Effects of gel volume on pharmacokinetics for vaginal and rectal applications of combination DuoGel-IQB4012, a dual chamber-dual drug HIV microbicide gel, in pigtailed macaques.

This study evaluated effects of differing gel volumes on pharmacokinetics (PK). IQB4012, a gel containing the non-nucleoside reverse transcriptase inhibitor IQP-0528 and tenofovir (TFV), was applied to the pigtailed macaque vagina and rectum. Vaginal gel volumes (1% loading of both drugs) were 0.5 or 1.5 ml; following wash-out, 1 or 4 ml of gel were then applied rectally. Blood, vaginal, and rectal fluids were collected at 0, 2, 4, and 24 h. Vaginal and rectal tissue biopsies were collected at 4 and 24 h. There were no statistically significant differences in concentrations for either drug between gel volumes within compartments at matched time points. After vaginal gel application, median IQP-0528 concentrations were ~ 104-105 ng/g, 105-106 ng/ml, and 103-105 ng/ml in vaginal tissues, vaginal fluids, and rectal fluids, respectively (over 24 h). Median vaginal TFV concentrations were 1-2 logs lower than IQP-0528 levels at matched time points. After rectal gel application, median IQP-0528 and TFV concentrations in rectal fluids were ~ 103-105 ng/ml and ~ 102-103 ng/ml, respectively. Concentrations of both drugs sampled in rectal tissues were low (~ 101-103 ng/g). For 1 ml gel, half of sampled rectal tissues had undetectable concentrations of either drug, and over half of sampled rectal fluids had undetectable TFV concentrations. These results indicate differences in drug delivery between the vaginal and rectal compartments, and that smaller vaginal gel volumes may not significantly compromise microbicide PK and prophylactic potential. However, effects of rectal gel volume on PK for both drugs were less definitive.

Expanding the Antiviral Spectrum of 3-Fluoro-2-(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates: Diamyl Aspartate Amidate Prodrugs.

Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein†7.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC50 values ranging from 1 to 100 µm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure-activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity.

Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9-[(2-Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

Current Concepts for the IND-Directed Development of Microbicide Products to Prevent the Sexual Transmission of HIV.

In the absence of an approved and effective vaccine, topical microbicides have become the strategy of choice to provide women with the ability to prevent the sexual transmission of HIV. Topical microbicides are chemical and physical agents specifically developed and formulated for use in either the vaginal or rectal environment to prevent the sexual transmission of infectious organisms. Although a microbicide product will have many of the same properties as other anti-infective therapeutic agents, the microbicide development pathway has significant differences which reflect the complex biological environment in which the products must act. These challenges to the development of an effective microbicide are reflected in the recently released FDA Guidance document which defines the microbicide development algorithm and includes the evaluation of preclinical efficacy and toxicity, and safety and toxicology, and indicates the necessity of testing of the active pharmaceutical product as well as an optimal formulation for delivery of the microbicide product. The microbicide development algorithm requires evaluation of the potential microbicidal agent and final formulated product in assays which mimic the microenvironment of the vagina and rectum during the sexual transmission of HIV, including the evaluation of activity and cytotoxicity in the appropriate biological matrices, toxicity testing against normal vaginal flora and at standard vaginal pH, testing in ectocervical and colorectal explant tissue, and irritation studies to vaginal, rectal and penile tissue. Herein, we discuss currently accepted practices required for the development of a successful microbicide product which will prevent virus transmission in the vaginal and rectal vaults.

The rational design and development of a dual chamber vaginal/rectal microbicide gel formulation for HIV prevention.

