Vaginal film for prevention of HIV: using visual and tactile evaluations among potential users to inform product design.

Topical prevention of HIV and other STIs is a global health priority. To provide options for users, developers have worked to design safe, effective and acceptable vaginal dissolving film formulations. We aimed to characterize user experiences of vaginal film size, texture and color, and their role in product-elicited sensory perceptions (i.e. perceptibility), acceptability and willingness to use. In the context of a user-centered product evaluation study, we elicited users' 'first impressions' of various vaginal film formulation designs via visual and tactile prototype inspection during a qualitative user evaluation interview. Twenty-four women evaluated prototypes. Participants considered size and texture to be important for easy insertion. Color was more important following dissolution than prior to insertion. When asked to combine and balance all properties to arrive at an ideal film, previously stated priorities for individual characteristics sometimes shifted, with the salience of some individual characteristics lessening when multiple characteristics were weighted in combination. While first impressions alone may not drive product uptake, users' willingness to initially try a product is likely impacted by such impressions. Developers should consider potential users' experiences and preferences in vaginal film design. This user-focused approach is useful for characterizing user sensory perceptions and experiences relevant to early design of prevention technologies.

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein.

Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents. toward that end, we have identified a number of antiviral chemotypes that electrophilically attack the sulfur atoms of the zinc-coordinating cysteine residues of the domains. Such nucleocapside inhibitors were directly virucidal by preventing the initiation of reverse transcription and blocked formation of infectious virus from cells through modification of CCHC domains within Gag precursors. Herein we report that azodicarbonamide (ADA) represents a new compound that inhibits HIV-1 and a broad range of retroviruses by targeting the the nucleocapsid CCHC domains. Vandevelde et al. also recently disclosed that ADA inhibits HIV-1 infection via an unidentified mechanism and that ADA was introduced into Phase I/II clinical trials in Europe for advanced AIDS. These studies distinguish ADA as the first known nucleocapsid inhibitor to progress to human trials and provide a lead compound for drug optimization.

Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS.

Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.

Preclinical evaluation of a mercaptobenzamide and its prodrug for NCp7-targeted inhibition of human immunodeficiency virus.

Although the effective use of highly active antiretroviral therapy results in the suppression of virus production in infected individuals, it does not eliminate the infection and low level virus production in cells harboring virus in sanctuary sites. Thus, the continued search for new antiretroviral agents with unique and different mechanisms of HIV inhibition remains critical, and compounds that can reduce the level of virus production from cells already infected with HIV, as opposed to preventing de novo infection, would be of great benefit. A mercaptobenzamide (MDH-1-38) and its prodrug (NS1040) are being developed as potential therapeutic compounds targeting the zinc finger of HIV nucleocapsid. In the presence of esterase enzymes, NS1040 is designed to be converted to MDH-1-38 which has antiviral activity. While we presume that NS1040 is rapidly converted to MDH-1-38 in all experiments, the two compounds were tested side-by-side to determine whether the presence of a prodrug affects the antiviral activity or mechanism of action. The two compounds were evaluated against a panel of HIV-1 clinical isolates in human PBMCs and monocyte-macrophages and yielded EC50 values ranging from 0.7 to 13 µM with no toxicity up to 100 µM. MDH-1-38 and NS1040 remained equally active in human PBMCs in the presence of added serum proteins as well as against HIV-1 isolates resistant to reverse transcriptase, integrase or protease inhibitors. Cell-based and biochemical mechanism of antiviral action assays demonstrated MDH-1-38 and NS1040 were virucidal at concentrations of 15 and 50 µM, respectively. Cell to cell transmission of HIV in multiple passages was significantly reduced in CEM-SS and human PBMCs by reducing progeny virus infectivity at compound concentrations greater than 2 µM. The combination of either MDH-1-38 or NS1040 with other FDA-approved HIV drugs yielded additive to synergistic antiviral interactions with no evidence of antiviral antagonism or synergistic toxicity. Serial dose escalation was used in attempts to select for HIV strains resistant to MDH-1-38 and NS1040. Virus at several passages failed to replicate in cells treated at increased compound concentrations, which is consistent with the proposed mechanism of action of the virus inactivating compounds. Through 14 passages, resistance to the compounds has not been achieved. Most HIV inhibitors with mechanism of antiviral action targeting a viral protein would have selected for a drug resistant virus within 14 passages. These studies indicate that these NCp7-targeted compounds represent new potent anti-HIV drug candidates which could be effectively used in combination with all approved anti-HIV drugs.

