Patent Abstract:
the viral macrophage inflammatory protein - ii is a chemokine that interacts with the cc and cxc chemokine receptors , including the ccr5 and cxcr4 chemokine receptors . ccr5 and cxcr4 are the principal coreceptors required for cell entry of human immunodeficiency virus type 1 . the present invention describes a peptide fragment of the vmip - ii that prevents the hiv - 1 virus from interacting with the coreceptor cxcr4 , thereby preventing viral infection of that cell . these peptide fragments will serve as lead compounds for the development of therapeutic agents against hiv - 1 infections .

Detailed Description:
recombinant human chemokines sdf - 1 , mip - 1β and vmip - ii ( r & amp ; d systems , minneapolis , minn .) were lyophilized and dissolved as 1 μg / μl or 2 . 5 μg / μl stock solutions in sterile phosphate - buffered saline ( pbs ) and stored at — 20 ° c . in aliquots . the radioiodinated sdf - 1α and mip - 1β were purchased from dupont nen . the specific activity of 125 i - sdf - 1α and 125 i - mip - 1β were 2200 ci / mmol . cell culture media and g418 were purchased from life technologies , inc . the anti - cxcr4 monoclonal antibody ( mab ) 12g5 ( endres , m . j ., et al ., cell , 87 : 745 - 756 , 1996 ) was purchased from pharmingen ( san diego , calif .). 293 and nih / 3t3 cells were kindly provided by robert w . doms of university of pennsylvania and maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium plus 10 % fetal bovine serum . the human pccxcr4 and recombinant vaccinia viruses encoding two envs of hiv - 1 , vsc60 ( bh10 ) ( s . chakrabarti and b . moss , personal communication ) and vbd3 ( 89 . 6 ), and t7 rna polymerase , vtf1 . 1 ( alexander , w ., et al ., j virol , 66 : 2934 - 2942 , 1992 ), were also generous gifts from robert w . doms . the peptides were prepared by solid phase synthesis using fmoc - strategy on a 430a peptide synthesizer ( applied biosystems , foster city , calif .) and a 9050 pepsynthesizer plus ( perseptive biosystems , cambridge , mass . ), as described previously ( satoh , t ., et al ., j biol chem , 272 : 12175 - 12180 , 1997 ; li , s ., et al , j biol chem , 273 : 16442 - 16445 , 1998 ). the side chain protecting groups of n • - fmoc ( n -( 9 - fluorenyl ) methoxycarbonyl ) amino acids were : arg , pmc ; asp , otbu ; cys , trt ; gln , trt ; his , trt ; lys , boc ; ser , tbu , tyr , tbu ; and trp , boc ( pmc = 2 , 2 , 5 , 7 , 8 - pentamethyl - chroman - 6 - sulfonyl , otbu = tert - butyl ester , trt = trityl , boc = tert - butyloxycarbonyl and tbu = tert - butyl ester ). in every coupling reaction step , a 4 - fold excess of n • - fmoc amino acid , o - benzotriazol - 1 - yl - n , n , n ′, n ′- tetramethyluronium hexafluorophosphate , and 1 - hydroxybenzotriazole , and 10 - fold excess of diisopropylethylamine were used . the cleavage of peptides from the resin was carried out with the cleavage reagent ( trifluoroacetic acid : thioanisole : phenol : water : ethandithiol : triisopropylsilane / 81 . 5 : 5 : 5 : 5 : 2 . 5 : 1 ) for 2 h at room temperature with gentle stirring . crude peptides were precipitated in ice - cold methyl - t - butyl ether , centrifuged , and lyophilized . the crude peptides were then purified by preparative hplc using a dynamax - 300 å c 18 25 cm × 21 . 4 mm i . d . column with two solvent systems of 0 . 1 % tfa / h 2 o and 0 . 