Barrett, R.W., and Dower, W.J. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A 87: 6378Π6382.

Cowman A.F. et al. (1994) Cloning and characterization of the vacuolar ATPase B subunit from Plasmodium falciparum. Molecular and Biochemical Parasitology Volume 65, Issue 1 , May 1994, Pages 123-133.

Deitsch, K.W., and Hviid, L. (2004) Variant surface antigens, virulence genes and the pathogenesis of malaria. Trends Parasitol 20: 562□ 566.

DeLano, W.L., Ultsch, M.H., de Vos, A.M., and Wells, J.A. (2000) Convergent solutions to binding at a protein□ protein interface. Science 287: 1279□ 1283.

Fairlie, W.D., Spurck, T.P., McCoubric, J.E., Gilson, P.R., Miller, S. ., McFadden, G.I., Malby, R., Crabb, B.S., and Hodder, A.N. (2008) Inhibition of malaria parasite development by a cyclic peptide that targets the vital parasite protein SERA5. Infect Immun 76: 4332Π4344.

Ghosh, A.K., Ribolla, P.E., and Jacobs DLorena, M. (2001) Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library. Proc Natl Acad Sci U S A 98: 13278□ 13281. Hayashi M, Yamada H, Mitamura T, Horii T, Yamamoto A, Moriyama Y. (2000) Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes. J Biol Chem. Nov 3; 275(44):34353-8.

Hiller, N.L., Bhattacharjee, S., van Ooij, C, Liolios, K., Harrison, T., Lopez DEstrano, C.,~ and Haldar, . (2004) A hosttargeting signal in virulence proteins reveals a secretome in malarial infection. Science 306: 1934□ 1937.

Hisaeda, H., Maekawa, Y., Iwakawa, D., Okada, H., Himeno, ., Kishihara, K., Tsukumo, S., and Yasutomo, . (2004) Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nat Med 10: 29□ 30.

Hisaeda, H., Yasutomo, K., and Himeno, K. (2005) Malaria: immune evasion by parasites. Int J Biochem Cell Biol 37: 700□ 706.

Kyes, S., Horrocks, P., and Newbold, C. (2001) Antigenic variation at the infected red cell surface in malaria. Annu Rev Microbiol 55: 673 Π707.

Ladner, R.C., Sato, A.K., Gorzelany, J., and de Souza, M. (2004) Phage display□ derived peptides as therapeutic alternatives to antibodies. Drug Discov Today 9: 525□ 529.

Li, F., Dluzewski, A., Coley, A.M., Thomas, A., Tilley, L., Anders, R.F., and Foley, M. (2002) Phage□ displayed peptides bind to the malarial protein apical membrane antigen□ 1 and inhibit the merozoite invasion of host erythrocytes. J Biol Chem 277: 50303 D50310.

Maier, A.G., Rug, M., O'Neill, M.T., Brown, M., Chakravorty, S., Szestak, T., Chesson, J., Wu, Y., Hughes, ., Coppel, R.L.,Newbold, C, Beeson, J.G., Craig, A., Crabb, B.S., and Cowman, A.F. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum□ infected human erythrocytes. Cell 134: 48D61.

Moriyama, Y., Hayashi, M., Yatsushiro, S., and Yamamoto, A. (2003) Vacuolar proton pumps in malaria parasite cells. J Bioenerg Biomembr 35: 367D375. Marchesini, N., Vieira, M., Luo, S., Moreno, S.N., and Docampo, R. (2005) A malaria parasite□ encoded vacuolar Η(+)Π ATPase is targeted to the host erythrocyte. J Biol Chem 280: 36841□ 36847.

Nishi, T., and Forgac, M. (2002) The vacuolar (H+)ClATPasesCl□ nature's most versatile proton pumps. Nat Rev Mol Cell Biol3: 94D 103.

Smith, J.D., and Craig, A.G. (2005) The surface of the Plasmodium falciparum□ infected erythrocyte. Curr Issues. Mol Biol 7:81□ 93.

Smith, J.D., Gamain, B., Baruch, D.I., and Kyes, S. (2001) Decoding the language of var genes and Plasmodium falciparum sequestration. Trends Parasitol 17: 538D545.

Sam DYellowe, T.Y., Florens, L., Johnson, J.R., Wang, T., Drazba, J.A., Le Roch, K.G., Zhou, Y., Batalov, S., Carucci, D.J., Winzeler, E.A., and Yates, J.R., 3rd (2004) A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling. Genome Res 14: 1052D 1059.

Petter, M., Haeggstrom, M., hattab, A., Fernandez, V., Klinkert, M.Q., and Wahlgren, M. (2007) Variant proteins of the Plasmodium falciparum RIFI family show distinct subcellular localization and developmental expression patterns. Mol Biochem Parasitol 156: 51□ 61.

SamDYellowe, T.Y., Florens, L., Johnson, J.R., Wang, T., Drazba, J.A., Le Roch, K.G., Zhou, Y., Batalov, S., Carucci, D.J., Winzeler, E.A., and Yates, J.R., 3rd (2004) A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling. Genome Res 14: 1052D 1059.

Sun D Wada, G.H., Wada, Y., and Futai, M. (2003b) Vacuolar H+ pumping ATPases in luminal acidic organelles and extracellular compartments: common rotational mechanism and diverse physiological roles. J Bioenerg Biomembr 35: 347 D358.

Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., SunO Wada, G.H., Wada, Y., and Futai, M. (2003) From lysosomes to the plasma membrane: localization of vacuolar□ type H+ DATPase with the a3 isoform during osteoclast differentiation. J Biol Chem 278: 22023 D22030.