Cross-resistance analysis and molecular modeling of nonnucleoside reverse transcriptase inhibitors targeting drug-resistance mutations in the reverse transcriptase of human immunodeficiency virus.

Oxathlin carboxanilide analogs (UC) and alpha APA, compounds recognized as nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI), were evaluated for activity against the human immunodeficiency virus (HIV-1) and drug-resistant variants. These NNRTIs are structurally diverse but potent inhibitors of HIV-1 with efficacy in the nanomolar to low micromolar concentrations. They interact at a specific site in the pain domain of the p66 subunit of RT. Treatment of HIV-1 infected cell cultures with UC compounds resulted in the selection of drug-resistant viruses bearing specific amino acid changes at 100, 101, 103, 106, and/or 181. Since Y181C and L1001 are the most commonly observed resistance-engendering mutations, RT enzymatic analysis was correlated with molecular modeling to glean information on the structural interactions between these NNRTIs and RT. Information derived from these studies will facilitate rational drug design and the selection of complementary anti-HIV drugs for combination therapy.

Characteristics of a group of nonnucleoside reverse transcriptase inhibitors with structural diversity and potent anti-human immunodeficiency virus activity.

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

Isolation and characterization of adociavirin, a novel HIV-inhibitory protein from the sponge Adocia sp.

Aqueous extracts of the New Zealand sponge Adocia sp. (Haplosclerida) displayed potent anticytopathic activity in CEM-SS cells infected with HIV-1. Protein fractions of the extract bound both to the viral coat protein gp120 and to the cellular receptor CD4, but not to other tested proteins. The purified active protein, named adociavirin, was characterized by isoelectric focusing, amino acid analysis, MALDI-TOF mass spectrometry and N-terminal sequencing. Adociavirin, a disulfide-linked homodimer with a native molecular weight of 37 kDa, was active against diverse strains and isolates of HIV-1, as well as HIV-2, with EC50 values ranging from 0.4 nM to > 400 nM. The anti-HIV potency of adociavirin appears dependent on host cell type, with macrophage cultures being the most sensitive and peripheral blood lymphocytes the most resistant.

Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.

Unusual single-stranded polyribonucleotides as potent anti-HIV agents.

Polyribonucleotides (PTMG and PMTI) containing 1-methyl-6-thioguanosine or 1-methyl-6-thioinosine, respectively, as the sole nucleoside component are shown to be potent inhibitors of various strains of HIV-1 and HIV-2 in a number of human lymphocyte and macrophage cell lines in tissue culture as well as in fresh human peripheral blood lymphocytes and macrophages. PMTI and PMTG exhibit potencies in the range of 10(-7)-10(-8) M in these systems. The polynucleotides are active against virus strains resistant to AZT and pyridinone derivatives. Both PMTI and PMTG are synergistic with AZT and with ddI, and both inhibit HIV reverse transcriptase at nanomolar concentrations. The polymers show little or no toxicity in human cell lines at the highest doses tested (100 micrograms/mL, or about 0.2-1 microM). This class of compounds represents a new lead in AIDS therapeutic drug discovery.

Antiviral oligo- and polyribonucleotides containing selected triazolo[2,3-a]purines.

Several amphipathic (hydrophobic base, hydrophilic backbone) polyribonucleotides have recently been shown to have potent antiviral activity against HIV and human cytomegalovirus (HCMV). The working hypothesis developed during these studies was that the ability to form an ordered, non-hydrogen-bonded array in solution was an important criterion for activity. To explore further the role of structure and molecular size on the inhibition of virus replication, one new polynucleotide and two 32-mer oligonucleotides based on the triazolo[2,3-a]purine ring system have now been prepared. High-molecular-weight polynucleotide 4a (PTPR) and sulfur-containing 32-mer 5b (TTPR) were moderately active against HIV but showed greater potency against HDMV than ganciclovir. Both 4a and 5b gave clear evidence of cooperative melting behavior, whereas inactive 32-mer 5a showed no such behavior.

Synthesis and biological activity of novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 2-Aryl-substituted benzimidazoles.

