Anti-HIV-1 peptides derived from partial amino acid sequences of CC-Chemokine RANTES

Fifteen acetyl-peptide-amides with partial amino acid sequences of RANTES (regulated upon activation, normal T-cell expressed and secreted), all Cys residues of which were substituted by Ala, were synthesized, and screened for anti-HIV-1 activity. Peptides corresponding to 1–10, 37–46, and 57–68 showed marked activity against CC-chemokine receptor 5-using HIV-1_JR-CSF (% inhibition at 100 nM 69, 82, 76%, respectively). The results indicate that multiple regions, including the N-terminal part responsible for chemotactic activity, are involved in anti-HIV-1 activity of RANTES, yielding possible lead compounds for anti-HIV-1 agents.     

Low-molecular-weight anti-HIV-1 peptides from the amino-terminal sequence of RANTES: possible lead compounds for coreceptor-directed anti-HIV-1 agents.

A series of small peptides corresponding to the amino-terminal sequence of RANTES were synthesized, and their anti-HIV-1 activity was evaluated. Pentapeptides, H-(10Ala-RANTES 6-10)-OH and Ac-(10Ala-RANTES 6-10)-NH2, were the smallest anti-HIV-1 peptides so far developed, and would be potentially important lead compounds for coreceptor-directed anti-HIV-1 drugs.     

The superior folding of a RANTES analogue expressed in lactobacilli as compared to mammalian cells reveals a promising system to screen new RANTES mutants

Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral coreceptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulfide bridge between non-natural cysteine residues at positions 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulfide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity.     

Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.     

Stronger anti-HIV-1 activity of C-peptide derived from HIV-1 89.6 gp41 C-terminal heptad repeated sequence

C34-LAI containing amino acids 118 to 151 of the HIV-1(LAI) gp41 ectodomain exhibits potent anti-HIV-1 activity. However, the N-terminal halves of C34 peptides vary more according to the HIV-1 strain than the C-terminal halves. Therefore, an analysis was conducted on the anti-HIV-1 activities of the C34 peptides derived from various HIV-1 strains. C34-89.6 exhibited the strongest anti-HIV-1 activity among the C34 peptides tested. Interestingly, its N-terminal half was more acidic than those of the other C34 peptides, whereas its C-terminal half was more basic. Since the C-peptides derived from the HIV-1(LAI) strain are used extensively, the anti-HIV-1 activities of these peptides were compared between the HIV-1 strains 89.6 and LAI. When using chimeric peptides, it was found that the C-terminal basic region of C34-89.6 was more critical than its N-terminal basic region. The anti-HIV-1 activity of T20-89.6 and C28-89.6 was also stronger than that of T20-LAI and C28-LAI, respectively. The anti-HIV-1 activity of C28-89.6 was weakened when the C-terminal basic residues were changed to the corresponding residues of C28-LAI. However, no conformational differences were found among the C28 peptides. Accordingly, these results imply that introducing the C-terminal basic residues of the HIV-1 89.6 C-peptide may be useful for developing potent anti-HIV-1 drugs.     

Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1α (MIP-1α), MIP-1β, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1α against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4⁺ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1β, MIP-1α. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1β, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1β, MIP-1α.     

Anti-HIV-1 activity of elafin is more potent than its precursor's, trappin-2, in genital epithelial cells

