Heterodimer formation between the antimicrobial peptides magainin 2 and PGLa in lipid bilayers: a cross-linking study.

The antimicrobial peptides magainin 2 and PGLa, isolated from the skin of the African clawed frog Xenopus laevis, show marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D. (1995) Eur. J. Biochem. 228, 257-264]. We suggested previously that these peptides form a potent heterodimer composed of either parallel or antiparallel helices in membranes [Matsuzaki, K., Mitani, Y., Akada, K., Murase, O., Yoneyama, S., Zasloff, M., and Miyajima, K. (1998) Biochemistry 37, 15144-15153]. To detect the putative heterodimer by chemical cross-linking, analogues of magainin 2 and PGLa with a Cys residue at either terminus were synthesized. These cross-linking experiments suggested that both peptides form a parallel heterodimer in membranes composed of phosphatidylglycerol/phosphatidylcholine but not in either buffer or a helix-promoting 2,2,2-trifluoroethanol/buffer mixture. The isolated parallel heterodimers exhibited an order of magnitude higher membrane permeabilization activity compared with the monomeric species, indicating that the observed synergism is due to heterodimer formation.

Synthesis and evaluation of bifunctional anti-HIV agents based on specific CXCR4 antagonists-AZT conjugation.

We have previously found that T140, a 14-amino acid residue peptide, inhibits infection of target cells by T cell-line-tropic strains of HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. Here, we report synthesis and evaluation of bifunctional anti-HIV compounds, which are composed of T140 analogues and a reverse transcriptase inhibitor, 3'-azido-3'-deoxythymidine (AZT). Novel conjugated analogues have been proved to have the ability for controlled release of AZT in neutral aqueous media as well as mouse and feline sera, and high selectivity indexes (SIs, 50% cytotoxic concentration/50% effective concentration) caused by a synergistic effect of two different regenerating agents. Thus, these bifunctional compounds have several potential advantages. T140 analogues can possibly work as a carrier of AZT targeting T cells due to their specific affinity for CXCR4 on T cells. A synergistic effect by two types of regenerating agents may enable drug dosage to be reduced, and thus it may effectively suppress toxic side effects and the appearance of drug-resistant virus.

Development of specific CXCR4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-HIV peptide T140.

We previously reported a truncated polyphemusin peptide analogue, T140, which efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. We have found that T140 is not stable in feline serum due to the cleavage of the C-terminal Arg,(14) indispensable for anti-HIV activity. On the other hand, a C-terminally amidated analogue of T140, TZ14004, has been found to be completely stable in incubation in the serum for 2 days. The C-terminal amide is thought to be needed for stability in serum. However, TZ14004 does not have fairly strong anti-HIV activity, but has relatively strong cytotoxicity, probably due to an increase by +1 charge from total +7 charges of T140. In our previous study, the number of total +6 charges seemed to be a suitable balance between activity and cytotoxicity. In this study, we have conducted a double-L-citrulline (Cit)-scanning study on TZ14004 based on the C-terminally amidated form in due consideration of the total net charges in the whole molecule to find novel effective CXCR4 inhibitors, TN14003 ([Cit(6)]-T140 with the C-terminal amide) and TC14012 ([Cit(6), D-Cit(8)]-T140 with the C-terminal amide), which possess high selectivity indexes (SIs) and complete stability in feline serum.

Phospho-Azatyrosine, a less effective protein-tyrosine phosphatase substrate than phosphotyrosine.