1 % tfa / acetonitrile . fractions containing the appropriate peptide were pooled together and lyophilized . the purity of the final product was assessed by analytical reverse phase high performance liquid chromatography , capillary electrophoresis and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry . all peptides were at least 95 % pure . v1 ( vmip - ii , 1 - 21 ) lgaswhrpdkcclgyqkrplp v2 ( vmip - ii , 6 - 18 ) hrpdkcclgyqkr v3 ( vmip - ii , 1 - 10 ) lgaswhrpdk v4 ( vmip - ii , 13 - 34 ) lgyqkrplpqvllsswyptsql v5 ( sdf - 1 , 1 - 4 , vmip - ii , 6 - 18 ) kpvshrpdkcclgyqkrplp v6 ( vmip - ii , 22 - 44 ) qvllsswyptsqlcskpgviflt v7 ( vmip - ii , 36 - 57 ) skpgvifltkrgrqvcadkskd v8 ( vmip - ii , 51 - 71 ) adkskdwvkklmqqlpvtar v9 ( vmip - ii , 30 - 40 , cyclic -) ctsqlaskpgc v10 ( vmip - ii , 41 - 51 , cyclic -) cfltkrgrqvc av1 ( v1 mutant , c11a & amp ; c12a ) lgaswhrpdkaalgyqkrplp v1 - 1 ( v1 mutant , l1a ) agaswhrpdkcclgyqkrplp v1 - 2 ( v1 mutant , w5a ) lgasahrpdkcclgyqkrplp v1 - 3 ( v1 mutant , r7a ) lgaswhapdkcclgyqkrplp v1 - 4 ( v1 mutant , k9a ) lgaswhrpdacclgyqkrplp v1 - 5 ( v1 mutant , c11a ) lgaswhrpdkaclgyqkrplp v1 - 6 ( v1 mutant , q15a ) lgaswhrpdkcclgyakrplp v1 - 7 ( v1 mutant , r17a ) lgaswhrpdkcclgyqkaplp rv1 ( vmip - ii , 1 - 21 , reserved ) plprkqyglcckdprhwsagl dv1 ( vmip - ii , 1 - 21 , all d - amino acid ) lgaswhrpdkcclgyqkrplp rdv1 ( vmip - ii , 1 - 21 , reserved , all d - plprkqyglcckdprhwsagl amino acid ) v1 - dcl ( vmip - ii , 1 - 1o , d - amino scid , 12 - lgaswhrpdkcclgyqkrplp 21 l - amino acid ) v1 - lcd ( vmip - ii , 1 - 1o , l - amino acid , 11 - lgaswhrpdkcclgyqkrplp 21 d - amino acid ) d1 ( dv1 mutant , l1a ) agaswhrpdkcclgyqkrplp d2 ( dv1 mutant , w5a ) lgasahrpdkcclgyqkrplp d3 ( dv1 mutant , r7a ) lgaswhapdkcclgyqkrplp d4 ( dv1 mutant , k9a ) lgaswhrpdacclgyqkrplp d5 ( dv1 mutant , c11a ) lgaswhrpdkaclgyqkrplp d6 ( dv1 mutant , q15a ) lgaswhrpdkcclgyakrplp d7 ( dv1 mutant , r17a ) lgaswhrpdkcclgyqkaplp ad1 ( dv1 mutant , c11a & amp ; c12 a ) lgaswhrpdkaalgyqkrplp l - 10 ( dv1 deletion , 1 - 10 ) lgaswhrpdk sup t1 cells ( 2 × 10 5 ) were washed with facs buffer ( 0 . 5 % bovine serum albumin , 0 . 05 % sodium azide in pbs ) and incubated with an anti - cxcr4 monoclonal antibody ( mab ) 12g5 ( 10 μg / ml ) for 30 min at 4 ° c . after washing with facs buffer , cells were incubated with 10 μg fitc conjugated goat anti - mouse igg ( southern biotechnology associates , inc . birmingham , ala .) for 30 min at 4 ° c . after washing twice with facs buffer , cells were fixed in the fixing buffer ( 2 % palaformaldehyde in pbs ) and then analyzed on a facscan flow cytometer ( coulter epics elite , coolten corp ., hialeah , fla .). cem - t4 cells were harvested and washed twice with pbs . competition binding experiments were performed using a single concentration ( 0 . 2 nm ) of 125 i - sdf - 1 • in the presence of increasing concentrations of unlabeled ligands in a final volume of 100 μl of binding buffer ( 50 nm hepes ph 7 . 4 , 1 nm cacl 2 , 5 nm mgcl 2 , 0 . 1 % bovine serum albumin ) containing 2 × 10 5 cells . nonspecific binding was determined by the addition of 100 nm unlabeled sdf - 1α samples were incubated for 60 min at room temperature . the incubation was terminated by separating the cells from the binding buffer by centrifugation and washing once with 500 μl of cold binding buffer . bound ligands were quantitated by counting γ emissions . following a similar experimental procedure as described above , 293 cells transfected with ccr5 and 125 i - mip - 1β were used to determine the specific binding activity of peptides to ccr5 . following a modified procedure published by our lab ( zhou , n ., et al ., eur j immunol , 30 : 164 - 173 , 2000 ) and others ( doranz , b . j ., et al ., cell , 85 : 1149 - 1158 , 1996 ; doranz , b . j ., et al ., j virol , 71 : 6305 - 6314 , 1997 ; rucker , j ., et al ., methods enzymol , 288 : 118 - 133 , 1997 ) a gene reporter fusion assay was used to determine the inhibition of the peptides on coreceptor activity of cxcr4 and ccr5 in mediating hiv - 1 viral entry . hiv - 1 env proteins and t7 rna polymerase were introduced into effector 293 cells by infection with recombinant vaccinia virus and incubated overnight at 32 ° c . in the presence of rifampicin ( 100 μg / ml ). nih / 3t3 target cells were co - transfected in 6 - well plates with plasmids encoding cd4 , cxcr4 or ccr5 and luciferase under control of t7 promotor by capo 4 transfection and incubated at 37 ° c . overnight . to initiate fusion , 10 5 effector cells were added to each well and incubated at 37 ° c . in the presence of ara - c and rifampicin . after 5 h of fusion , cells were lysed in 150 μl of reporter lysis buffer ( promega ) and assayed for luciferase activity by using commercially available reagents ( promega ). sup t1 cells and ccr5 transfected 293 cells were used to measure the intracellular calcium influx . [ ca 2 + ] i was measured using excitation at 340 and 380 nm on a fluorescence spectrometer ( perkin elmer ls50 ). calibration was performed using 10 % triton x - 100 for total fluorophore release and 0 . 5 m egta to chelate free ca 2 + . intracellular ca 2 + concentrations were calculated by using the fluorescence spectrometer measurement program . migration of sup t1 cells was assessed in disposable transwell trays ( costar , cambridge , mass .) with 6 . 5 - mm diameter chambers and membrane pore size of 3 μm . sdf - 1 at 100 nm ( kindly provided by elias lolis of yale university ) in 0 . 5 % bsa rpmi 1640 was added to the lower well . 100 μl of sup t1 cells at 1 × 10 7 cells / ml in the same medium without sdf - 1 was added to the upper well . for peptide inhibition experiments , the cells were preincubated with various concentrations of the peptide for 15 min at 25 ° c . the peptide at the same concentration was also added to the lower well . after incubation at 37 ° c . and 5 % co 2 for 4 h , cells that migrated to the lower well were counted . chemotactic migration was determined by subtraction of cells migrated in medium alone ( blank control experiment ). the v1 peptide ( seq . id . no : 2 ) was synthesized corresponding to residues 1 - 21 of the n - terminal region of vmip - ii ( seq . id . no : 1 ) ( table 1 ). since vmip - ii interacts with cxcr4 and ccr5 ( kledal , t . n ., et al ., science , 277 : 1656 - 1659 , 1997 ), we tested the binding activity of v1 peptide ( seq . id . no : 2 ) with both receptors . for cxcr4 binding , the peptide , together with native vmip - ii and sdf - 1α as controls , were examined by using both 125 i - sdf - 1α and anti - cxcr4 mab 12g5 competitive binding assays ( fig1 and table 2 ). the v1 peptide ( seq . id . no : 2 ) strongly competes with the cxcr4 binding of 125 i - sdf - 1α in a concentration dependent manner with an ic 50 of 190 nm . thus , the v1 peptide ( seq . id . no : 2 ) appears to have much higher cxcr4 binding affinity than other reported peptides derived from sdf - 1 n - terminus ( loetscher , p ., et al ., j biol chem , 273 : 22279 - 22283 , 1998 ; heveker , n ., et al ., current biology , 8 : 369 - 376 , 1998 ). since the dimerization of a cysteine - containing peptide derived from sdf - 1 n - terminus has been reported to contribute to receptor binding ( loetscher , p ., et al ., j biol chem , 273 : 22279 - 22283 , 1998 ), dimer formation of the v1 peptide ( seq . id . no : 2 ), which contains two cysteines , was examined . analysis by mass spectrometry demonstrated a pure monomer with no dimer detectable , thereby excluding the contribution of dimerization to the strong cxcr4 binding by the v1 peptide ( seq . id . no : 2 ). to further characterize residues within the n - terminus of vmip - ii ( seq . id . no : 1 ) important for cxcr4 recognition , truncated v1 analogs were synthesized ( table 1 ). the v2 peptide ( residues 6 - 18 of vmip - ii , seq . id . no : 3 ) containing truncation on both ends of v1 ( seq . id . no : 2 ) showed a significant loss in cxcr4 binding , whereas the v3 peptide ( residues 1 - 10 of vmip - ii , seq . id . no : 4 ) containing the first half of v1 sequence ( seq . id . no : 2 ) retained some activity ( fig1 ). the interaction of these peptides with ccr5 receptor was tested in a competitive binding assay using radiolabeled mip - 1β . these peptides did not show any binding activity with ccr5 . these results demonstrated that the peptides derived from the n - terminus