Natural product-derived compounds in HIV suppression, remission, and eradication strategies.

While combination antiretroviral therapy (cART) has successfully converted HIV to a chronic but manageable infection in many parts of the world, HIV continues to persist within latent cellular reservoirs, which can become reactivated at any time to produce infectious virus. New therapies are therefore needed not only for HIV suppression but also for containing or eliminating HIV reservoirs. Compounds derived from plant, marine, and other natural products have been found to combat HIV infection and/or target HIV reservoirs, and these discoveries have substantially guided current HIV therapy-based studies. Here we summarize the role of natural product-derived compounds in current HIV suppression, remission, and cure strategies.

Synthetic access to spacer-linked 3,6-diamino-2,3,6-trideoxy-alpha-D-glucopyranosides--potential aminoglycoside mimics for the inhibition of the HIV-1 TAR-RNA/Tat-peptide complex.

The synthesis of spacer-linked neoaminoglycoside 5 is described. Key steps of the synthesis are the introduction of nitrogen functionalities at C-3 and C-6 and the olefin cross metathesis of allyl glycoside 16. Although it is known that Grubbs catalysts tolerate nitrogen functionalities, difficulties were encountered in the cross metathesis reaction. Factors that govern this dimerization are the steric and electronic demands of the catalyst and the substrate. Preliminary biological evaluation of homodimer 5, by studying the inhibition of HIV-1 TAR-RNA/Tat-peptide complex using a method based on fluorescence titration, revealed an inhibitory effect of 5.

Acute exposure to ethanol potentiates human immunodeficiency virus type 1 Tat-induced Ca(2+) overload and neuronal death in cultured rat cortical neurons.

A significant number of human immunodeficiency virus type 1 (HIV-1)-infected patients are alcoholics. Either alcohol or HIV alone induces morphological and functional damage to the nervous system. HIV-1 Tat is a potent transcriptional activator of the viral promoter, with the ability to modulate a number of cellular regulatory circuits including apoptosis and to cause neuronal injury. To further evaluate the involvement of alcohol in neuronal injury, the authors examined the effect of ethanol on Tat-induced calcium responses in rat cerebral cortical neurons, using microfluorimetric calcium determination. HIV Tat protein (10 or 500 nM) elicited two types of calcium responses in cortical neurons: a fast-onset, short-lasting response and a slow-onset, sustained response. The responses were concentration-dependent and diminished in calcium-free saline. A short exposure to ethanol (50 mM) potentiated both types of calcium response, which was markedly decreased when the cells were pretreated with BAPTA-AM (20 microM). In addition, an increase in the neurotoxic effect of Tat, which was assessed by trypan blue exclusion assay, was observed. The result led the authors to conclude that alcohol exposure significantly potentiates Tat-induced calcium overload and neuronal death.

Design and synthesis of a Tat-related gene transporter: a tool for carrying the adenovirus vector into cells.

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.

Combinatorial chemistry as a tool for targeting different stages of the replicative HIV-1 cycle.

In the present era, acquired immunodeficiency syndrome (AIDS) is the most fatal disorder for which no completely successful chemotherapy has been developed so far. The pandemic spread of this disease has prompted an unprecedented scientific and clinical effort to understand and combat it. A number of targets has been identified to stop the replication of the virus at different stages of its life cycle: Reverse Transcriptase (RT), protease (PR) and CCR5 are the most promising targets. Although highly active antiretroviral therapy (HAART) has been effective in reducing the mortality and morbidity in recent years, adverse side effects of the chemotherapy, patient non-compliance and the development of viral resistance remain major problems. With the aim to find better drug candidates with minor adverse side effects in recent years, several groups have investigated combinatorial approaches for the generation of libraries of HIV PR inhibitors while only few contributions to the preparation of libraries of HIV Reverse Transcriptase (RT) and CCR5 inhibitors are available. This review summarizes the recent developments of combinatorial chemistry in this area.

Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR interaction inhibitors.

Four new isoquinoline derivatives bearing guanidinium group or amino group-terminated side chain were synthesized to target the HIV-1 TAR element. Their abilities to bind TAR RNA and inhibit Tat-TAR RNA interaction were determined by CE analysis, a Tat-dependent HIV-1 LTR-driven CAT assay and SIV-induced syncytium evaluation.

Effects of insulin-like growth factor binding protein-3 and farnesyltransferase inhibitor SCH66336 on Akt expression and apoptosis in non-small-cell lung cancer cells.

