Potent Inhibitor Of HIV Replication Research Articles

HIV-1 3'-Polypurine Tract Mutations Confer Dolutegravir Resistance by Switching to an Integration-Independent Replication Mechanism via 1-LTR Circles.

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

Targeting ABL1 or ARG Tyrosine Kinases to Restrict HIV-1 Infection in Primary CD4+ T-Cells or in Humanized NSG Mice.

Previous studies support dasatinib as a potent inhibitor of HIV-1 replication. However, a functional distinction between 2 kinase targets of the drug, ABL1 and ARG, has not been assessed. We used primary CD4 T-cells, CD8-depleted peripheral blood mononuclear cells (PBMCs) from a treatment naïve HIV-1 patient, and a humanized mouse model of HIV-1 infection. We assessed the roles of ABL1 and ARG during HIV-1 infection and use of dasatinib as a potential antiviral against HIV-1 in humanized mice. Primary CD4 T-cells were administered siRNA targeting ABL1 or ARG, then infected with HIV-1 containing luciferase reporter viruses. Quantitative polymerase chain reaction of viral integration of 4 HIV-1 strains was also assessed. CD8-depleted PBMCs were treated for 3 weeks with dasatinib. NSG mice were engrafted with CD34 pluripotent stem cells from human fetal cord blood, and infected with Ba-L virus after 19 weeks. Mice were treated daily with dasatinib starting 5 weeks after infection. siRNA knockdown of ABL1 or ARG had no effect on viral reverse transcripts, but increased 2-LTR circles 2- to 4-fold and reduced viral integration 2- to 12-fold. siRNA knockdown of ARG increased SAMHD1 activation, whereas knockdown of either kinase reduced RNA polymerase II activation. Treating CD8-depleted PBMCs from a treatment-naïve patient with 50 nM of dasatinib for 3 weeks reduced p24 levels by 99.8%. Ba-L (R5)-infected mice injected daily with dasatinib showed a 95.1% reduction in plasma viral load after 2 weeks of treatment. We demonstrate a novel nuclear role for ABL1 and ARG in ex vivo infection experiments, and proof-of-principle use of dasatinib in a humanized mouse model of HIV-1 infection.

APOBEC3G expression and HIV-1 infection in Burkina Faso.

APOBEC3G is a potent inhibitor of HIV-1 replication, and act by deaminating cytidines in uracil on the negative strand of the viral cDNA. In this case-control study, APOBEC3G expression in subjects’ naïve to HAART infected by HIV-1 and the effect of APOBEC3G polymorphism on its expression were evaluated. The results show that the HIV-1 infected carriers of the G minor alleles of the variant rs8177832 had a higher expression of APOBEC3G mRNA than the controls carriers of the G minor allele. APOBEC3G polymorphisms could play an important role in the modulation of the HIV-1 dissemination.

Syntheses of (-)-Tripterifordin and (-)-Neotripterifordin from Stevioside.

We report short syntheses of (-)-tripterifordin and (-)-neotripterifordin, potent inhibitors of HIV replication, from stevioside, a natural sweetener used worldwide. The key transformations are reduction at C13 through the formation of a tertiary chloride and subsequent three-step lactonization including a selective iodination at C20 by the photoreaction of the C19-alcohol. The title compounds were reliably obtained from stevioside in 9 and 11 steps (with 5-7 isolation steps), respectively. Additionally, the related lactone-containing ent-kaurenes, doianoterpenes A and B, and two more natural products were synthesized.

Potent Inhibition of HIV-1 Replication in Resting CD4 T Cells by Resveratrol and Pterostilbene.

HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health-promoting benefits. Resveratrol has been shown to inhibit the replication of several viruses, including herpes simplex viruses 1 and 2, papillomaviruses, severe acute respiratory syndrome virus, and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at a low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, an HIV-1 and simian immunodeficiency virus protein that increases deoxynucleoside triphosphate (dNTP) levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene, or related compounds as adjuvants in anti-HIV preexposure prophylaxis (PrEP) formulations.

Novel indole sulfides as potent HIV-1 NNRTIs.