Azatyrosine (AzaTyr, 4) is a natural product isolated from Streptomyces chibanesis, whose structure is characterized by a nitrogen atom in the aryl ring of a tyrosyl residue. This seemingly minor modification to the tyrosyl residue results in profound physiological effects, as AzaTyr has been shown to promote permanent reversion of ras-dependent transformed cells to the normal phenotype in culture and to inhibit chemical induction of carcinogenesis in transgenic mice bearing oncogenic human ras. The mechanisms underlying these effects are not known, however ras-pathways involve an intricate balance between both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). The present study was undertaken to examine the general utility of AzaTyr as a structural motif for PTP inhibitor design by examining the phospho-azatyrosine (pAzaTyr)-containing peptide Ac-Asp-Ala-Asp-Glu-pAzaTyr-Leu-amide (8) in a PTP1 enzyme system. Kinetic analysis indicated that 8 binds with a Km value of 210 microM and a catalytic turnover rate, kcat of 52 s(-1). This represents a greater than 50-fold reduction in binding affinity relative to the parent phosphotyrosine-containing peptide, indicating that the aryl nitrogen adversely affects binding affinity. The much lower PTP affinity of the pAzaTyr-containing peptide reduces the potential utility of the AzaTyr pharmacophore for PTP inhibitor design. These results are discussed from the point of view that incorporation of AzaTyr residues into proteins could result in perturbation of protein-tyrosine phosphorylation,dephosphorylation cascades that control signal transduction processes, including ras-dependent pathways.

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity.

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.

Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of effective anti-HIV agents with very high selectivity indexes.

A polyphemusin peptide analogue, T22 ([Tyr(5,12), Lys7]-polyphemusin II), and its shortened potent analogues, T134 (des-[Cys(8,13), Tyr(9,12)]-[D-Lys10, Pro11, L-citrulline16]-T22 without C-terminal amide) and T140 [[L-3-(2-naphthyl)alanine3]-T134], strongly inhibit the T-cell line-tropic (T-tropic) HIV-1 infection through their specific binding to a chemokine receptor, CXCR4. T22 is an extremely basic peptide possessing five Arg and three Lys residues in the molecule. In our previous study, we found that there is an apparent correlation in the T22-related peptides between the number of total positive charges and anti-HIV activity or cytotoxicity. Here, we have conducted the conventional Ala-scanning study in order to define the anti-HIV activity pharmacophore of T140 (the strongest analogue among our compounds) and identified four indispensable amino acid residues (Arg2, Nal3, Tyr5, and Arg14). Based on this result, a series of L-citrulline (Cit)-substituted analogues of T140 with decreased net positive charges have been synthesized and evaluated in terms of anti-HIV activity and cytotoxicity. As a result, novel effective inhibitors, TC14003 and TC14005, possessing higher selectivity indexes (SIs, 50% cytotoxic concentration/50% effective concentration) than that of T140 have been developed.

Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells.

We investigated the involvement of Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca(2+)-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca(2+)-independent activity. The autophosphorylation and appearance of Ca(2+)-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (alphaT286A kinase) or Asp (alphaT286D kinase) was examined. alphaT286A kinase was not converted to a Ca(2+)-independent form, and alphaT286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alphaT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing alphaT286D kinase had much longer neurites than Nb2a/alpha cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca(2+)-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.

Interactions between local anesthetics and Na+ channel inactivation gate peptides in phosphatidylserine suspensions as studied by 1H-NMR spectroscopy.

Interactions between local anesthetics and a sodium channel inactivation gate peptide (Ac-GGQDIFMTEEQK-NH2, MP-1A), which was dissected from the cytoplasmic linker between domains III and IV of the sodium channel alpha-subunit (G1484-K1495 in rat brain type IIA), have been studied by 1H-NMR spectroscopy. Changes in 1H-NMR chemical shifts of the aromatic proton resonances of dibucaine (pH 7.0) and lidocaine (pH 6.0 and 9.0) in phosphatidylserine (PS) suspensions were observed. The effects of substitution of glutamine (F1489Q; MP-2A) or D-phenylalanine (MP-1A') for L-phenylalanine (F1489) in MP-1A and the effects of substitution of neutral amino acid residues for the corresponding acidic amino acid residues (D1487N, MP-1NA; E1492Q, MP-IQEA; E1493Q, MP-IEQA) in MP-1A, on the aromatic 1H-NMR chemical shift changes of dibucaine and lidocaine were also investigated. From these results it was concluded that: the aromatic ring of phenylalanine of MP-1A and the aromatic ring of the cationic form of dibucaine or lidocaine are interacting by pi-pi stacking; the tertiary amine nitrogen of dibucaine is interacting electrostatically with D1487, whereas that of lidocaine is interacting with E1492.

