bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance Profiles of Cyclic and Linear Inhibitors of HIV-1 Protease
Authors: Ahlsén G , Hultén J , Shuman CF , Poliakov A , Lindgren MT , Alterman M , Samuelsson B , Hallberg A , Danielson UH
Published: March 2015

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, I84V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1–30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, I84V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the I84V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.
Coumarins and Pyranocoumarins, Potential Novel Pharmacophores for Inhibition of Measles Virus Replication
Authors: Barnard Dale L , Xu Ze-Qi , Stowell Valerie D , Yuan Hongwei , Smee Donald F , Samy Raghu , Sidwell Robert W , Nielsen M Kim , Sun Lihui , Cao Hua , Li Ailing , Quint Clay , Deignan Jeffrey , Crabb Jennifer , Flavin Michael T
Published: March 2015

A series of coumarin and pyranocoumarin analogues were evaluated in vitro for antiviral efficacy against measles virus (MV), strain Chicago. Of the 22 compounds tested for inhibition, six were found to have selectivity indices greater than 10. These were compounds 5-hydroxy-7-propionyloxy-4-propylcoumarin (2a), 5,7-bis(tosyloxy)-4-propylcoumarin (7); 5-hydroxy-4-propyl-7-tosyloxy-coumarin (8); 6,6-dimethyl-9-propionyloxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b‘]dipyran-2-one (9); 6,6-dimethyl-9-pivaloyloxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b’]dipyran-2-one (10); and 7,8-cis-10,11,12-trans-4-propyl-6,6,10,11-tetramethyl-7,8,9-trihydroxy-2H,6H,12H-benzo[1,2-b:3,4-b‘:5,6- b“]tripyran-2-one (18). Three of the active drugs were propyl coumarin analogues (2a, 7 and 8), two were dipyranone or chromeno-coumarins (9 and 10), and one was a benzotripyranone with a coumarin nucleus (18). Some appeared to be rather specific and potent inhibitors of MV with EC50 values ranging from 0.2 to 50 μg/ml and the majority of the EC50 values being less than 5 μg/ml. The compounds inhibited an additional nine strains of MV, and in virucidal tests the drugs did not physically disrupt the virion to inhibit virus replication. The inhibitory activity for one of the compounds tested (7) was somewhat dependent on virus concentration and it was still active when added to cells up to 24 h after virus exposure. When used in combination with ribavirin, compound 7 appeared not to profoundly affect the antiviral efficacy of ribavirin or its cell-associated toxicity. However, a slightly antagonistic MV-inhibitory effect was observed at the highest concentration of ribavirin used in combination with most concentrations of compound 7 tested. This and related compounds may be valuable leads in the development of a potent and selective class of MV inhibitors that could be used in future in the clinic.
Alkyloxyphenyl Furano Pyrimidines as Potent and Selective Anti-VZV Agents with Enhanced Water Solubility
Authors: McGuigan Christopher , Blewett Sally , Siccardi Dario , Erichsen Jonathan T , Andrei Graciela , Snoek Robert , De Clercq Erik , Balzarini Jan
Published: March 2015

We have previously reported bicyclic furanopyrimidines as potent and selective inhibitors of varicella zoster virus (VZV) with subnanomolar activity for p-alkylphenyl substituted analogues. These compounds are highly lipophilic and of limited water solubility. In an effort to address this issue, and with a view to oral dosing, we have sought to enhance water solubility whilst retaining high antiviral potency and we herein report a novel series of p-alkyloxyphenyl compounds which contain a phenolic ether atom intended to boost hydrophilicity. We report the synthesis, characterisation and antiviral evaluation of this series and note the retention of extremely high antiviral potency, with EC50 values as low as 1 nanomolar.
Molecular Mechanism of DAPD/DXG against Zidovudine- and Lamivudine- Drug Resistant Mutants: A Molecular Modelling Approach
Authors: Chong Youhoon , Borroto-Esoda Katyna , Furman Phillip A , Schinazi Raymond F , Chu Chung K
Published: March 2015