The development of new nonnucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) active against the drug-induced mutations in RT continues to be a very important goal of AIDS research. We used a known inhibitor of HIV-1 RT, 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-alpha]benzimidazole (TZB), as the lead structure for drug design with the objective of making more potent inhibitors against both wild-type (WT) and variant RTs. A series of structurally related 1,2-substituted benzimidazoles was synthesized and evaluated for their ability to inhibit in vitro polymerization by HIV-1 WT RT. A structure-activity study was carried out for the series of compounds to determine the optimum groups for substitution of the benzimidazole ring at the N1 and C2 positions. The best inhibitor, 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimida zole (35), has an IC50 = 200 nM against HIV-1 WT RT in an in vitro enzyme assay. Cytoprotection assays utilizing HIV-infected MT-4 cells revealed that 35 had strong antiviral activity (EC50 = 440 nM) against wild-type virus while retaining broad activity against many clinically observed HIV-1 strains resistant to nonnucleoside inhibitors. Overall, the activity of 35 against wild-type and resistant strains with amino acid substitution in RT is 4-fold or greater than that of TZB and is comparable to that of other nonnucleoside inhibitors currently undergoing clinical trials, most of which do not have the capacity to inhibit the variant forms of the enzyme.

Structural analogues of the calanolide anti-HIV agents. Modification of the trans-10,11-dimethyldihydropyran-12-ol ring (ring C).

(+)-Calanolide A is a potent inhibitor of reverse transcriptase from human immunodeficiency virus type 1 (HIV-1), which was isolated from an extract of Calophyllum lanigerum, along with seven related compounds. In order to examine the structure-activity relationships of the trans-10,11-dimethyldihydropyran-12-ol ring (designated ring C), a series of structural analogues were prepared and evaluated using a whole cell cytopathicity assay (XTT). Removal of the 10-methyl group resulted in decreased activity, with only one epimer exhibiting anti-HIV activity. Substituting the 10-methyl group with an ethyl chain maintained anti-HIV activity, with only a 4-fold reduction in potency relative to racemic calanolide A. Substitution of the 10-methyl group with an isopropyl moiety completely eliminated the anti-HIV activity. Addition of an extra methyl group at either the 10- or 11-position maintained the basic stereochemical features of the parent calanolide system while removing the chirality at the respective carbon, but resulted in decreased activity relative to calanolide A. In all the above examples, analogues containing a cis relationship between the 10- and 11-alkyl moieties were completely devoid of activity. Synthetic intermediates in which the 12-hydroxyl group was in the ketone oxidation state exhibited suppressing anti-HIV activity, with EC50 values only 5-fold less potent than that of calanolide A for both the 10,11-cis (6) and -trans (5) series. These ketones represent the first derivatives in the calanolide series to exhibit anti-HIV activity while not containing a 12-hydroxyl group. Likewise, ketone derivative 6 was the first example of a compound in the calanolide series having a cis relationship between the 10- and 11-methyl groups found to exhibit anti-HIV activity. Analogues which showed anti-HIV activity in the CEM-SS cytoprotection assay were further confirmed to be inhibitors of HIV-1 reverse transcriptase.

Synthesis and biological evaluation of certain alkenyldiarylmethanes as anti-HIV-1 agents which act as non-nucleoside reverse transcriptase inhibitors.

Several novel alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized. The most potent of these proved to be 3',3"-dibromo-4',4"-dimethoxy-5'5"-bis(methoxycarbonyl)-1,1-diphenyl-1-+ ++heptene (8) ADAM 8 inhibited the cytopathic effect of HIV-1 in CEM cell culture with an EC50 value of 7.1 microM and was active against an array of laboratory strains of HIV-1 in CEM-SS and MT-4 cells, but was inactive as an inhibitor of HIV-2. In common with the other known non-nucleoside reverse transcriptase inhibitors, ADAM 8 was an effective inhibitor of HIV-1 reverse transcriptase (IC50 1 microM) with poly(rC).oligo(dG), but not with poly(rA).oligo(dT), as the template/primer. ADAM 8 was inactive against HIV-1 reverse transcriptases containing non-nucleoside reverse transcriptase inhibitor resistance mutations at residues 101, 106, 108, 139, 181, 188, and 236, while it remained active against enzymes with mutations at residues 74, 98, 100, 103, and at 103/181. An AZT-resistant virus having four mutations in reverse transcriptase was more sensitive to inhibition by ADAM 8 than the wild-type HIV-1. In addition, ADAM 8 displayed synergistic activity with AZT, but lacked synergy with ddI. ADAM 8 or a structurally related analog may therefore be useful as an antiviral agent in combination with AZT or with other NNRTIs that are made ineffective by mutations at residues which do not confer resistance to ADAM 8.