Cervicovaginal lavage fluid (CVL) is a natural source of anti-HIV-1 factors; however, molecular characterization of the anti-HIV-1 activity of CVL remains elusive. In this study, we confirmed that CVLs from HIV-1-resistant (HIV-R) compared to HIV-1-susceptible (HIV-S) commercial sex workers (CSWs) contain significantly larger amounts of serine antiprotease trappin-2 (Tr) and its processed form, elafin (E). We assessed anti-HIV-1 activity of CVLs of CSWs and recombinant E and Tr on genital epithelial cells (ECs) that possess (TZM-bl) or lack (HEC-1A) canonical HIV-1 receptors. Our results showed that immunodepletion of 30% of Tr/E from CVL accounted for up to 60% of total anti-HIV-1 activity of CVL. Knockdown of endogenous Tr/E in HEC-1A cells resulted in significantly increased shedding of infectious R5 and X4 HIV-1. Pretreatment of R5, but not X4 HIV-1, with either Tr or E led to inhibition of HIV-1 infection of TZM-bl cells. Interestingly, when either HIV-1 or cells lacking canonical HIV-1 receptors were pretreated with Tr or E, HIV-1 attachment and transcytosis were significantly reduced, and decreased attachment was not associated with altered expression of syndecan-1 or CXCR4. Determination of 50% inhibitory concentrations (IC(50)) of Tr and E anti-HIV-1 activity indicated that E is ～130 times more potent than its precursor, Tr, despite their equipotent antiprotease activities. This study provides the first experimental evidence that (i) Tr and E are among the principal anti-HIV-1 molecules of CVL; (ii) Tr and E affect cell attachment and transcytosis of HIV-1; (iii) E is more efficient than Tr regarding anti-HIV-1 activity; and (iv) the anti-HIV-1 effect of Tr and E is contextual.     

Synthesis, antitumor and anti-HIV activities of benzodithiazine-dioxides

Several 8-chloro-7-R1-6-R2-3-R3-imidazo[1,2-b][1,4,2]benzodithiazine 5,5-dioxide derivatives (9-11, 16-19, and 21-24) have been synthesized as potential antitumor or anti-HIV agents. The in vitro antitumor and anti-HIV-1 activities of the compounds were determined in a panel of cell lines. The benzodithiazine-dioxide 10 showed 50% growth inhibitory activity in low micromolar against most cells. It was particularly effective in leukemia, lung, melanoma, ovarian, and renal cancer cells with GI50 values of 1-2 microM. Interestingly, benzodithiazine-dioxide 16 showed remarkable anti-HIV-1 activity with 50% effective concentration EC50 value of 0.94 microM and no significant cytotoxicity at 200.0 microM.     

Structural basis for the interaction of CCR5 with a small molecule, functionally selective CCR5 agonist

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.     

Synthesis and antiviral activities of novel gossypol derivatives

In this study, a series of novel gossypol derivatives were synthesized and screened in vitro for their anti-HIV-1 and anti-H(5)N(1) activities, respectively. Replacing the aldehyde groups of gossypol with some amino acids not only reduced the cytotoxicity but also enhanced the activities against HIV-1 and H(5)N(1). Compounds 13-17 showed more potent activities against HIV-1 and H(5)N(1) than the other gossypol derivatives. Meanwhile, these compounds also exhibited more potent activities against H(5)N(1) than 1-adamantylamine. The absence of the COONa group in gossypol derivatives resulted in a loss of anti-HIV-1 activity, suggesting that this group might play an important role in mediating the antiviral activity. Time-of-addition assays indicated that compounds 13-17 had the similar mechanism of anti-HIV-1 action with T20. Molecular modeling analysis demonstrated that compounds 13-17 could fit inside the gp41 hydrophobic pocket through hydrogen bonding network, hydrophobic contacts and strong electrostatic interactions.     

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

A simple, rapid, and sensitive system for the evaluation of anti-viral drugs in rats

The lack of small animal models for the evaluation of anti-human immunodeficiency virus type 1 (HIV-1) agents hampers drug development. Here, we describe the establishment of a simple and rapid evaluation system in a rat model without animal infection facilities. After intraperitoneal administration of test drugs to rats, antiviral activity in the sera was examined by the MAGI assay. Recently developed inhibitors for HIV-1 entry, two CXCR4 antagonists, TF14016 and FC131, and four fusion inhibitors, T-20, T-20EK, SC29EK, and TRI-1144, were evaluated using HIV-1(IIIB) and HIV-1(BaL) as representative CXCR4- and CCR5-tropic HIV-1 strains, respectively. CXCR4 antagonists were shown to only possess anti-HIV-1(IIIB) activity, whereas fusion inhibitors showed both anti-HIV-1(IIIB) and anti-HIV-1(BaL) activities in rat sera. These results indicate that test drugs were successfully processed into the rat sera and could be detected by the MAGI assay. In this system, TRI-1144 showed the most potent and sustained antiviral activity. Sera from animals not administered drugs showed substantial anti-HIV-1 activity, indicating that relatively high dose or activity of the test drugs might be needed. In conclusion, the novel rat system established here, "phenotypic drug evaluation", may be applicable for the evaluation of various antiviral drugs in vivo.     

