Potent and selective activity of a new carbocyclic nucleoside analog (carbovir: NSC 614846) against human immunodeficiency virus in vitro

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir: NSC 614846), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a large screening program conducted by the National Cancer Institute and its contractors. Its hydrolytic stability and its ability to inhibit the infectivity and replication of HIV in T-cells at concentrations of approximately 200- to 400-fold below toxic concentrations make carbovir a top-priority candidate for development as a potential antiretroviral agent in the treatment of AIDS patients.     

Resolution of racemic carbovir and selective inhibition of human immunodeficiency virus by the (-) enantiomer

(+-) Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which is undergoing preclinical evaluation for the treatment of AIDS. Racemic carbovir was separated into its D and L enantiomers by the action of adenosine deaminase on the 2,6-diaminopurine precursor. Subsequent evaluation of the enantiomers against human immunodeficiency virus type 1 revealed that the antiviral activity of carbovir resides in the (-) isomer that is analogous to the nucleoside, beta-D-2',3'-didehydro-2',3'-dideoxyguanosine.     

Carbovir: A Carbocyclic Nucleoside with Potent and Selective Activity Against Human Immunodeficiency Virus (HIV) in Vitro

Abstract

Carbocyclic 2′, 3′-didehydro-2′,3′-dideoxyquanosine (carbovir), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a primary screen of a large number of carbocyclic nucleosides.¹ Carbovir inhibited the infectivity and replication of HIV in T-cells at concentrations 200 to 400-fold below toxicity to host cells. Carbovir was also evaluated for its Inhibitory effects on the expression of viral antigen in HIV-infected CEM cells. Production of p 24 core antigen at optimal inhibitory concentrations of the antiviral agents indicated comparable results for AZT, ddA and carbovir.     

Sequence specific interaction of the photoactive drug hypericin depends on the structural arrangement and the stability of the structure containing its specific 5AG3 target: a resonance Raman spectroscopy study.

The resonance Raman spectra of three oligonucleotides with different lengths containing a specific 5'AG3' target doublet for hypericin - a potent antiretroviral and anticancer photoactive agent, and their 1:1 and 1:2 (oligonucleotide: hypericin) complexes are reported. It is shown that the structural arrangement of the oligonucleotides, their structural stability and the local structural arrangement around the 5'AG3' hypericin target, are the factors which determine the formation of a stable, specifically bounded DNA-hypericin complexes.     

Targeted therapy of human immunodeficiency virus-related disease.

Since the discovery of human immunodeficiency virus (HIV) as a pathogenic retrovirus linked to acquired immunodeficiency syndrome (AIDS), a number of potentially useful strategies for antiretroviral therapy of AIDS and its related diseases have emerged. One such strategy involves use of the broad family of 2',3'-dideoxynucleosides, to which 3'-azido-2',3'-dideoxythymidine (AZT) belongs. AZT has been shown to reduce the replication of HIV in vivo and to confer significant clinical benefits in patients in both early and advanced stages of infection. Other members of the family, 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have also been reported to be active against HIV in short-term clinical trials. The armamentarium of antiretroviral agents is rapidly growing. Various nonnucleoside agents have recently been identified to be active against HIV in vitro. HIV-1 protease inhibitors are notable as possible new therapies for HIV-1-related diseases. However, we have faced several new challenges in the antiretroviral therapy in AIDS. These include long-term drug-related toxicities; emergence of drug-resistant HIV variants; and development of various cancers, particularly as effective therapies prolong survival. Progress in understanding structure-activity relations and clinical effectiveness will continue with dideoxynucleoside analogs. However, it seems certain that a variety of nonnucleoside analogs affecting multiple steps in viral replication will become available before long, and combination therapies using multiple antiretroviral drugs will be available. Such therapies will exert major effects against the moribidity and mortality caused by HIV.     