BACKGROUND: Overexpression of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in non-small-cell lung cancer (NSCLC) cells in vitro and in vivo. However, Ras-mediated signaling pathways could develop resistance to apoptotic activities of IGFBP-3 in NSCLC cells. We thus evaluated the therapeutic potential of the combination of IGFBP-3 and SCH66336, a farnesyltransferase inhibitor that blocks Ras activation, in NSCLC cell lines. METHODS: The effects of the combination of adenoviral IGFBP-3 (Ad-IGFBP3) and SCH66336 on proliferation and apoptosis of NSCLC cell lines (H1299, H596, A549, H460, H358, H322, and H226B) were assessed in vitro and in vivo by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometry-based terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, western blot analyses, and an NSCLC xenograft tumor model. The specific effects of Ad-IGFBP 3 and SCH66336 on mitogen-activated protein kinase and Akt were assessed by using adenoviral vectors that express constitutively active MEK1 or constitutively active Akt. Synergy was assessed by median effect analysis. RESULTS: The combination of Ad-IGFBP3 and SCH66336 had synergistic antiproliferative effects in five cell lines (H1299, H596, A549, H460, and H322). Antiproliferative effects were accompanied by increased apoptosis in H460 cells in vitro. Overexpression of a constitutively active Akt but not a constitutively active MEK-1 rescued H460 cells from apoptosis induced by single or combined treatment of Ad-IGFBP3 and SCH66336. In H1299 tumor xenografts, Ad-IGFBP3 and SCH66336 was associated with decreased tumor volume, increased apoptosis, and decreased Akt levels. CONCLUSIONS: The combination of Ad-IGFBP3 and SCH66336 decreased Akt expression and increased apoptosis in NSCLC cells in vitro and in vivo. Simultaneous treatment with IGFBP-3 and SCH66336 may have the potential to be an effective therapeutic strategy in NSCLC.

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev.

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.

Inhibition of hematopoietic progenitor cell proliferation by ethanol in human immunodeficiency virus type 1 tat-expressing transgenic mice.

BACKGROUND: A number of hematological abnormalities are associated with both human immunodeficiency virus type 1 (HIV-1) infection and alcohol abuse. There is little information on how alcohol abuse might further influence the survival and growth of hematopoietic progenitors in HIV-infected individuals in the presence of immune system abnormalities and anti-HIV drugs. Because there is evidence that viral transactivator Tat itself can induce hematopoietic suppression, in this study we examined the role of ethanol as a cofactor in transgenic mice that expressed HIV-1 Tat protein. METHODS: Tat transgenic mice and nontransgenic littermates were given ethanol (20% v/v) and the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT; 1 mg/ml) in drinking water. Immunosuppression in mice was induced by weekly intraperitoneal injections of anti-CD4 antibody. Hematopoiesis was examined by erythroid colony forming unit (CFU-E) and granulocyte/macrophage colony-forming unit (CFU-GM) assays of the bone marrow progenitor cells. RESULTS: Administration of ethanol for 7 weeks resulted in a 50% decrease in the proliferative capacity of CFU-E- and CFU-GM-derived progenitors from transgenic mice compared with that of ethanol-treated nontransgenic controls. Similar decreases also were observed in transgenic mice treated with AZT or a combination of AZT and ethanol. Furthermore, ethanol and AZT were significantly more toxic to the granulopoietic progenitors (40-50% inhibition) than to the erythropoietic progenitors (10-20% inhibition) in Tat transgenic mice. Although a 10 day exposure of Tat transgenic and nontransgenic mice to a combination of ethanol and AZT had no suppressive effect on the erythropoietic and granulopoietic progenitor cells, there was a marked decrease (40-60%) in CFU-GM in mice made immunodeficient by CD4+ T-lymphocyte depletion. The ethanol-treated Tat transgenic mice but not the nontransgenic litter-mates also showed a significant decrease (25%) in CFU-GM. CONCLUSION: Our in vivo study strongly suggests that ethanol ingestion in HIV-1-infected individuals, particularly those on antiretroviral drugs, might increase bone marrow toxicity and contribute to HIV-1-associated hematopoietic impairment.

Inhibition of HIV-1 transcription by cyclopentenone prostaglandin A1 in Jurkat T lymphocytes.