In a previous communication we described a series of indole based NNRTIs which were potent inhibitors of HIV replication, both for the wild type and K103N strains of the virus. However, the methyl ether functionality on these compounds, which was crucial for potency, was susceptible to acid promoted indole assisted SN1 substitution. This particular problem did not bode well for an orally bioavailable drug. Here we describe bioisosteric replacement of this problematic functional group, leading to a series of compounds which are potent inhibitors of HIV replication, and are acid stable.

Designing Stem-Cell Based Anti-HIV Therapies using Molecular-Detailed Multiscale Models

Although HAART ensures normal lives for HIV-infected individuals, it does not lead to a functional cure. In 2008, the “Berlin patient”, a HIV-infected cancer patient, underwent a bone marrow transplant from a CCR5-/- donor. Since then, he has shown no signs of active HIV-1 replication in the absence of HAART. However, finding such a matched donor for each HIV patient is challenging; instead, inserting anti-HIV genes into patients’ or matched donors’ stem cells before transplantation could provide HIV-resistance to the progeny target cells and lead to a functional cure. We developed a molecular-detailed, mechanistic model of HIV infection to design stem-cell therapies using endogenous anti-HIV genes and to identify logistics of successful treatment. The model simulates the dynamics of HIV infection to track key restriction factors and their interactions with HIV-encoded proteins, the virus, and multiple immune cell types: CD4+ T-cells, macrophages, latently-infected T-cells, and CTLs. Using viral load and T-cell count data from HIV-infected individuals, we explored interpatient variability of stem-cell therapies by creating a virtual population of HIV patients. Our model predicted that APOBEC3G overexpression at medium or high transduction efficiency could effectively stop HIV replication and increase the T-cell count to normal levels. SAMHD1 overexpression in macrophages even at high transduction efficiency is not by itself a potent inhibitor of HIV replication; it stabilizes the viral load and slightly improves the T-cell count. However, the model predicted APOBEC3G-SAMHD1 synergy: faster viral decay and return to normal T-cell counts at lower overexpression levels. Surprisingly, HIV-triggered pro-apoptosis gene circuits combined with APOBEC3G overexpression were predicted to negatively impact HIV replication blockade if expressed at low transduction efficiency. This is because some infected T-cells die at a faster rate, weakening the effect of immune response in killing the remaining infected cells.

Molecular modeling, synthesis and biological evaluation of N-heteroaryl compounds as reverse transcriptase inhibitors against HIV-1.

Different N-heteroaryl compounds bearing pyrimidine and benzimidazole moieties have been designed in silico using Discovery studio 2.5 software, synthesized and evaluated for their inhibitory activity as reverse transcriptase inhibitors against HIV-1 replication using laboratory adapted strains HIV-1IIIB (X4, subtype B) and HIV-1Ada5 (R5, subtype B), and the primary isolates HIV-1UG070 (X4, subtype D) and HIV-1VB59 (R5, subtype C). Cell-based assay showed that compounds were active at 1.394 μm concentrations (Selectivity Index: 1.29-38.39). The studies on structure-activity relationship clearly suggested anti-HIV activity of pyrimidine and benzimidazole derivatives and these findings were consistent with the in vitro cell-based experimental data. The results of molecular modeling and docking confirmed that all compounds assumed a butterfly-like conformation and showed H-bond, 'π-π' and 'π-+' and hydrophobic interactions within flexible non-nucleoside inhibitor binding pocket of HIV-1 reverse transcriptase, similar to known non-nucleoside reverse transcriptase inhibitors, such as nevirapine. In view of the results obtained, it can be said that the chemical skeletons of N, N'-bis-(pyridin-2-yl)-succinamide (14 and 15) and 1, 4-bis-benzoimidazol-1-yl-butane-1, 4-dione (16 and 17) may be used for developing potent inhibitors of HIV-1 replication, with suitable structure/pharmacophore modifications.