of vmip - ii ( seq . id . no : 1 ) interact with cxcr4 but not ccr5 . this is in contrast with native vmip - ii which recognizes both receptors . a cell - cell fusion assay was used to determine the ability of cxcr4 and ccr5 peptides in their ability to block the coreceptor function , which mediates cell entry of various hiv - 1 isolates . the v1 peptide ( seq . id . no : 2 ) showed inhibition of both t - and dual - tropic hiv - 1 gp120 - mediated cell - cell fusion via cxcr4 ( fig2 ). as expected from its significant loss in cxcr4 binding ( fig1 ), the truncated v2 peptide ( seq . id . no : 3 ) did not show any activity . on the other hand , both v1 ( seq . id . no : 2 ) and v2 ( seq . id . no : 3 ) peptides displayed no effect on m - tropic hiv - 1 gp120 - mediated cell - cell fusion via ccr5 . these results were consistent with binding studies and demonstrated that the v1 peptide ( seq . id . no : 2 ) selectively inhibited cxcr4 coreceptor function in mediating hiv - 1 entry . the v1 peptide blocks the signaling and chemotaxis of sdf - 1 via cxcr4 as the v1 peptide ( seq . id . no : 2 ) can bind cxcr4 receptor , its ability to induce an intracelluar signal or interfere with sdf - 1 signaling via cxcr4 was studied by measuring intracellular calcium influx in sup t1 cells expressing the receptor . at various concentrations , the peptide did not show any signaling activity via cxcr4 , thereby revealing it &# 39 ; s activity as an antagonist ( fig3 a ). in addition , this peptide interfered with the signaling of sdf - 1 , a natural cxcr4 ligand , and almost completely blocked sdf - 1 signal at the concentration of 200 μm ( fig3 a ). the effect of the v1 peptide ( seq . id . no : 2 ) on signal transduction via ccr5 was also tested in 293 cells transfected with ccr5 . as expected from its lack of binding to ccr5 , the peptide neither displayed signaling activity nor blocked the signal induced by mip - 1β via ccr5 ( fig3 b ). the v2 peptide ( seq . id . no : 3 ), which does not bind cxcr4 or ccr5 ( fig1 ), did not show any effect on cxcr4 or ccr5 signal transduction . in addition to calcium influx , the v1 peptide ( seq . id . no : 2 ) was tested in assays of chemotaxis of sup t1 cells . consistent with its ability to interfere with sdf - 1 signaling via cxcr4 , the v1 peptide ( seq . id . no : 2 ) was found to inhibit the chemotactic activity of sdf - 1 in a concentration dependent manner ( fig4 ). the present invention provides methods for treating hiv - 1 infection by inhibiting viral entry into cells expressing the cxcr4 receptor . such cxcr4 - expressing cells include , for example , t - cells . accordingly , one or more vmip - ii peptides according to the invention is administered to a patient in need of such treatment . a therapeutically effective amount of the drug may be administered as a composition in combination with a pharmaceutically carrier . pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents , excipients , solvents , adjuvants , or vehicles , for parenteral injection , for intranasal or sublingual delivery , for oral administration , for rectal or topical administration or the like . the compositions are preferably sterile and nonpyrogenic . examples of suitable carriers include but are not limited to water , saline , dextrose , mannitol , lactose , or other sugars , lecithin , albumin , sodium glutamate cysteine hydrochloride , ethanol , polyols ( propyleneglycol , ethylene , polyethyleneglycol , glycerol , and the like ), vegetable oils ( such as olive oil ), injectable organic esters such as ethyl oleate , ethoxylated isosteraryl alcohols , polyoxyethylene sorbitol and sorbitan esters , microcrystalline cellulose , aluminum methahydroxide , bentonite , agar - agar and tragacanth , or mixtures of these substances , and the like . the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting agents , emulsifying agents , ph buffering agents , antibacterial