2',3'-didehydro-2',3'-dideoxy-5-chlorocytidine is a selective anti-retrovirus agent

2',3'-Didehydro-2',3'-dideoxy-5-chlorocytidine (D4CC) is, in contrast with 2',3'-dideoxy-5-chlorocytidine (ddClCyd) and 2',3'-didehydro-2',3'-dideoxy-5-chlorouridine (D4CU), a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) types 1 and 2, simian immunodeficiency virus (SIV) and simian AIDS related virus (SRV). D4CC is a poor inhibitor of the phosphorylation of [5-3H]2'-deoxycytidine (dCyd) by partially purified MT-4 cell dCyd kinase (Ki: 612 microM). The findings that (i) D4CC has little, if any, affinity for MT-4 cell Cyd/dCyd deaminase, (ii) D4CU is not antivirally active and (iii) the antiretroviral action of D4CC can be reversed by dCyd, but not dThd, indicate that D4CC is antivirally active as its Cyd metabolite (D4CC 5'-triphosphate) and does not need to be deaminated (to the corresponding Urd metabolite) to exert its antiretroviral action.     

In vitro toxicity and metabolism of 2',3'-dideoxycytidine, an inhibitor of human immunodeficiency virus infectivity.

U937 human monoblastoid cell growth was inhibited in a concentration-dependent manner by 2',3'-dideoxycytidine (ddCyd) (an antiretroviral drug) up to 500 microM. Cell growth inhibition was associated with a pronounced increase in cell volume, however this was not due to cell ATP or NAD+ depletion that could effect osmotic balance or DNA repair. This ddCyd toxicity paralleled the accumulation of ddCyd into acid soluble material where 2',3'-dideoxycytidine-5'-triphosphate (ddCTP) was the predominant labelled nucleotide up to an extracellular ddCyd concentration of 150 microM. At higher ddCyd concentrations, the amount of 2',3'-dideoxycytidine-5'-diphosphate (ddCDP) became predominant over ddCTP. This increase of phosphorylated dideoxycytidine in U937 cells was also associated with an increased incorporation of the drug into cell DNA suggesting a possible toxicity mechanism. That ddCyd does indeed become cytotoxic to human cell by incorporation into DNA was shown by incubating human resting and stimulated lymphocytes with ddCyd. While the drug does not affect cell viability in resting cells it strongly affects cell proliferation upon phytohemagglutinin (PHA) addition.     

Assessment of developmental toxicity of antiretroviral drugs using a rat whole embryo culture system

BACKGROUND: Previous guidelines for HIV-infected pregnant women have recommended zidovudine (ZDV) monotherapy during the second and third trimesters of pregnancy to prevent fetal HIV infection. New guidelines suggest that women should continue or be offered combination antiretroviral therapy (including protease inhibitors) during pregnancy. Nevertheless, little animal or human toxicity data underlie these recommendations. METHODS: We used an in vitro rat whole embryo culture system to assess the embryo toxicity of various nucleoside analogues, namely, ZDV, dideoxyinosine (ddI), and 2', 3'-dideoxycytidine (ddC), and the HIV-1 protease inhibitor, indinavir, both alone and in combination. RESULTS: Although human fetal concentrations of these compounds are unknown, no gross abnormalities were detected after incubation with these agents, either alone or in combination at concentrations that would be expected to be achievable in human maternal serum (1-50 microM). ZDV in combination with ddC at >100 microM, resulted in severe growth retardation and morphologic abnormalities not seen with either agent singly. CONCLUSIONS: We conclude that the combination of ZDV/ddC results in severe concentration-dependent embryo toxicity. No growth retardation or gross morphologic abnormalities were found for any of the agents, either singly or in combination, at clinically relevant concentrations.     

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.     

Lipid conjugates of antiretroviral agents: release of antiretroviral nucleoside monophosphates by a nucleoside diphosphate diglyceride hydrolase activity from rat liver mitochondria.