The DuoGel™ was developed for safe and effective dual chamber administration of antiretroviral drugs to reduce the high incidence of HIV transmission during receptive vaginal and anal intercourse. The DuoGel™s containing IQP-0528, a non-nucleoside reverse transcriptase inhibitor (NNRTI), were formulated from GRAS excipients approved for vaginal and rectal administration. The DuoGel™s were evaluated based upon quantitative physicochemical and biological evaluations defined by a Target Product Profile (TPP) acceptable for vaginal and rectal application. From the two primary TPP characteristics defined to accommodate safe rectal administration three DuoGel™ formulations (IQB3000, IQB3001, and IQB3002) were developed at pH 6.00 and osmolality ⩽400mmol/kg. The DuoGel™s displayed no in vitro cellular or bacterial toxicity and no loss in viability in ectocervical and colorectal tissue. IQB3000 was removed from consideration due to reduced NNRTI delivery (∼65% reduction) and IQB3001 was removed due to increase spread resulting in leakage. IQB3002 containing IQP-0528 was defined as our lead DuoGel™ formulation, possessing potent activity against HIV-1 (EC50=10nM). Over 12month stability evaluations, IQB3002 maintained formulation stability. This study has identified a lead DuoGel™ formulation that will safely deliver IQP-0528 to prevent sexual HIV-1 transmission in the vagina and rectum.

Comparative analysis of the capacity of elite suppressor CD4+ and CD8+ T cells to inhibit HIV-1 replication in monocyte-derived macrophages.

UNLABELLED: Elite controllers or suppressors (ESs) are HIV-1-infected individuals who are able to maintain viral loads below the limit of detection of clinical assays without antiretroviral therapy. The mechanisms of virologic control are not fully understood, but ESs have been shown to have a more effective CD8+ T cell response to infected CD4+ T cells than chronic progressors (CPs). While macrophages are another cell type productively infected by HIV-1, few studies have examined the ability of primary effector T cells to suppress HIV-1 replication in these target cells. Here, we compared the ability of unstimulated primary CD4+ and CD8+ effector T cells to suppress viral replication in monocyte-derived macrophages (MDMs) in ESs and CPs. While CD4+ effector T cells were capable of inhibiting viral replication in MDMs, the magnitude of this response was not significantly different between ESs and CPs. In contrast, the CD8+ T cells from ESs were significantly more effective than those from CPs at inhibiting viral replication in MDMs. The CD4+ T cell response was partially mediated by soluble factors, while the CD8+ T cell response required cell-to-cell interaction. Our results suggest that the individual contributions of various effector cells should be considered in rational vaccine design and in ongoing eradication efforts. IMPORTANCE: Elite suppressors are individuals capable of maintaining low-level viremia in HIV-1 infection without antiretroviral drugs. Their T cell responses have been implicated in eliminating infected CD4+ T cells, and as such, elite suppressors may represent a model of a functional cure of HIV-1 infection. Here, we sought to determine whether the suppressive T cell responses against infected CD4+ T cells also apply to infected macrophages by comparing the responses of elite suppressors and HIV-1-positive individuals on highly active antiretroviral therapy (HAART). Our results show that the CD8+ cells but not CD4+ T cells from elite suppressors have a response against infected macrophages superior to the response of CD8+ cells from patients on HAART. Our results suggest that the induction of a CD8+ T cell response effective against infected macrophages is an outcome to consider in rational vaccine design.

HIV and HCV activate the inflammasome in monocytes and macrophages via endosomal Toll-like receptors without induction of type 1 interferon.

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-ß, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1ß. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.

Novel preclinical models of topical PrEP pharmacodynamics provide rationale for combination of drugs with complementary properties.