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles.

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.

Inhibition of pRb phosphorylation and cell cycle progression by an antennapedia-p16(INK4A) fusion peptide in pancreatic cancer cells.

In this study, we examined whether or not a small peptide derived from p16(INK4A) protein with the antennapedia carrier sequence could inhibit the growth of pancreatic cancer cells through the inhibition of cell cycle progression. Growth inhibition by the p16-derived peptide was observed in a time- and dose-dependent manner in AsPC-1 and BxPC-3 cells (p16-negative and pRb-positive), whereas Saos-2 cells (p16-positive and pRb-negative) showed no inhibitory effect. In AsPC-1 and BxPC-3 cells, the proportion of cells in the G(1) phase markedly increased 48 h after treatment with 20 microM p16-derived peptide. Cell-cycle analysis of Saos-2 cells showed little change during the entire period of treatment. Immunoblot analysis showed inhibition of pRb phosphorylation after treatment of BxPC-3 with 10 microM p16 peptide. Furthermore, the p16 peptide caused a decrease in cyclin A at later times of treatment. These results demonstrate that the p16-derived peptide can inhibit the growth of p16-negative and pRb-positive pancreatic cancer cells by means of G(1) phase cell cycle arrest resulting from the inhibition of pRb phosphorylation. Restoration of p16/pRb tumor-suppressive pathway by re-expression of p16(INK4A) may play a therapeutic role in the treatment of pancreatic cancer.

Stereoselective synthesis of CF(2)-substituted phosphothreonine mimetics and their incorporation into peptides using newly developed deprotection procedures.

Stereoselective syntheses of all four stereoisomers of CF(2)-substituted nonhydrolyzable phosphothreonine derivatives (33, 39, and their enantiomers) and their incorporation into peptides are described herein. Key to the synthesis of these amino acids was construction of secondary phosphate-mimicking difluoromethylphosphonate units along with generation of two stereocenters. The former was achieved using a Cu(I)-mediated cross-coupling reaction of BrZnCF(2)P(O)(OEt)(2) (8) and beta-iodo-alpha,beta-unsaturated ester 12, with stereochemistry of both alpha- and beta-stereocenters being established using bornane-10,2-sultam as a chiral auxiliary. Diastereoselective hydrogenation of a chiral alpha,beta-unsaturated acylsultam (for the beta-center) (e.g., 16a) and subsequent stereoselective bromination (for the alpha-center of the threo derivative) or amination (for the alpha-center of erythro (allo) derivative) were utilized. Transesterification of the bromide to the benzyl ester followed by azide displacement of the halogen, then reduction of the resulting azide, followed by Boc-protection and finally removal of the benzyl group, afforded protected both L- and D-phosphothreonine mimetics (39 and its enantiomer). On the other hand, protected both L- and D-allo-phosphothreonine mimetics (33 and its enantiomer) were synthesized via transesterification of the above-mentioned amination product, followed by hydrogenolytic removal of the benzyl group. Key to utilization of these amino acid analogues in peptide synthesis was removal of ethyl protection from the difluoromethylphosphonate moiety. A two-step deprotection methodology, consisting of a combination of a first-step reagent [0.3 M BSTFA-TBAI in CH(2)Cl(2), BF(3).Et(2)O] followed by a second-step reagent [1 M TMSOTf-thioanisole in TFA, m-cresol, EDT] was developed for use in solid-phase protocols. A 12-residue Cdc (cell division cycle) 2-peptide 41, possessing two nonhydrolyzable phosphoamino acid mimetics (F(2)Pmab 6 and F(2)Pmp 4), was subjected to this deprotection procedure and was obtained in 25% yield based on the protected resin. The present synthetic method affords nonhydrolyzable phosphoamino acid mimetics-containing peptides in high yield without accompanying side reactions.