The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3'-deoxythymidine, 3'-deoxythymidine and 2',3'-dideoxycytidine from the corresponding nucleoside diphosphate diglycerides as a result of rat liver mitochondrial enzymatic activity is shown. The three analogs appeared to be about equally active as substrate for this pyrophosphatase activity which showed maximum conversion rates of 3-6 nmol min-1 mg protein-1 at substrate concentrations between 500 to 800 microM. These results may contribute to the biochemical explanation for the observed anti-HIV activity of this type of phospholipid conjugates in vitro.     

Metabolism of the carbocyclic nucleoside analogue carbovir, an inhibitor of human immunodeficiency virus, in human lymphoid cells

Carbovir (CBV) is a highly selective carbocyclic nucleoside inhibitor of HIV replication in human lymphocytes and is potentially useful in the treatment of AIDS [Vince et al. (1988) Biochem. Biophys. Res. Commun. 156, 1046-1053]. Using human lymphoid cells severely deficient in nucleoside kinases, we were able to identify the route of activation of CBV metabolism. The present studies have demonstrated that CBV is anabolized to the mono-, di-, and triphosphates and to guanosine 5'-triphosphate in CCRF-CEM cells. Conversion to GTP amounted to 15-20% of the total analogue nucleotides formed in the cells and may arise from CBV through depurination and salvage via HGPRT. Evidence was obtained that neither deoxycytidine kinase, adenosine kinase, or mitochondrial deoxyguanosine kinase is primarily involved in the initial step of phosphorylation of CBV in CCRF-CEM cells. In contrast, earlier studies [Johnson & Fridland (1989) Mol. Pharmacol. 36, 291-295] showed that a cytosolic 5'-nucleotidase catalyzes the activation of CBV to the monosphosphate. Other biochemical effects examined showed that the nucleobases hypoxanthine and adenine, but not guanine, their respective nucleosides, and the dideoxynucleosides 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and 3'-azido-3'-deoxythymidine produced significant increased accumulation of CBV nucleotides in CEM cells. The exact mechanism for this potentiation of CBV phosphorylation has not been elucidated but may be due to a modulating effect of intracellular nucleotides on 5'-nucleotidase activity.     

Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli

Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity.     

Montmorillonite: A multifunctional mineral catalyst for the prebiological formation of phosphate esters

Reaction of diiminosuccinonitrile (DISN) with 3'-AMP in the presence of alkali- and alkaline earth-montmorillonites results in the formation of 2',3'-cAMP in aqueous solution. Little or no 2', 3'-cAMP is produced when metal ion concentrations equivalent to that of the metal ion associated with the homoionic clays are used instead of mobntmorillionite. Yields comparable to those obtained with DISN are obtained when diaminomaleonitrile (DAMN) is used in place of DISN as the condensing agent. DAMN, a compound which is more stable than DISN in aqueous solution, is oxidized to DISN on the surface of the clay by Fe+3 in the clay lattice. DISN, the true condensing agent, is thus generated in the presence of the bound 3'-AMP on the montmorillonite surface. The montmorillonite catalyzes the DISN-mediated formation of 2', 3'-cAMP and this product, which binds much less strongly than does the 3'-AMP, is desorbed from the clay surface. This research established that the montmorillonite performs four different functions in its role as catalyst: (1) Binding one of the substrate molecules (3'-AMP) (2) Activating the second substrate (DAMN) (3) Catalyzing the formation of 2', 3'-cAMP (4) Releasing the reaction product so another substrate molecules can bind to the montmorillonite.     