New alkenyldiarylmethanes with enhanced potencies as anti-HIV agents which act as non-nucleoside reverse transcriptase inhibitors.

Twenty-two new alkenyldiarylmethanes (ADAMs) were synthesized and evaluated for inhibition of HIV-1 replication. The most potent compound proved to be methyl 3',3"-dichloro-4',4"-dimethoxy-5', 5"-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (ADAM II), which displayed an EC50 of 13 nM for inhibition of the cytopathic effect of HIV-1RF in CEM-SS cells. ADAM II inhibited HIV-1 reverse transcriptase with an IC50 of 0.3 microM but was inactive as an inhibitor of HIV-1 attachment/fusion to cells, protease, integrase, and the nucleocapsid protein. Molecular target-based and cell-based assays revealed that ADAM II acted biologically as a nonnucleoside reverse transcriptase inhibitor (NNRTI). ADAM II inhibited replication of a wide variety of laboratory, clinical, and clade-representative isolates of HIV-1 in T cell lines and cultures of peripheral blood mononuclear cells or monocyte/macrophages. Mutations that conferred resistance to ADAM II clustered at residues 101, 103, 108, 139, 179, 181, and 188, which line the nonnucleoside binding pocket of HIV-1 reverse transcriptase. However, HIV-1 NL4-3 strain expressing a mutation at residue 100 of reverse transcriptase, and an AZT-resistant virus, displayed increased sensitivity to ADAM II. Thus, ADAM II could serve as an adjunct therapy to AZT and NNRTIs that select for L100I resistance mutations.

5,5-disubstituted hydantoins: syntheses and anti-HIV activity.

A series of 5,5-disubstituted hydantoin derivatives was synthesized by alkylating 5,5-bis(mercaptomethyl)-2,4-imidazolidinedione (3) with various halomethylaromatic or halomethylheteroaromatic precursors, or by using the Buchener-Berg procedure on the required ketone. When evaluated for their ability to inhibit HIV-induced cell killing and virus production in CEM or MT-2 cells only compounds 2, 4n, 4o, and 4i demonstrated modest activity, the latter with an IC50 = 53 microM.

Correlation of anti-HIV activity with anion spacing in a series of cosalane analogues with extended polycarboxylate pharmacophores.

Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed to involve ionic interactions of the negatively charged carboxylates of the ligands with positively charged arginine and lysine amino acid side chains of the protein. To investigate the effect of anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates was synthesized in which the two proximal and two distal carboxylates are separated by 6--12 atoms. Maximum activity was observed when the proximal and distal carboxylates are separated by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine, aspartic acid, beta-alanine, leucine, and phenylalanine residues, maximum activity was displayed by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are more potent against HIV-1(RF) in CEM-SS cells than they are vs HIV-1(IIIB) in MT-4 cells, and they are least potent vs HIV-2(ROD) in MT-4 cells.

The biological effects of structural variation at the meta position of the aromatic rings and at the end of the alkenyl chain in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors.