BACKGROUND: The limited success of recent HIV topical pre-exposure prophylaxis clinical trials highlights the need for more predictive models of drug efficacy that better simulate what may happen during sexual exposure. To address this gap, we developed complementary in vitro models to evaluate the ability of drugs to retain anti-HIV activity if cells were washed with seminal plasma (simulating what may happen following exposure to ejaculate), and to protect drug-naive T cells (representing newly recruited immune cells) co-cultured with explants that had been pretreated with drug. We focused on tenofovir disoproxil fumarate (TDF), the non-nucleoside reverse transcriptase inhibitors dapivirine (DPV) and IQP-0528, and the entry inhibitors maraviroc (MVC) and the D-peptide chol-PIE-12 trimer (PIE12). Studies were extended to macaques and the ability of cervical biopsies obtained from animals treated with an intravaginal ring formulation of IQP-0528 to protect ex vivo co-cultured T cells was determined. The antiviral activity of cervicovaginal lavage samples against a primary Clade C isolate was also measured and correlated with drug levels. RESULTS: Cells exposed to TDF were equally protected from HIV whether or not the drug-treated cells were washed with medium or seminal plasma prior to challenge. In contrast, several-fold higher concentrations of NNRTIs and entry inhibitors were needed to attain similar levels of HIV inhibition following a wash with seminal plasma. Conversely, the NNRTIs and PIE12, but not TDF or MVC, were effectively transferred from ex vivo treated explants and protected co-cultured T cells. Biopsies obtained from IQP-0528 ring-treated macaques also protected co-cultured T cells with viral inhibition ranging from 42-72%. Antiviral activity correlated with the concentration of drug recovered. Combinations of TDF with IQP-0528 protected in both in vitro models. CONCLUSIONS: Together, these models suggest that intracellularly retained drugs such as TDF may protect resident immune cells following coitus but sustained delivery may be required to protect immune cells subsequently recruited into the genital tract. Sustained delivery may also be critical for NNRTIs, which are rapidly transported out of cells and could be lost following sexual intercourse. An ideal approach may be a combination of drugs with complementary bioavailability profiles formulated for sustained delivery.

Safety and efficacy of tenofovir/IQP-0528 combination gels - a dual compartment microbicide for HIV-1 prevention.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a non-nucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24h exposure. The ED(50) calculated from the formulations for IQP-0528 was ~32nM and for TFV was ~59nM and their inhibitory activity was not affected by semen. The ED(50) of TFV in the combination gels was ~100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ≥1log(10) and ≥2log(10), respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.

Development of dual-acting pyrimidinediones as novel and highly potent topical anti-HIV microbicides.

In the absence of an effective vaccine against the human immunodeficiency virus (HIV), topical microbicides to prevent the sexual transmission of HIV represent an important strategy to prevent the continued spread of infection. The recent trend in the development of new microbicide candidates includes the utilization of FDA-approved therapeutic drugs that target the early stages of the HIV life cycle, including entry inhibitors and reverse transcriptase inhibitors. We have investigated 12 pyrimidinedione compounds with potent HIV activities and their abilities to inhibit both virus entry and reverse transcription, in an effort to determine a lead microbicide for product development. The candidate compounds were evaluated for efficacy against subtype B, C, and E clinical virus strains in fresh human peripheral blood mononuclear cells and against CCR5-tropic virus strains in both monocyte-macrophages and dendritic cells. Microbicide-specific biological assays and toxicity evaluations were also performed in a variety of established and fresh human cells as well as against Lactobacillus strains common to the vaginal environment. These evaluations resulted in the identification of congeners with cyclopropyl and cyclobutyl substituents at the N-1 of the pyrimidinedione as the most active molecules in the structure-activity relationship series. The pyrimidinediones represent excellent microbicide candidates in light of their significantly high efficacies against HIV-1 (subnanomolar concentration range), potencies (therapeutic index, >1 million), solubility profiles, and dual mechanism of antiviral action that includes two early steps of virus replication prior to the integration of the virus that are considered most important for microbicidal activity.

Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles.

This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 h in the presence of HSP at 37°C and pSS released from the microgels within 30 min reached a concentration of 10 µg/mL, equivalent to the published IC(90) for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described.

1-Benzyl derivatives of 5-(arylamino)uracils as anti-HIV-1 and anti-EBV agents.

Pyrimidine analogs have long found use over a broad chemotherapeutic spectrum. In an effort to further explore the antiviral potential of several uracil derivatives previously synthesized in our laboratories, a series of benzylated pyrimidines were designed and synthesized. Introduction of the benzyl residue onto the 5-phenylaminouracil scaffold was carried out using 2,4-bis(trimethylsilyloxy)pyrimidine with the corresponding benzyl bromides. Similarly, 1-benzyl-5-(benzylamino)- and 1-benzyl-5-(phenethylamino)uracils were obtained via amination of 1-benzyl-5-bromouracils with benzylamine or phenylethylamine. The results of the broad screen antiviral studies revealed that compounds 5 and 11 exhibit promising inhibitory activity against HIV-1 in CEM-SS culture. A 50% protective effect was observed at concentrations of 11.9 and 9.5 µÐœ, respectively. Moreover, compounds 8 and 3 exhibited good inhibitory effects against EBV in АKАТА cell culture with EC50 values of 2.3 and 12 µM, respectively. The synthesis and biological studies are detailed herein.