In order to understand molecular mechanism of antiviral drug resistance of HIV-1 reverse transcriptase (RT) as well as potent antiviral activity of 2,6-diaminopurine dioxolane (DAPD) [prodrug of (–)-β-D-dioxolane guanine (DXG)] against drug-resistant RTs, molecular modelling studies of three structurally distinct nucleoside RT inhibitor (NRTI)-triphosphates (TP) [zidovudine (AZT)-TP, lamivudine (3TC)-TP and DXG-TP] complexed with the wild-type (WT) and mutated RT were conducted. The computational analyses indicated that the antiviral activity and the calculated relative binding energy of the RT inhibitor triphosphates can be correlated, and the minimized structures gave information on the molecular mechanism of drug resistance conferred by mutations. The interactions between the NRTI-TP and adjacent amino acid residues (Lys65, Lys70, Arg72, Tyr115 and/or Gln151) played important roles in stabilizing the enzyme—inhibitor complex. Particularly, Arg72 was found to stabilize the dioxolane and oxathiolane sugar moiety through hydrogen bonding, which was responsible for favourable binding affinity of DXG-TP to AZT- as well as 3TC-resistant mutants. The conformational changes in these amino acid residues caused by mutation always affected the changes in the tertiary structures of enzyme-inhibitor complexes through either closing or opening the gap between the fingers and palm domains. The enzyme-inhibitor complexes with good binding affinity showed tight binding modes by closing the gap between the two domains, whereas weak inhibitors gave open and loose complexes.
Review Hepatitis B Virus Resistance to Lamivudine and its Clinical Implications
Authors: Liu Xuefeng , Schinazi Raymond F
Published: March 2015

Lamivudine is the first orally available drug approved for treatment of chronic hepatitis B, but hepatitis B virus (HBV) resistance to lamivudine appears to be a sine qua non in the therapy of HBV. The mutations at the FLLA and YMDD motif in the domains B and C of HBV polymerase contribute to this resistance. These mutations are found at codon (or AA) rtL180M and rtM204V/I in the reverse transcriptase (RT) domain of the HBV polymerase for all genotypes according to a new standardized RT domain numbering system. The resistant HBV may be less replication-competent in vitro and in vivo, and it is rarely associated with markedly increased HBV replication or liver injury. Therefore, certain physicians favour continuing lamivudine therapy even after emergence of HBV resistance with the expectation of maintaining lower-than baseline HBV DNA, alanine aminotransferase, and histological improvement, and avoiding reversion to wild-type HBV until additional antiviral strategies are developed. Ultimately, once several antiviral agents are approved, combination strategy is likely to be incorporated in antiviral treatment for chronic HBV to suppress, prevent or minimize the emergence of resistant virus.
Sulphated and Sulphonated Polymers Inhibit the Initial Interaction of Hepatitis B Virus with Hepatocytes
Authors: Ying C , Van Pelt JF , Van Lommel A , Van Ranst M , Leyssen P , De Clercq E , Neyts J
Published: March 2015