In an effort to elucidate a set of structure-activity relationships in the alkenyldiarylmethane (ADAM) series of non-nucleoside reverse transcriptase inhibitors, a number of modifications were made at two locations: (1) the meta positions of the two aromatic rings and (2) the end of the alkenyl chain. Forty-two new ADAMs were synthesized and evaluated for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cell culture and for inhibition of HIV-1 reverse transcriptase. The size of the aromatic substituents was found to affect anti-HIV activity, with optimal activity appearing with Cl, CH(3), and Br substituents and with diminished activity occurring with smaller (H and F) or larger (I and CF(3)) substituents. The substituents at the end of the alkenyl chain were also found to influence the antiviral activity, with maximal activity associated with methyl or ethyl ester groups and with diminished activity resulting from substitution with higher esters, amides, sulfides, sulfoxides, sulfones, thioesters, acetals, ketones, carbamates, ureas, and thioureas. Twelve of the new ADAMs displayed submicromolar EC(50) values for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cells. Selected ADAMs, 19 and 21, were compared to previously published ADAMs 15 and 17 for antiviral efficacy and activity against the HIV-1 reverse transcriptase enzyme. All four ADAMs were found to inhibit HIV-1 reverse transcriptase enzyme activity, to inhibit the replication of a variety of HIV-1 clinical isolates representing syncytium-inducing, nonsyncytium-inducing, and subtype representative isolates, and to inhibit HIV-1 replication in monocytes. Subsequent assessment against a panel of site-directed reverse transcriptase mutants in NL4-3 demonstrated no effect of the K103N mutation on antiviral efficacy and a slight enhancement (6- to 11-fold) in sensitivity to AZT-resistant viruses. Additionally, ADAMs 19 (44-fold) and 21 (29-fold) were more effective against the A98G mutation (found in association with nevirapine resistance in vitro), and ADAM 21 was 5-fold and 2-fold more potent against the Y181C inactivation mutation than the previously reported ADAMs 15 and 17, respectively. All four ADAMs were tested for efficacy against a multidrug-resistant virus derived from a highly experienced patient expressing resistance to the reverse transcriptase enzyme inhibitors AZT, ddI, 3TC, d4T, foscarnet, and nevirapine, as well as the protease inhibitors indinavir, saquinavir, and nelfinavir. ADAM 21 was 2-fold more potent than ADAM 15 and 6-fold more potent than ADAMs 17 and 19 at preventing virus replication. Thus, we have identified a novel series of reverse transcriptase inhibitors with a favorable profile of antiviral activity against the primary mutation involved in clinical failure of non-nucleoside reverse transcriptase inhibitors, K103N, and that retain activity against a multidrug-resistant virus.

Novel modifications in the alkenyldiarylmethane (ADAM) series of non-nucleoside reverse transcriptase inhibitors.

In an effort to obtain more insight into the interaction between HIV-1 reverse transcriptase and the alkenyldiarylmethanes (ADAMs), a new series of compounds has been synthesized and evaluated for inhibition of HIV-1 replication. The modifications reported in this new series include primarily changes to the alkenyl chain. The most potent compound proved to be methyl 3',3' '-dibromo-4',4' '-dimethoxy-5',5' '-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (28), which displayed an EC(50) of 1.3 nM for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cells. ADAM 28 inhibited HIV-1 reverse transcriptase with an IC(50) of 0.3 microM. Mutations that conferred greater than 10-fold resistance to ADAM 28 clustered at residues Val 106, Val 179, Tyr 181, and Tyr 188. Results derived from this series indicate that ADAMs containing chlorines in the aromatic rings might bind to HIV-1 reverse transcriptase in a slightly different mode when compared with those analogues incorporating bromine in the aromatic rings.

The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum.

Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.

Inhibition of in vitro and in vivo HIV replication by a distamycin analogue that interferes with chemokine receptor function: a candidate for chemotherapeutic and microbicidal application.

Select chemokine receptors act as coreceptors for HIV-1 entry into human cells and represent targets for antiviral therapy. In this report we describe a distamycin analogue, 2,2'-[4, 4'-[[aminocarbonyl]amino]bis[N,4'-di[pryrrole-2-carboxamide- 1, 1'-dimethyl]]-6,8-naphthalenedisulfonic acid]hexasodium salt (NSC 651016), that selectively inhibited chemokine binding to CCR5, CCR3, CCR1, and CXCR4, but not to CXCR2 or CCR2b, and blocked chemokine-induced calcium flux. Inhibition was not due to nonspecific charge interactions at the cell surface, but was based on a specific competition for the ligand receptor interaction sites since the inhibitory effect was specific for some but not all chemoattractant receptors. NSC 651016 inhibited in vitro replication of a wide range of HIV-1 isolates, as well as HIV-2 and SIV, and exhibited in vivo anti-HIV-1 activity in a murine model. In contrast, a distamycin analogue with similar structure and charge and the monomeric form of NSC 651016 demonstrated no inhibitory effects. These data demonstrate that molecules which interfere with HIV-1 entry into cells by targeting specific chemokine coreceptors can provide a viable approach to anti-HIV-1 therapy. NSC 651016 represents an attractive candidate for the chemotherapeutic treatment of HIV-1 infection and as a microbicide to prevent the sexual transmisssion of HIV-1. Moreover, NSC 651016 can serve as a template for medicinal chemical modifications leading to more effective antivirals.