A new practical strategy for the synthesis of long-chain phosphopeptide.

A new practical strategy has been developed for the synthesis of long-chain phosphopeptide. Both the 2-chlorobenzyloxycarbonyl (CIZ) group for Lys and methyl (Me) for phosphoamino acids remained intact, while other commonly used side-chain protecting groups were cleaved quantitatively, during the reaction using a highly acidic trifluoromethanesulfonic acid (TFMSA)-based reagent system (High TFMSA: TFMSA-TFA-m-cresol=1:9:1, v/v). Selective deprotection of the CIZ and Me group-containing protected phosphopeptide resin with the High TFMSA gave a partially protected phosphopeptide fragment suitable for thioester-mediated fragment condensation. A deprotection protocol of the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which evades significant side reaction toward the protected phosphoamino acid, was also developed. These two new findings enabled us to synthesize long-chain phosphopeptide via thioester-mediated fragment condensation.

Cell adhesive sequences in mouse laminin beta1 chain.

Laminin-1, a major component of the basement membrane, consists of three different chains, alpha1, beta1, and gamma1. We sought to identify cell adhesive sequences from the mouse laminin beta1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin beta1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin beta1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.

[Development of new deprotecting methodologies for peptides and application to studies on signaling mechanism].

This review summarizes the development of deprotecting methodologies for peptides and their practical application to the synthesis of disulfide bond- or phosphoamino acid-containing peptides. Acidic deprotecting systems utilizing Brønsted acid (HF, trifluoromethanesulfonic acid (TFMSA) and HBr etc.) have been used for the removal of protecting groups in peptide chemistry; however, these reagents are not always applicable to all of the peptides including cystine- or phosphoamino acid-containing peptides. Our attempt to utilize Lewis acid for the deprotective reaction resulted in the development of efficient and practical reagent systems (1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf)-sulfide in trifluoroacetic acid (TFA) and 1 M trimethylsilyl bromide (TMSBr)-sulfide in TFA) suitable for peptide synthesis. A new disulfide bond-forming reaction using Tl(OCOCF3)3 was developed for the synthesis of cystine peptides. The use of TMSOTf or TMSBr-mediated deprotecting system in conjunction with the disulfide bond-forming reaction utilizing Tl (III) provides a procedure for the practical synthesis of cystine peptides. A two-step deprotection method consisting of high acidic (1 M TMSOTf-thioanisole in TFA, m-cresol, ethanedithiol) and low acidic (high acidic system + dimethyl sulfide--TMSOTf) treatments was successfully applied to the deprotection of protected phosphopeptide with dimethyl-protected phosphoamino acids. Furthermore, we synthesized phosphatase-resistant phosphoamino acid isosters bearing the substitution of a phosphate oxygen with a difluoromethylene. The syntheses of peptides possessing these nonhydrolyzable phosphoamino acids were achieved utilizing two-step deprotecting methodologies. Additionally, we demonstrated the usefulness of phosphatase-resistant phosphopeptides as biochemical tools for understanding signal transduction.

1H-NMR and circular dichroism spectroscopic studies on changes in secondary structures of the sodium channel inactivation gate peptides as caused by the pentapeptide KIFMK.

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional (1)H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by approximately 11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent ( approximately 6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content ( approximately -4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.

Induction of human leukocyte antigen (HLA)-A2-restricted and MAGE-3-gene-derived peptide-specific cytolytic T lymphocytes using cultured dendritic cells from an HLA-A2 esophageal cancer patient.