The initial step during hepatitis B virus (HBV) infection is the specific attachment of the virus to the hepatocyte. Here we studied whether the binding of HBV to hepatocytes can, as is the case with most other enveloped viruses, be blocked by polyanionic compounds. Viral particles produced by HepAD38 cells were used as inoculum and HBV-negative HepG2 cells, as well as primary human hepatocytes, as target cells. Three sulphated polymers, that is, PAVAS (a co-polymer of acrylic acid with vinyl alcohol sulphate), heparin and dextran sulphate (DS) (MW 5000), and the sulphonated polymer PAMPS [poly(2-acryl-amido-2-methyl-1-propanesulfonic acid] (MW ≈7000–12000), proved strong inhibitors of the binding of HBV to HepG2 cells and primary hepatocytes. The 50% effective concentration (EC50) for inhibition of HBV binding to HepG2 cells by PAVAS, heparin, DS and PAMPS was 1.3 μg/ml, 1.6 μg/ml, 1.8 μg/ml and 3.3 μg/ml, respectively, and to primary hepatocytes 1.6 μg/ml (PAVAS), 1.6 μg/ml (heparin), 2.6 μg/ml (DS) and 4.1 μg/ml (PAMPS). These values are in the same range as the concentrations required for these compounds to prevent such viruses as herpesviruses and HIV from binding to cells. These findings may be helpful in elucidating the mechanism of the initial interaction of HBV with hepatocytes.
Viral Pharmacodynamic Model for (–)-β-2′,3′-Dideoxy-5-Fluoro-3′-Thiacytidine (Emtricitabine) in Chronically Infected Woodchucks
Authors: Hurwitz Selwyn J , Tennant Bud C , Korba Brent E , Liberman Irina , Gerin John L. , Schinazi Raymond F
Published: March 2015

There is a need for adequate models of virus depletion in animals and humans as a function of drug dose in order to plan starting dose regimens in the clinic for new antiretroviral nucleoside agents. An indirect response pharmacodynamic model was fitted to link the plasma pharmacokinetics from a 28 day treatment with the nucleoside reverse transcription inhibitor emtricitabine [(–)-FTC], with the resulting virus depletion and recovery profiles in woodchucks chronically infected with woodchuck hepatitis B virus. In this approach it is assumed that the virus is eliminated from serum in a first order fashion and that the fraction of serum virus load produced per day is inhibited by the accumulation of nucleoside triphosphate in a manner that could be described using a Hill equation. Nadir virus load values were inversely related to pretreatment virus load levels within each dose group. A median inhibitory concentration value of 1.5 μM for (–)-FTC triphosphate, previously measured against the isolated viral polymerase of woodchuck hepatitis, was used in model fitting. The fitted value for concentration exponent η of 3.46 indicated a greater than linear sensitivity of virus inhibition with dose. Since the post-treatment virus rebound was much greater than predictions of an initial model, a dose-dependent rebound factor was incorporated in the final model. The rebound factor was maximal at the end of (–)-FTC treatment and decayed mono-exponentially with a rate constant Kreb of 0.11/day. The model inferred decay half-life of (–)-FTC triphosphate in the apparent ‘effect compartment’ of the model was similar to the half-life value previously estimated for human hepatitis B virus-infected hepatocytes. The model described adequately the virus depletion and recovery profiles for the dose range tested and could be adapted for the selection of starting doses for future animal and human studies with emtricitabine and other nucleoside analogues in development.
Inhibition of Human T-Lymphotropic Virus Type I Gene Expression by the Streptomyces-Derived Substance EM2487
Authors: Wang X , Okamoto M , Kawamura M , Izumo S , Baba M
Published: March 2015

EM2487, a Streptomyces-derived substance, has previously been shown to inhibit HIV-1 replication in both acutely and chronically infected cells. In this study, we found that EM2487 was also a selective inhibitor of human T-lymphotropic virus type I (HTLV-I) replication in persistently infected cells. Its 50% effective concentrations for HTLV-I p19 antigen production were 3.6 and 1.2 μM in MT-2 and MT-4 cells, respectively. However, the compound did not reduce cell proliferation and viability at these concentrations. The 50% cytotoxic concentrations of EM2487 were 30.6 and 5.7 μM in MT-2 and MT-4 cells, respectively. The compound also displayed selective inhibition of HTLV-I production in peripheral blood mononuclear cells obtained from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis. Quantitative reverse transcription PCR analysis revealed that EM2487 selectively suppressed HTLV-I mRNA synthesis in MT-2 cells in a dose-dependent fashion. However, the compound did not inhibit endogenous Tax-induced HTLV-I long terminal repeat-driven reporter gene expression. Furthermore, intracellular Tax accumulation was not suppressed in MT-2 cells exposed to EM2487. These results suggest that the inhibition occurred at the viral transcription level, but it cannot be attributed to the inhibition of the Tax function.
Polysulfonates Derived from Metal Thiolate Complexes as Inhibitors of HIV-1 and Various other Enveloped Viruses In Vitro
Authors: Bergstrom Donald E , Lin Xiaoping , Wood Troy D , Witvrouw Myriam , Ikeda Satoru , Andrei Graciela , Snoeck Robert , Schols Dominique , De Clercq Erik
Published: March 2015