and antifungal agents ( such as parabens , chlorobutanol , phenol , sorbic acid , and the like ). if desired , absorption enhancing or delaying agents ( such as liposomes , aluminum monostearate , or gelatin ) may be used . the compositions can be prepared in conventional forms , either as liquid solutions or suspensions , solid forms suitable for solution or suspension in liquid prior to injection , or as emulsions . compositions containing the vmip - ii peptides are administered by any convenient route which will result in delivery to the site of infection of cxcr4 - expressing cells by hiv - 1 , in an amount effective for inhibiting that infection from proceeding . modes of administration include , for example , orally , rectally , parenterally ( intravenously , intramuscularly , intraarterially , or subcutaneously ), intracisternally , intravaginally , intraperitoneally , locally ( powders , ointments or drops ), or as a buccalor nasal spray or aerosol . the pharmaceutical compositions are most effectively administered parenterally , preferably intravenously or subcutaneously . for intravenous administration , they may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances , such as sodium chloride , glycine , and the like , having a buffered ph compatible with physiologic conditions . such intravenous delivery vehicles are known to those skilled in the art . in a preferred embodiment , the vehicle is a sterile saline solution . if the peptides are sufficiently small , other preferred routes of administration are intranasal , sublingual , and the like . intravenous or subcutaneous administration may comprise , for example , injection or infusion . the vmip - ii - derived peptides according to the invention can be administered in any circumstance in which inhibition of hiv infection is desired . the peptides of the invention may be used for treatment of subjects as a preventative measure to avoid hiv infection , or as a therapeutic to treat patients already infected with hiv . the viruses whose transmission may be inhibited by the peptides of the invention include strains of hiv - 1 , but is most useful for those strains which gain entry via the cxcr4 , such as t - tropic and dual - tropic strains . t - tropic strains utilize cxcr4 for entry , while dual - tropic strains utilize cxcr4 or ccr5 ( simmons et al ., j . virol . 70 : 8355 - 60 , 1996 ). the peptides of the invention may be used prophylactically in uninfected individuals after exposed to an hiv virus . examples of such uses include in the prevention of viral transmission from mother to infant , and following accidents in healthcare wherein workers may become exposed to hiv - contaminated blood , syringes and the like . the peptides may be administered to other individuals at risk of contracting hiv , such as homosexuals , prostitutes and intravenous drug users . the vmip - ii - derived peptides may be administered alone or in combination with other peptides or other anti - hiv pharmaceutical agents . the effective amount and method of administration will vary based upon the sex , age , weight and disease stage of the patient , whether the administration is therapeutic or prophylactic , and other factors apparent to those skilled in the art . based upon the studies described herein , a suitable dosage of peptide is a dosage which will attain a tissue concentration of from about 1 to about 100 • m , more preferably from about 10 to about 50 • m , most preferably about 25 • m . it is contemplated that lower or higher concentrations would also be effective . the tissue concentration may be derived from peptide blood levels . the amount of active agent administered depends upon the degree of the infection . those skilled in the art will derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient . doses are contemplated on the order of from about 0 . 01 to about 1 , preferably from about 0 . 1 to about 0 . 