Ueda, T., Ugawa, S., and Shimada, S. (2003) A novel putative M9.2 isoform of VDATPase expressed in the nervous system.Neuroreport 14: 25 D 30.

PATENT REFERENCES

US 6,774,21 1 B l 10/2010 Henkin et o/ 514/15

US 6,998,237 B 1 2/2006 Ferrone et al 530/300

US 7,452,965 B2 1 1/2008 Kelly et al 514/12

US 7,569,537 B2 8/2009 Das Gupta et al None

US 7,81 1,578 B2 10/2010 Eckert et al None

SEQUENCE LISTING

Seq ID No. 1

Cys Asp Leu Ser Gly He Pro Arg Cys

Seq ID No. 2

Cys Thr Thr Lys His Val He Ala Cys

Seq ID No. 3

Met Ser Lys Glu Val Val Asn Thr Lys Ala Glu Ala Ser Arg Val Asn

Ala Leu Ala Ala Val Arg Asn Tyr Lys Val Cys Pro Arg Leu Glu Tyr

Lys Thr He Ser Gly Val Gin Gly Pro Leu Val He He Glu Asp Val

Lys Phe Pro Lys Tyr Ser Glu He Val Thr He His Leu Ser Asp Asn

Thr Thr Arg Gin Gly Gin He Leu Glu Val Cys Gly Lys Lys Ala Val

He Gin Val Phe Glu Gly Thr Ser Gly He Asp Asn Lys Asn Ser Tyr

Val Glu Val Ser Gly Asp He Leu Lys Met Pro Met Ser Asp Glu Met

Leu Gly Arg Val Phe Asn Gly Ser Gly Lys Pro He Asp Lys Gly Pro

Asn He Leu Ala Asp Asp Tyr Leu Asp He Asn Gly Asn Pro He Asn

Pro Gin Cys Arg Val Tyr Pro Lys Glu Met He Gin Thr Gly He Ser

Thr He Asp Val Met Asn Ser He Val Arg Gly Gin Lys He Pro Leu

Phe Ser Ala Ala Gly Leu Pro His Asn Glu He Gly Ala Gin He Cys Arg Gin Ala Ser Leu Val Gin Gly Lys Asp Val Leu Asp His Ser Asp Asp Asn Phe Ala Val Val Phe Gly Ala Met Gly Val Asn Met Glu Thr Ala Arg Tyr Phe Arg Gin Asp Phe Glu Glu Asn Gly Lys Met Glu Arg Val Cys Leu Phe Leu Asn Leu Ala Asn Asp Pro Thr He Glu Arg He Leu Thr Pro Arg He Ala Leu Thr Thr Ala Glu Tyr Leu Ala Phe Glu Lys Glu Met His Val Phe Val He Leu Thr Asp Met Ser Ser Tyr Ala Asp Ala Leu Arg Glu Val Ser Ser Ala Arg Glu Glu Val Pro Gly Arg Arg Gly Tyr Pro Gly Tyr Met Tyr Ser Asp Leu Ser Thr He Tyr Glu Arg Ala Gly Arg Val Glu Gly Arg Asn Gly Ser He Thr Gin Phe Pro He Leu Thr Met Pro Asn Asp Asp He Thr His Pro He Pro Asp Leu Thr Gly Tyr He Thr Glu Gly Gin He Phe Val Asp Arg Asn Leu Tyr Asn Arg Gin lie Tyr Pro Pro He Asn Val Leu Pro Ser Leu Ser Arg Leu Met Lys Ser Gly He Gly His Asn Met Thr Arg He Asp His Pro Tyr Val Ser Asp Glh Leu Tyr Ser Asn Tyr Ala He Ala Gin Asp Val Lys Ala Met Lys Ala Val He Gly Glu Glu Ala Leu Ser Asn Asp Asp He Leu Tyr Leu Glu Phe Leu Asp Lys Phe Glu Lys Arg Phe He Thr Gin Asn Thr Tyr Glu Cys Arg Asp He Tyr Gin Ser Leu Asp He Ala Trp Glu Leu Leu Arg He Phe Pro Glu Asp Met Leu Lys Lys lie Lys Thr Asp He Leu Ser Lys Tyr Tyr Pro Arg His His Ala Asn

Seq ID No. 4

gtaggatcca tgagtaaaga agtagtaaat acaaaagctg

Seq ID No. 5

cctgcacacc tgaaatggtt ttatattcta gtcg

Seq ID No. 6

ccatttcagg tgtgcagggg cccctag

Seq ID No. 7

gttctcgagt tagttggcat ggtgacg

Claims

We Claim:

1. A cyclic peptide having an amino acid sequence selected from the group consisting of Seq ID No. 1 and 2 useful as a therapeutic.

2. The peptide as claimed in claim 1, wherein the peptide is a 7 mer cysteine constrained cyclic peptide.

3. The peptide as claimed in claim 1, wherein the peptide targets the highly conserved B subunit of V-^H+ ATPase.

4. The peptide of claim 1 inhibits P. falciparum growth in vitro.

5. The peptide of claim 1 , wherein the peptide is useful for setting of screens to identify other potent therapeutic agents .

6. The peptide of claim 1 , wherein the peptide is selected for setting of screens to identify chemical analogues.

7. A method of inhibiting B subunit of V-^H+ ATPase by using the cyclic peptides as claimed in claim 1.

8. A pharmaceutical composition comprising a peptide as claimed in claim 1 for use as a therapeutic including pharmaceutically acceptable carrier.

9. Use of the peptide as claimed in claim 1 for treatment of Malaria caused by pathogen having similar B subunit of V-H⁺ ATPase.

10. A kit comprising the pharmaceutical composition as claimed in claim 8, further comprising means for delivering said composition to a subject.