Rational design and synthesis of novel thiazolidin-4-ones as non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel thiazolidin-4-one analogues, characterized by different substitution patterns at positions C-2 and N-3 of the thiazolidin-4-one scaffold for anti-HIV-1 activity has been investigated. Most of the compounds showed anti-HIV-1 activity at micromolar concentrations when tested in TZM-bl cells in vitro. Among the thirty-three compounds tested, compound 16 was the most potent inhibitor of HIV-1 replication against HIV-1IIIB, HIV-1ADA5, HIV-1UG070 and HIV-1VB59 (EC50=0.02, 0.08, 0.08 and 0.08μM, respectively) with selectivity index (SI=6940, 1735, 1692 and 1692) against tested viral strains, respectively. The results of the present study suggested that the substitution of the nitro group at 6′ position of the C-2 phenyl ring and 4,6-dimethylpyridin-2-yl at the N-3 position of thiazolidin-4-one had a major impact on the anti-HIV-1 activity and was found to lower cytotoxicity. The substitution of the heteroaryl ring with bromo group and bicyclic heteroaryl ring at N-3 thiazolidin-4-one was found to lower anti-HIV-1 activity and increase cytotoxicity. The undertaken docking studies thus facilitated the identification of crucial interactions between the HIV-1 RT enzyme and thiazolidin-4-one inhibitors, which can be used to design new potential inhibitors.

Inhibition of HIV-1 Replication by Secondary Metabolites From Endophytic Fungi of Desert Plants

Most antiretroviral drugs currently in use to treat an HIV-1 infection are chemically synthesized and lead to the development of viral resistance, as well as cause severe toxicities. However, a largely unexplored source for HIV-1 drug discovery is endophytic fungi that live in a symbiotic relationship with plants. These fungi produce biologically active secondary metabolites, which are natural products that are beneficial to the host. We prepared several hundred extracts from endophytic fungi of desert plants and evaluated the inhibitory effects on HIV-1 replication of those extracts that showed less than 30% cytotoxicity in T-lymphocytes. Those extracts that inhibited viral replication were fractionated in order to isolate the compounds responsible for activity. Multiple rounds of fractionation and antiviral evaluation lead to the identification of four compounds, which almost completely impede HIV-1 replication. These studies demonstrate that metabolites from endophytic fungi of desert plants can serve as a viable source for identifying potent inhibitors of HIV-1 replication.

A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+T Cells

Here we show potent inhibition of HIV-1 replication in a human T cell line and primary human CD4(+) cells by expressing a single antiviral protein. Nullbasic is a mutant form of the HIV-1 Tat protein that was previously shown to strongly inhibit HIV-1 replication in nonhematopoietic cell lines by targeting three steps of HIV-1 replication: reverse transcription, transport of viral mRNA, and trans-activation of HIV-1 gene expression. Here we investigated gene delivery of Nullbasic, using lentiviral and retroviral vectors. Although Nullbasic could be delivered by lentiviral vectors to target cells, transduction efficiencies were sharply reduced primarily because of negative effects on reverse transcription mediated by Nullbasic. However, Nullbasic did not inhibit transduction of HEK293T cells by a murine leukemia virus (MLV)-based retroviral vector. Therefore, MLV-based virus-like particles were used to transduce and express Nullbasic-EGFP or EGFP in Jurkat cells, a human leukemia T cell line, and Nullbasic-ZsGreen1 or ZsGreen1 in primary human CD4(+) cells. HIV-1 replication kinetics were similar in parental Jurkat and Jurkat-EGFP cells, but were strongly attenuated in Jurkat-Nullbasic-EGFP cells. Similarly, virus replication in primary CD4(+) cells expressing a Nullbasic-ZsGreen1 fusion protein was inhibited by approximately 8- to 10-fold. These experiments demonstrate the potential of Nullbasic, which has unique inhibitory activity, as an antiviral agent against HIV-1 infection.

Small organic molecules targeting PCAF bromodomain as potent inhibitors of HIV-1 replication

Human p300/CBP associated factor bromodomain (PCAF BRD), a relatively conserved host cell protein, can selectively bind with Tat-AcK50 (tat acetylated lys50). This interaction is essential for the transcription activation of HIV-1 viral gene, therefore PCAF BRD provides a potential targeting protein to inhibit the transcription. We synthesized a series of N1-aryl-propane-1,3-diamine compounds to bind to PCAF BRD. And cellular level anti-HIV-1 assay showed that these small molecules had good inhibitory potency on HIV-1 replication, which was consistent with the molecular assay. Also, it was found that 3-(2-nitrophenoxy) propan-1-amine derivatives had strong bioactivity against HIV-1 replication.