BACKGROUND AND OBJECTIVES: Using peripheral blood mononuclear cells (PBMCs) from a 10-year survivor with established human leukocyte antigen (HLA)-A2(+) and MAGE-3(+) esophageal cancer cell line (KYSE-170), we examined the induction of HLA-A2-restricted and MAGE-3-gene-derived peptide (FLWGPRALV, amino acids 271-279)-specific cytolytic T lymphocytes (CTLs). METHODS: Autologous dendritic cells (DCs) cultured with granulocyte-macrophage colony stimulating factor and interleukin-4 were used as antigen presenting cells. PBMCs were stimulated by peptide-pulsed DCs in vitro. RESULTS: PBMC cocultured with FLWGPRALV-pulsed DCs could induce the relevant peptide-specific CTLs, which had tumor necrosis factor production and specific cytotoxicity against relevant peptide-pulsed autologous DCs (34%, effector:target ratio = 40:1). Moreover, they showed specific cytotoxicity against the autologous esophageal cancer cell line KYSE-170 (17%, effector:target ratio = 40:1). CONCLUSIONS: These results suggest that FLWGPRALV-pulsed cultured DCs would be a potent candidate for peptide vaccine against HLA-A2(+) and MAGE-3(+) esophageal cancer.

Cell binding sequences in mouse laminin alpha1 chain.

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.

Pharmacophore identification of a chemokine receptor (CXCR4) antagonist, T22 ([Tyr(5,12),Lys7]-polyphemusin II), which specifically blocks T cell-line-tropic HIV-1 infection.

We have previously found that T22 ([Tyr(5,12), Lys7]-polyphemusin II) has strong anti-human immunodeficiency virus (HIV) activity, and that T22 inhibits T cell-line-tropic HIV-1 infection mediated by CXCR4/fusin. T22 is an 18-residue peptide amide, which takes an antiparallel beta-sheet structure that is maintained by two disulfide bridges. Structure-activity relationship (SAR) studies on T22 have disclosed the contributions of each region of T22 to activity or cytotoxicity, and have provided the following useful information to develop new CXCR4 antagonists: The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related to anti-HIV activity. Addition of a variety of functional groups at the N-terminal end results in increases in activity. Disulfide rings, especially the major disulfide loop, are indispensable for anti-HIV activity and maintenance of the beta-sheet structure. Trp3 can be replaced by other aromatic residues (Tyr, Phe and L-2-naphthylalanine). Between two repeats of Tyr-Arg-Lys, which are a characteristic structure in T22, Tyr-Arg-Lys in the N-terminal portion is more closely associated with anti-HIV activity and maintenance of the beta-sheet structure. A positive charge in the side chain at the (i + 1) position of the beta-turn region is necessary for strong activity. Through these studies, we have found several compounds having higher selectivity indexes (50% cytotoxic concentration/50% effective concentration) than that of T22.

Downsizing of an HIV-cell fusion inhibitor, T22 ([Tyr5,12, Lys7]-polyphemusin II), with the maintenance of anti-HIV activity and solution structure.

T22 ([Tyr5,12,Lys7]-polyphemusin II) has been shown to have strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2',3'-dideoxythymidine (AZT). T22, an 18-residue peptide amide, takes an antiparallel beta-sheet structure that is maintained by two disulfide bridges. Herein we synthesized several shortened analogs of T22 in order to search for a more suitable lead compound. A 14-residue analog having one disulfide bridge, TW70 (des-[Cys8,13, Tyr9,12]-[D-Lys10, Pro11]-T22), was found to have highly potent activity comparable to that of T22, and to take an antiparallel beta-sheet structure similar to that of T22. This indicates that the molecular size of T22 can be reduced without loss of activity or significant change in the secondary structure, and that TW70 may represent a novel lead compound. Furthermore, modifying the N-terminal alpha-amino group of TW70 with a fluoresceinthiocarbamoyl group, and the epsilon-amino group of D-Lys8 at the turn portion with a 5-aminopentanoyl group remarkably increased the selectivity index (50% cytotoxic concentration/50% effective concentration).