5 , mg / kg of body weight . the active agent may be administered by injection daily , over a course of therapy lasting two to three weeks , for example . alternatively , the agent may be administered by continuous infusion , such as via an implanted subcutaneous pumps . the viral chemokine vmip - ii differs from all known human chemokines in that vmip - ii binds with high affinity to a number of both cc and cxc chemokine receptors ( kledal , t . n ., et al ., science , 277 : 1656 - 1659 , 1997 ). this unique property of vmip - ii presents an intriguing avenue to probe the structural basis for the promiscuous receptor interaction . the present invention relates to determining if the common binding sites of vmip - ii have been optimized by the virus for multiple receptor interactions , or if distinctive binding determinants have evolved for different receptors . a synthetic peptide approach was used to study the role of the n - terminus of vmip - ii ( seq . id . no : 1 ) in the recognition with two important chemokine receptors , cxcr4 and ccr5 . the n - terminal region is most diverse among vmip - ii and other chemokines and , on the basis of the importance of n - termini in other chemokines ( clark - lewis , i ., et al ., j leuk biol , 57 : 703 - 11 , 1995 ), critical for the unique function of vmip - ii . the v1 peptide ( seq . id . no : 2 ) of the present invention corresponds to this region and is shown to interact with cxcr4 , thereby blocking signal transduction and the coreceptor function that mediates hiv - 1 entry . this indicates that the n - terminus of vmip - ii is essential for its biological function through cxcr4 . in contrast to its potent activities via cxcr4 , v1 peptide ( seq . id . no : 2 ) did not display any interaction with ccr5 or inhibition of ccr5 signaling and coreceptor function . whereas the native vmip - ii ( seq . id . no : 1 ) binds and blocks the function of both receptors , the lack of interaction of the n - terminal fragment of vmip - ii with ccr5 implies that other domains , yet to be identified , mediate vmip - ii function via ccr5 . alternatively , the peptide without the other domains of the vmip - ii protein may fail to adopt conformations necessary for ccr5 recognition . however , this is unlikely given the strong interaction of this peptide with another receptor , cxcr4 , implying the peptide has the proper structural elements for receptor binding . taken together , the present invention describes distinctive determinants in vmip - ii ( seq . id . no : 1 ) that mediate biological function via different receptors . the important feature of the n - terminus of vmip - ii for cxcr4 recognition was further analyzed with truncated peptide analogs of v1 . it has been suggested that a spatial cluster of positive residues in sdf - 1 is critical for forming favorable electrostatic interaction with the negative charge surface of the extracellular domains of cxcr4 ( dealwis , c ., et al ., proc natl acad sci usa , 95 : 6941 - 6946 , 1998 ). a high positive charge is seen in several peptide and nonpeptide inhibitors of cxcr4 , such as t22 ( murakami , t ., et al ., j exp med , 186 : 1389 - 1393 , 1997 ), alx40 - 4c ( doranz , b . j ., et al ., j exp med , 186 : 1395 - 1400 , 1997 ), and amd3100 ( schols , d . et al ., j exp med , 186 : 1383 - 1388 , 1997 ). interestingly , vmip - ii ( seq . id . no : 1 ) has a high net positive charge like sdf - 1 , despite the very low sequence homology between them . since the v1 peptide ( seq . id . no : 2 ) derived from the n - terminus of vmip - ii also contains a number of positive charge residues , this raised the question whether these residues play a role in receptor interaction . the v2 peptide ( seq . id . no : 3 ) of the present invention , which retains all positive residues in the core region of the v1 peptide ( seq . id . no : 2 ), tested the function of these positively charged residues . the loss of