Enhanced anti-HIV-1 activity of CC-chemokine LD78beta, a non-allelic variant of MIP-1alpha/LD78alpha.

We compared the anti-HIV-1 activity of CC-chemokine LD78beta with that of MIP-1alpha, another CC-chemokine which shows 94% sequence homology with LD78beta. Despite its close similarity to MIP-1alpha, the anti-HIV-1 activity of LD78beta appeared to be nearly 10 times higher than that of MIP-1alpha. Mutagenesis of MIP-1alpha showed that the N-terminal additional tetrapeptide, which was present in LD78beta and absent in MIP-1alpha, is responsible for enhanced anti-HIV-1 activity. The N-terminal structure-function relationship of LD78beta described here will be of value in understanding the chemokine-receptor interactions and designing anti-HIV-1 compounds based on LD78beta.     

A beta-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity

A 30 kDa beta-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC(50) values were 0.0043 and 0.057 microM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC(50) of 0.073 microM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 microM and 0.07 microM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.     

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

Synthesis and antiviral activity of novel fluorinated 2',3'-dideoxynucleosides

A series of 5-(trifluoroethoxymethyl)-2',3'-dideoxyuridines and 5-[bis(trifluoroethoxy)-methyl]-2',3'-dideoxyuridines have been prepared and screened for antiviral activity. The conformations of these compounds are discussed on the bases of NOE studies and the MO calculations. Modelling and NOE studies suggest both syn- and anti conformations for these 5-(2,2,2-trifluoroethoxymethyl)- and 5-[bis(2,2,2-trifluoroethoxy)-methyl]- derivatives. The NOE parameters are also suggested to be more attributable to the nature of the fluorine atom than to structural or conformational changes. Compounds 17, 26 and 30 showed some activity in anti-HIV-1 and anti-HIV-2 assays, but the compounds were devoid of activity against HSV and human rhinovirus. The compounds tested exhibited low cytotoxicity and were inactive against a bank of cancer cells in vitro.     

Biologically active oligodeoxyribonucleotides. Part 11: The least phosphate-modification of quadruplex-forming hexadeoxyribonucleotide TGGGAG, bearing 3′- and 5′-end-modification, with anti-HIV-1 activity

We have found that a hexadeoxyribonucleotide (5′TGGGAG3′, R-95288), Koizumi, M. et al. Bioorganic & Medicinal Chemistry, 1997, 5, 2235, bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end and a 2-hydroxyethylphosphate at the 3′-end, has high anti-HIV-1 activity and the least cytotoxicity in vitro and in vivo. In order to synthesize more potent hexadeoxyribonucleotides, we substituted phosphodiester (P---O) bonds in the 6-mer with the least phosphorothioate (P---S), phosphoramidate (P---N), or methylphosphonate (P---Me) bonds. When more than two P---N or P---Me bonds were introduced into a 6-mer, the phosphate-modified 6-mers had weak or no anti-HIV-1 activity, in spite of quadruplex structure formation. However, when P---S bonds were substituted for P---O bonds, anti-HIV-1 activity of their 6-mers did not dramatically decrease, compared with compounds substituted with P---N or P---Me bonds. The results suggest that the formation of a quadruplex structure is not always sufficient for anti-HIV-1 activity of the 6-mer, and that net negative charges derived from P---O or P---S bonds in the quadruplex are important for anti-HIV-1 activity. Moreover, among various phosphate-modified ODNs, we found that the anti-HIV-1 activity of ODN PS7 with only one P---S bond was the same as that of R-95288, both having a high stability in human plasma.