Cyclopentenone prostaglandins inhibit virus replication in several DNA and RNA virus models. In this report we investigated the effect of prostaglandin A1 (PGA1) on HIV-1 transcription in human CD4+ Jurkat T lymphocyte cells. A dramatic reduction of HIV-1 RNA levels was detected up to seven days post infection in both unstimulated and phorbol 12-mystrate 13-acetate (PMA)-stimulated cells treated with PGA1- PGA1 treatment of cells was also effective in inhibiting the transcription of a chloramphenicol acetyltransferase (CAT) reporter gene, under the control of HIV-1 LTR, in Jurkat-Tat cells. We also show that PGA1 induced the synthesis of 70-kDa heat-shock protein (HSP70) in this cell system and the induction correlated with the drug-antiviral activity. PGA1 was also found to induce the loss of the tumor suppressor p53 protein, in the "proliferative" conformation, in a time correlation with the induction of the HSP70 As the "proliferative" p53 has been involved in the positive trans-activation of the HIV-1 LTR its depletion could contribute to the inhibitory mechanisms of PGA1 on virus transcription.

HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches.

Tat proteins (trans-activating proteins) are present in all known lentiviruses and are early RNA binding proteins that regulate transcription. Tat from the human immunodeficiency virus type-1 is a protein comprising 86 amino acids and encoded by 2 exons. The first 72 amino acids are encoded by exon 1 and exhibit full trans-activating activity. The second exon encodes a 14-amino-acid C-terminal sequence that is not required for trans-activation but does contain an RGD motif, which is important in binding to alphavbeta3 and alpha5beta1 integrins. Tat has an unusual property for a transcription factor; it can be released and enter cells freely, yet still retain its activity, enabling it to up-regulate a number of genes. Tat also has an angiogenic effect; it is a potent growth factor for Kaposi sarcoma-derived spindle cells, and, separately, it has been shown to bind to a specific receptor, Flk-1/KDR, on vascular smooth muscle cells, as well as to integrin-like receptors present on rat skeletal muscle cells and the lymphocyte cell line H9. It appears that the basic domain of tat is important, not only for translocation but also for nuclear localisation and trans-activation of cellular genes. As such, targeting of tat protein or, more simply, the basic domain provides great scope for therapeutic intervention in HIV-1 infection. There is also opportunity for tat to be used as a molecular tool; the protein can be manipulated to deliver non-permeable compounds into cells, an approach that already has been employed using ovalbumin, beta-galactosidase, horseradish peroxidase, and caspase-3.

Characterization of the effects of two polysulfonated distamycin A derivatives, PNU145156E and PNU153429, on HIV type 1 Tat protein.

We examined whether two sulfonated distamycin A derivatives, PNU145156E and PNU153529, inhibit the trans-activating and angiogenic effects of HIV-1 Tat protein. The study was carried out by analyzing the activity of the two drugs on: (1) extracellular and intracellular Tat protein, introduced into HL3T1 cells containing an integrated HIV-1 LTR/CAT plasmid; (2) binding of Tat to 3H-labeled heparin and to 14C-labeled PNU145156E; and (3) the angiogenic response induced in vivo by culture medium conditioned by T53c14 cells, which release extracellular Tat. PNU145156E and PNU153429 interacted with extracellular Tat in the culture medium and physically bound the Tat protein, most likely sequestering it in the extracellular space. As a consequence, the two drugs inhibited trans-activation of the HIV-1 LTR on addition of the free Tat protein to HL3T1 cells. However, the two compounds inhibited the activity of intracellular Tat when they were introduced into the cells by lipofection. In vivo experiments showed that the two drugs blocked the neoangiogenesis induced by Tat released in the conditioned medium of T53c14 cells. Owing to the critical role of intracellular and extracellular Tat in HIV-1 replication, these drugs show promise as a means to control the progression of HIV-1 infection as well as the neoplastic and angiogenic effects induced by Tat in the course of AIDS.

Inhibition of acute-, latent-, and chronic-phase human immunodeficiency virus type 1 (HIV-1) replication by a bistriazoloacridone analog that selectively inhibits HIV-1 transcription.