Synthesis of 3′-O-Propargylthymidine as a Candidate Antiretroviral Agent

Abstract

3′-O-Propargylthymidinc, which may be viewed as a stnictural analogue of the potent antiretroviral agent 3′-azido-3′-deoxythymidine (AZT), was synthesized from 5′-O-(4,4′-dimethoxytritylthymidine by reaction with propargyl bromide followed by gentle acidolysis. The 3′-O-propargyl derivative was tested for antiretroviral activity in SC-1 mouse fibroblasts infected with Rauscher murine leukemia virus (MuLV). No inhibition of MuLV proliferation was observed at concentrations of 3′-O-propargylthymidine from 0.001 to 100 μM. whereas the IC₅₀ against the host cells was 30 μM. By comparison, AZT had an IC₅₀ for MuLV growth of 0.01 μM and an IC₅₀ for cell growth of >100 μM. Thus, replacement of the 3′-N-N≡N group in AZT by a 3′-OCH₂C≡CH group increased cytotoxicity but decreased antiretroviral activity relative to AZT.     

Use of thiopental in man. Determination of this drug and its metabolites in plasma and urine by liquid phase chromatography and mass spectrometry

Thiopental is an anaesthetic drug which is currently used for cerebral resuscitation. In this last indication the drug is administrated at high doses over a period of several days. Under clinical trials thiopental and two metabolites namely 5 ethyl-5 (1' methyl-3' hydroxy-butyl) 2 thiobarbituric acid and 5 ethyl-5 (1' methyl-3' carboxy-propyl) 2 thiobarbituric acid have been determined by high performance liquid chromatography and mass spectrometry. In human plasma high concentrations of thiopental and 5 ethyl-5 (1' methyl-3' hydroxy-butyl) 2 thiobarbituric acid and a lower concentration of 5 ethyl-5 (1' methyl-3' carboxy-propyl) 2 thiobarbituric acid have been found. In urine samples these metabolites are excreted in large and approximately equal quantities, whereas small amounts of thiopental were recovered.     

Synthesis and anti-HIV evaluation of 2',3'-dideoxy imidazo- and nu-triazolo[4,5-d]pyridazine nucleosides

The syntheses of the 2'-deoxy and 2',3'-dideoxynucleosides of 2,8-diaza-3-deazainosine and the 2',3'-dideoxynucleoside of 2-aza-3-deazainosine were achieved and the pathways leading to these novel nucleosides are described. The preparation of the 2',3'-dideoxynucleoside (1) of 2-aza-3-deazainosine involved deoxygenation of the 2'-deoxy-3'-imidazolide intermediate with n-Bu3SnH and AlBN. The latter nucleoside was synthesized from the known 2'-deoxy derivative of 2-aza-3-deazainosine. The three-step synthesis of 1 from the 2'-deoxy analogue was accomplished in 40% overall yield. Rather than synthesize the corresponding 2',3'-dideoxynucleoside (2) of 2,8-diaza-3-deazainosine in the same manner, i.e. deoxygenation of the 2'-deoxynucleoside, a more cost-effective route was chosen. This pathway involved reductive cleavage of the 5'-protected, 2',3'-thiocarbonate derivative to furnish a mixture of the 2'- and 3'-deoxy isomers. This mixture was not separated, but was deoxygenated by the aforementioned imidazolide method. Using this methodology, 2 was prepared in 23% overall yield from 2,8-diaza-3-deazainosine. Nucleosides 1 and 2 were evaluated for antiretroviral activity and were found to be inactive.     

ddC- and 3TC-bis(SATE) monophosphate prodrugs overcome cellular resistance mechanisms to HIV-1 associated with cytidine kinase deficiency.

A 2',3'-dideoxycytidine (ddC)-resistant T-lymphoid cell line (MOLT-4/8rddC250), in which deoxycytidine kinase (dCK) gene-expression was decreased when compared with parental cells, has been selected. Cytotoxic and antiretroviral activity of ddC and 3TC was significantly lower in MOLT-4/8rddC250-than in parental MOLT-4/8 cells. ddC- and 3TC-bis(SATE)phosphotriesters completely overcame cellular resistance mechanisms and showed comparable both cytotoxic and antiretroviral activity in parental and ddC-resistant cells.     

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.