activity in the v2 peptide ( seq . id . no : 3 ) argued against a primary role of the positive residues in receptor binding . alternatively , the first five residues of vmip - ii are more critical and their removal in the v2 peptide ( seq . id . no : 3 ) explains the loss of activity . this is consistent with observations made for other chemokines where the first several residues at the n - terminus are most important for biological function ( heveker , n ., et al ., current biology , 8 : 369 - 376 , 1998 ; hèbert , c . a ., et al ., j biol chem , 266 : 18989 - 18994 , 1991 ; crump , m . p ., et al ., embo journal , 16 : 6996 - 7007 , 1997 ). the role of the first five residues of the n - terminus of vmip - ii was further demonstrated by v3 ( seq . id . no : 4 ), a shorten analog containing only the n - terminal half of the v1 peptide ( seq . id . no : 2 ), which retained some activity in cxcr4 binding ( fig1 ). the v1 peptide ( seq . id . no : 2 ) of the present invention is a promising lead compound for the development of high affinity ligands for cxcr4 . although a direct comparison with other chemokine derived peptides can not be made due to the difference in binding assay protocols , the relative affinity of the v1 peptide ( seq . id . no : 2 ) as compared with other cxcr4 binding peptides is estimated by comparing these peptides with native sdf - 1 . in the present invention , the v1 peptide ( seq . id . no : 2 ) was shown to compete with the cxcr4 binding of anti - cxcr4 mab 12g5 and 125 i - sdf - 1α , with ic 50 values of 640 and 190 nm , respectively ( table 2 and fig1 ), which are about 33 - and 70 - fold less potent than sdf - 1 , respectively . this compares favorably with other reported peptides derived from the n - terminus of sdf - 1 which are about 82 - to 1000 - fold less potent than sdf - 1 ( loetscher , p ., et al ., j biol chem , 273 : 22279 - 22283 , 1998 ; heveker , n ., et al ., current biology , 8 : 369 - 376 , 1998 ). in addition to its relatively high cxcr4 affinity among chemokine derived peptides reported so far , the v1 peptide ( seq . id . no : 2 ) possesses other interesting biological properties , such as the induction of cxcr4 internalization . the potency of the v1 peptide ( seq . id . no : 2 ) in the cell - cell fusion assay ( fig2 ) was much lower than that in the competition binding assay ( fig1 and table 2 ). a similar discrepancy in potency between these two assays was also observed for sdf - 1 , which showed an ic 50 of 2 . 7 nm in 125 i - sdf - 1α competitive binding assay ( fig1 and table 2 ) but had only 45 % inhibition of cell - cell fusion even at 200 nm ( fig2 ). these are due to the relative insensitivity of the cell - cell fusion assay for the quantitative determination of the potency of anti - hiv agents , as previously reported by others ( rucker , j ., et al , methods enzymol , 288 : 118 - 133 , 1997 ). therefore , the activity of the v1 peptide ( seq . id . no : 2 ), as well as the control sdf - 1 , are underestimated in the cell - cell fusion assay . compared to the cell - cell fusion assay , the inhibitory activity of the v1 peptide ( seq . id . no : 2 ) in the chemotaxis assay was much higher with an ic 50 of about 1 μm , which was more consistent with its cxcr4 binding potency ( fig4 ). in summary , the characterization of precise binding sites within vmip - ii ( seq . id . no : 1 ) for cxcr4 and ccr5 is a critical step toward understanding the molecular mechanism of vmip - ii function and development of broad - spectrum hiv inhibitors . the present invention identifies the n - terminus , particularly the first five residues , of vmip - ii as an important binding site for cxcr4 . a synthetic peptide derived from this region displays widely different interactions with cxcr4 and ccr5 , thus providing experimental support for the notion that distinctive sites within vmip - 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