Nanomolar concentrations of temacrazine (1,4-bis[3-(6-oxo-6H-v-triazolo[4,5,1-de]acridin-5-yl)amino-propyl ]piperazine) were discovered to inhibit acute human immunodeficiency virus type 1 (HIV-1) infections and suppress the production of virus from chronically and latently infected cells containing integrated proviral DNA. This bistriazoloacridone derivative exerted its mechanism of antiviral action through selective inhibition of HIV-1 transcription during the postintegrative phase of virus replication. Mechanistic studies revealed that temacrazine blocked HIV-1 RNA formation without interference with the transcription of cellular genes or with events associated with the HIV-1 Tat and Rev regulatory proteins. Although temacrazine inhibited the in vitro 3' processing and strand transfer activities of HIV-1 integrase, with a 50% inhibitory concentration of approximately 50 nM, no evidence of an inhibitory effect on the intracellular integration of proviral DNA into the cellular genome during the early phase of infection could be detected. Furthermore, temacrazine did not interfere with virus attachment or fusion to host cells or the enzymatic activities of HIV-1 reverse transcriptase or protease, and the compound was not directly virucidal. Demonstration of in vivo anti-HIV-1 activity by temacrazine identifies bistriazoloacridones as a new class of pharmaceuticals that selectively blocks HIV-1 transcription.

Interaction of pregnancy steroid hormones and zidovudine in inhibition of HIV type 1 replication in monocytoid and placental Hofbauer cells: implications for the prevention of maternal-fetal transmission of HIV.

Zidovudine (AZT) has been shown to reduce maternal-fetal transmission of HIV-1 by more than two-thirds in a variety of clinical settings. However, the mechanism of action of AZT in this setting is unclear. Suppression of vertical transmission has occurred in the absence of an impact on maternal plasma viremia and no lower threshold of viral load for such transmission has been identified. We hypothesized that augmentation of the anti-HIV effect of AZT may occur locally, at the maternal-fetal interface. We report that the pregnancy hormone progesterone at broad concentrations has little effect on acute HIV-1 infection of a monocytic cell line or primary peripheral blood cells. However, the combination of physiologic concentrations of progesterone (10[-7] to 10[-6] M) and low-dose AZT (10[-8] to 10[-9] M) produced markedly synergistic inhibition of HIV-1 replication within acutely infected monocytic cell lines (U937), and additive inhibition of HIV-1 growth within chronically infected monocytic cells (U1) and primary placental macrophages (Hofbauer cells). Anti-HIV effects were not seen with another pregnancy steroid hormone, estrogen. In terms of possible mechanisms of action for progesterone, we demonstrated that it incompletely suppressed tat activation of long terminal repeat (LTR)-driven gene expression in monocytic cells. However, the progesterone-mediated suppession of tat activation was not affected by mutation of the three consensus progesterone/androgen/glucocorticoid response elements within the HIV-1 LTR, previously shown by our group to be involved in glucocorticoid-mediated suppression of LTR-driven transcription. It is likely that progesterone suppresses LTR-driven gene expression through a nontranscriptional mechanism, and augments the efficacy of AZT through enhancement of its phosphorylation.

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.

[Therapeutic potential of Ro 5-3335. New active benzodiazepine in vitro against HIV-1 viruses resistant or non-resistant to AZT]. / Potentiel thérapeutique du Ro 5-3335. Une nouvelle benzodiazépine active in vitro contre les virus HIV-1 résistants ou non à l'AZT.

Ro 5-3335 is a new benzodiazepine highly active in vitro (IC50 = 0.1-1.0 microM [corrected]) against HIV-1 viruses of AIDS resistant or non-resistant to zidovudine (AZT). It is also active against HIV-2. Ro 5-3335 is original by its mechanism of action, acting on the trans-activation factor of transcription (TAT) and non on the reverse transcriptase. Such as, it could prevent proviral DNA to express in both evolutive and silent AIDS resistant or non-resistant to AZT or to other anti-reverse transcriptase series. In addition, in antagonizing extracellular TAT's actions, Ro 5-3335 could alleviate the syndrome commonly associated with AIDS as Kaposi's syndrome. In rodent test, Ro 5-3335 has no diazepam-like central effects and presents in comparison to AZT a more favorable therapeutic index. In dog, the elimination half-life, peak concentration and availability are 2 h, 0.8 microM and 85% respectively, after a 1 mg.kg-1 oral dose of Ro 5-3335. Theoretically, Ro 5-3335 and now its analogue Ro 24-7429 seem to possess all virtues to antagonize evolutive and latent AIDS. Its arrival is timely to cope with the ever increasing resistance phenomena, lengthy development of AIDS vaccines, exponential contamination of populations worldwide and last but not least possibly to impede evolutions of the disease. Ability to manipulate TAT-mediated activation of HIV-1 genes paves the ways to study conceivable corrections of abnormal gene expressions of neurotransmitters, hormones, oncogenes and key enzymes.