Development of hexadecyloxypropyl tenofovir (CMX157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant HIV.

CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC(50)s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC(50)s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

Inhibition of human immunodeficiency virus type 1 by triciribine involves the accessory protein nef.

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

Novel targets for HIV therapy.

There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human immunodeficiency virus (HIV) infection. However, new anti-HIV agents are still needed to confront the emergence of drug resistance and various adverse effects associated with long-term use of antiretroviral therapy. The 21st International Conference on Antiviral Research, held in April 2008 in Montreal, Canada, therefore featured a special session focused on novel targets for HIV therapy. The session included presentations by world-renowned experts in HIV virology and covered a diverse array of potential targets for the development of new classes of HIV therapies. This review contains concise summaries of discussed topics that included Vif-APOBEC3G, LEDGF/p75, TRIM 5alpha, virus assembly and maturation, and Vpu. The described viral and host factors represent some of the most noted examples of recent scientific breakthroughs that are opening unexplored avenues to novel anti-HIV target discovery and validation, and should feed the antiretroviral drug development pipeline in the near future.

Comparative evaluation of virus transmission inhibition by dual-acting pyrimidinedione microbicides using the microbicide transmission and sterilization assay.

In the absence of a fully effective human immunodeficiency virus (HIV) vaccine, topical microbicides represent an important strategy for preventing the transmission of HIV through sexual intercourse, the predominant mode of HIV transmission worldwide. Although a comprehensive understanding of HIV transmission has not yet emerged in the microbicide field, it is likely the result of rapid infection of monocyte-derived cells in the vaginal mucosa by CCR5-tropic viruses. Inhibition of HIV transmission requires agents that prevent entry, fusion, reverse transcription, or other preintegrative replication events or agents which directly inactivate HIV or modulate the target cells to render them uninfectible. In vitro assays typically used to evaluate the ability of a microbicide to prevent virus transmission use epithelial or human osteosarcoma-derived cells or immune cells more relevant to the development of anti-HIV therapeutic agents and quantify virus production at short time intervals following infection. We have developed a microbicide transmission and sterilization assay (MTSA) to more sensitively and quantitatively evaluate virus transmission in cell culture in the presence of microbicidal compounds. Results obtained with the MTSA demonstrate that the inhibitory capacity of microbicides is often overestimated in short-term transmission inhibition assays, while some compounds yield equivalent inhibitory results, indicating a biological relevance for the MTSA-based evaluations to identify superior potent microbicides. The MTSA defines the concentration of the microbicide required to totally suppress the transmission of virus in cell culture and may thus help define the effective concentration of the microbicide required in a formulated microbicide product.

Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

Inhibition of tubulin polymerization by select alkenyldiarylmethanes.

During studies on the alkenyldiarylmethane (ADAM) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), analogues were discovered that exhibit low micromolar and submicromolar cytotoxicities. Since the ADAMs are structurally related to the tubulin polymerization inhibitor CC-5079, a set of 14 ADAMs were tested for inhibition of tubulin polymerization in an attempt to identify the biological target responsible for their cytotoxicity. The results indicate that, overall, the ADAMs are poor inhibitors of tubulin polymerization. However, the two most cytotoxic compounds, 15 and 16, are in fact active as inhibitors of tubulin assembly with IC(50) values of 3.7+/-0.3 and 2.8+/-0.2 microM, respectively, and they both inhibit the binding of colchicine to tubulin. Both compounds were investigated for anticancer activity in the National Cancer Institute's panel of 60 human cancer cell lines, and both compounds consistently displayed submicromolar cytotoxicities with mean-graph midpoint (MGM) values of 0.31+/-0.08 and 0.47+/-0.09 microM, respectively.