Functional In Vivo Delivery of Multiplexed Anti-HIV-1 siRNAs via a Chemically Synthesized Aptamer With a Sticky Bridge

One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3′ 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2′ OMe/2′ Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.

Nerium oleander derived cardiac glycoside oleandrin is a novel inhibitor of HIV infectivity

We evaluated the effectiveness of Anvirzel™, an aqueous extract of Nerium oleander on HIV infection of human peripheral blood mononuclear cells. Oleandrin, the principle cardiac glycoside (CG) in Anvirzel™ has been shown to exhibit anti-cancer properties but its efficacy against HIV is unknown. Treatment with Anvirzel™ significantly reduced the infectivity of virus produced from infected cells without any change in the total amount of virus produced. This is in contrast to treatment with AZT, a potent inhibitor of HIV replication that has been shown to significantly reduce virus production. Relative to untreated cultures, virus in cultures treated with oleandrin had significantly reduced expression of the envelope protein gp120, the sole determinant of virus infectivity, suggesting a novel mechanism underlying the impaired infectivity. These results support the potential utility of the Nerium oleander aqueous extract, containing the CG oleandrin as a novel candidate anti-HIV therapeutic.

The Strengths, Weaknesses, Opportunities and Threats (SWOT) Analysis of HIV Type-1

–I hereby bring into perspective, the Strengths, Weaknesses, Opportunities and Threats (SWOT) analysis of HIV Type -1 and the first proposal for the application of this innovative concept in the field of HIV research, to holistically devise strategies at undermining the prowess of the Human Immunodeficiency Virus and overcoming its scourge. The strengths of HIV are the biological attributes that facilitate the pathogenicity, establishment of infection and spread of the virus. They include , the possession of Reverse Transcriptase enzyme for reverse transcription of RNA genome to DNA, ability to maintain persistence by forming a provirus, ability to specifically target and replicate in the immune cells, antigenic mimicry and adoption of host derived glycan shield for immune evasion, HIV-1 strains harbor enhanced biological fitness upon recombination, HIV has exceptionally high rate of replication and disrupts the BloodBrain Barrier. The weaknesses of HIV are the internal attributes and requirements for survival of the virus that invariably present as opportunities and targets for elimination. These include: the requirement for CCR5 and CXCR4 chemokine co-receptors for fusion prior to internalization, presence on HIV-1 envelope of conserved determinants that mediate CD4 binding, the Achilles heel of HIV is a tiny stretch of amino acids numbered 421 to 423 on gp 120, the capsid proteins reveal potential weakness at the inner core of the virus, HIV proteins retain virus internal carbohydrate, and the virus thrives on human proteins. The various host factors that were harnessed as opportunities for infection by HIV include, the ability to establish infection via multiple portals of entry, Sexually Transmitted Infections(STIs) increase the susceptibility to HIV-1 infection, STIs boost HIV shedding in the genital tract and amplifies infectiousness, presence of hidden HIV reservoirs that serve as foci of dissemination, the genital anatomical structure of the female genital tract aids biological susceptibility, the immune status of the host, and underlying illness or subsequent exposure to a bandwagon of opportunistic pathogens. The Threats to HIV are the external conditions that are inimical to the virus establishment and which may be harnessed for elimination of the pathogen. They include: limited host reservoir, the virus is susceptible to a Lectin isolated from ripened banana fruits as potent inhibitor of HIV replication, the development and application of Abzymes, development of agents that can selectively target only HIV infected cells, sustenance of global AIDS funding, and evolution of a potent HIV / AIDS Vaccine.