Sodium 2-mercaptoethanesulfonate reacts with the metal ions Pd(II), Pt(II), Ag(I), Cd(II) and Zn(II) to yield complexes containing multiple anionic sulfonate sites. On the basis of spectroscopic and other analytical data the complexes were assigned the tentative molecular formulas: Pd6(SCH2CH2SO3Na)12, Ptn(SCH2CH2SO3Na)2n+2, Agn(SCH2CH2SO3Na)n, Na2Zn4(SCH2CH2SO3Na)10, and Na2Cd4(SCH2CH2SO3Na)10. The complexes displayed a variety of differences in activity towards DNA and RNA viruses. The platinum complex showed no measurable cytotoxicity and exhibited a spectrum of antiviral activity resembling that of dextran sulfate. It was active against HIV-1 and HIV-2, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine kinase-deficient HSV-1, human cytomegalovirus, vesicular stomatitis virus (VSV), influenza A virus, respiratory syncytial virus (RSV), Sindbis virus, Junin virus and Tacaribe virus. The palladium complex also showed no measurable cytotoxicity, but was completely inactive against most viruses, with one notable exception: both HIV-1 and HIV-2 were substantially inhibited by the palladium complex. The silver complex showed significantly less antiviral activity and greater cytotoxicity than the platinum complex but did show some selectivity against RSV. The zinc complex showed only modest activity against VSV, RSV, Junin virus, and Tacaribe virus, and like the silver compound was more cytotoxic than either the platinum or palladium complex. The cadmium complex was toxic to all of the cell lines used for in vitro evaluation of antiviral activity. Based on these results, the platinum and palladium compounds appear to be promising candidates for further studies, that is, as vaginal microbicides in the prevention of genital HIV and/or HSV transmission.
Effects of Aryl Substituents on the Anti-HIV Activity of the Arylphosphoramidate Derivatives of Stavudine
Authors: Uckun Fatih M , Samuel P , Qazi S , Chen C , Pendergrass S , Venkatachalam TK
Published: March 2015

We compared the anti-HIV activity of 13 phenyl phosphate derivatives of stavudine (2′,3′-didehydro-2′,3′-dideoxythymidine/d4T) by examining their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells. Our results show that the introduction of electron-withdrawing substituents enhances the activity of these phosphoramidate derivatives. The rate of chemical hydrolysis under alkaline conditions (but not the lipophilicity) predicted the potency of the compounds.
Synthesis of Methylenecyclobutane Analogues of Nucleosides with Axial Chirality and Their Phosphoralaninates: A New Pronucleotide Effective against Epstein-Barr Virus
Authors: Wang Ruifang , Kern Earl R , Zemlicka Jiri
Published: March 2015

Methylenecyclobutane analogues of 2′-deoxyadenosine, 2′-deoxyguanosine and 2′- deoxycytidine, and the corresponding phosphoralaninate pronucleotides comprising adenine and guanine bases, were synthesized as potential antiviral agents. Phosphoralaninate of adenine methylenecyclobutane was a potent inhibitor of replication of Epstein-Barr virus (EBV) in Daudi cell culture. Phosphoralaninate of guanine analogue was inactive but both pronucleotides were substrates for porcine liver esterase. Adenine methylenecyclobutane analogue was deaminated by adenosine deaminase.
Bicyclic Nucleoside Inhibitors of Varicella-Zoster Virus: Effect of Terminal Aryl Substitution in the Side-Chain
Authors: Carangio A , Srinivasan S , McGuigan C , Andrei G , Snoeck R , De Clercq E , Balzarini J
Published: March 2015