In vitro Anti-HIV Potency of Stampidine Alone and in Combination with Standard Anti-HIV Drugs

The purpose of the present study was compare the in vitro anti-HIV potency stampidine (CAS 217178-62-6), a novel aryl phosphate derivative of stavudine (CAS 3056-17-5), and drug combinations containing stampidine to the anti-HIV tency of the standard drugs zidovudine (CAS 30516-87-1), stavudine, lamivudine (CAS 134678-17-4), nelfinavir (CAS 159989-65-8), and nevirapine (CAS 129618-40-2) as well as their combinations. Stampidine inhibited the laboratory HIV-1 strain HTLV(IIIB) (B-envelope subtype) as well as the primary clinical HIV-1 isolates BR/92/025 (C-envelope subtype) and BR/93/20 (F-envelope sub-type) with subnanomolar IC50 values. Stampidine was as effective as zidovudine against HTLV(IIIB) and BR/92/025 and 3-logs more effective than zidovudine against BR/93/20. Stampidine was more effective than stavudine, lamivudine, nelfinavir, and nevirapine against all three HIV-1 isolates. The combination of stampidine with zidovudine + lamivudine was more effective than the combination of nelfinavir or nevirapine with zidovudine lamivudine against all three HIV-1 isolates. The combination of stampidine with nelfinavir was more effective than zidovudine + lamivudine as well as the combination of zidovudine + lamivudine with nelfinavir. The combination of stampidine with lamivudine + nelfinavir was more effective than the combination of zidovudine with lamivudine + nelfinavir. The combination of stampidine with lamivudine + nevirapine was more effective than the combination of stavudine with lamivudine + nevirapine. These findings demonstrate that (a) stampidine, as well as its combinations with the standard anti-HIV drugs zidovudine, lamivudine, nelfinavir or nevirapine, are potent inhibitors of HIV-1 replication in human peripheral blood mononuclear cells, and (b) replacement of either zidcovudine, zidovudine+lamivudine or stavudine in 3-drug cocktails with stampidine resulted in greater anti-HIV potency in vitro.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs

β-D-3′-Azido-2′,3′-dideoxyguanosine (3′-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3′-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3′-azido-ddG in primary cells. To gain insight into their structure–activity–resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3′-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3′-azido-2,6-diaminopurine >3′-azido-6-chloropurine; 3′-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure–activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

IFN-γ Mediates Enhancement of HIV Replication in Astrocytes by Inducing an Antagonist of the β-Catenin Pathway (DKK1) in a STAT 3-Dependent Manner

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous β-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished β-catenin signaling in astrocytes by 40%, as evaluated by both active β-catenin protein expression and β-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ-mediated inhibition of β-catenin signaling was dependent on its ability to induce an antagonist of the β-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the β-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and β-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.

Pharmacokinetics of 2',3'-dideoxycytidine in rats: application to interspecies scale-up.

The effects of dose on the pharmacokinetics of 2',3'-dideoxycytidine (DDC), a potent inhibitor of HIV replication, have been studied in rats. DDC was administered intravenously at doses of 10, 50, 100 and 200 mg kg-1. Plasma and urine drug concentrations were determined by HPLC. Non-compartmental pharmacokinetic parameters were calculated by area/moment analysis. DDC plasma concentrations declined rapidly with a terminal half-life of 0.98 +/- 0.18 h (mean +/- s.d.). No statistically significant differences were observed in pharmacokinetic parameters between the four doses. Total, renal and non-renal clearance values were independent of dose and averaged 1.67 +/- 0.24, 0.78 +/- 0.11, and 0.89 +/- 0.27 L h-1 kg-1, respectively. Approximately 50% of the dose was excreted unchanged in urine. Steady state volume of distribution was also independent of dose and averaged 1.2 +/- 0.21 L kg-1. Protein binding of DDC to rat serum proteins was independent of drug concentration with the fraction of drug bound averaging 0.45 +/- 0.12. Thus, the disposition pattern of DDC in the rat is independent of the administered dose even at high doses. Significant interspecies correlations were found for total, renal and non-renal clearance and steady state volume of distribution. Interspecies scaling resulted in superimposable plasma DDC concentration-time profiles from four laboratory animal species and man. Thus, plasma DDC concentrations in humans can be predicted from pharmacokinetic parameters obtained in laboratory animals.