We have previously reported the discovery and preliminary structure-activity relationships of a new class of inhibitors of varicella-zoster virus (VZV). These novel furanopyrimidine nucleosides bear unusual bicyclic base moieties and exhibit complete specificity for VZV. Limited in vitro cytotoxicity has been detected and the bicyclic nucleosides are now well established as a new family of potent antivirals. Our initial studies revealed an absolute requirement of a long alkyl side-chain, with an optimal chain length of C8-C10, for antiviral activity. Following further studies, we recently reported a significant enhancement of both antiviral potency and selectivity by the inclusion of a phenyl group within the alkyl side-chain of these compounds. The new lead p-alkylphenyl analogues displayed EC50 values below 1 nM versus VZV and selectivity index values >1000000. We herein report the synthesis and characterization of a further series of alkylaryl analogues bearing terminal phenyl groups with varying n-alkyl side-chain lengths. Synthesis of the target bicyclic systems involved the Pd-catalysed coupling of terminal acetylenes with 5-iodo-2′-deoxyuridine to give intermediate 5-alkynyl nucleosides which were then cyclized in the presence of copper (I) iodide. The current compounds display excellent selectivity for VZV with no detectable in vitro cytotoxicity but despite being chemically isomeric with the previous lead p-alkylphenyl analogues, the compounds reported herein exhibit only moderate antiviral activities. A possible correlation between antiviral activity and conformational freedom of the side-chain is discussed.
Inhibition of HIV-1 Replication by an HIV-1 Dependent Ribozyme Expression Vector with the Cre/loxP (ON/OFF) System
Authors: Habu Yuichiro , Miyano-Kurosaki Naoko , Nagawa Takashi , Matsumoto Norihiko , Takeuchi Hiroaki , Takaku Hiroshi
Published: March 2015

Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator ( tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAiMet promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudo-type viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.
A Novel Substance Purified from Perilla Frutescens Britton Inhibits an Early Stage of HIV-1 Replication without Blocking Viral Adsorption
Authors: Kawahata T , Otake T , Mori H , Kojima Y , Oishi I , Oka S , Fukumori Y , Sano K
Published: March 2015

Pf-gp6, a 6 kDa anti-degranulation glycoprotein purified from the extract of Perilla frutescens, was examined for its antiviral activity against HIV-1 and HIV-2 in vitro. HIV-1-induced cytopathic effect and proviral DNA synthesis were inhibited in the presence of Pf-gp6. The 50% inhibitory concentrations of Pf-gp6 for various HIV-1 strains, including clinical isolates and CCR5-using (R5) HIV-1, ranged between 1.3 and 71.0 μg/ml, depending on the combination of viral strain and host cell. Furthermore, Pf-gp6 did not directly inactivate infectious viral particles. A time-of-addition experiment revealed that Pf-gp6 lost its activity before zidovudine but after the CXCR-4 antagonist AMD3100 during the early stage of viral infection. Although the pinpoint target of Pf-gp6 remains to be elucidated, it may interfere with a step between viral entry and reverse transcription.
Selection of a Thiazole Urea-Resistant Variant of Bovine Viral Diarrhoea Virus That Maps to the RNA-Dependent RNA Polymerase
Authors: King Robert W , Scarnati Helen T , Priestley E Scott , De Lucca Indawati , Bansal Anu , Williams J Kendall
Published: March 2015

By passing wild type bovine viral diarrhoea virus (BVDV) in increasing concentrations of DPC-A69280–29, a thiazole urea class compound that inhibits BVDV replication, we were able to select several variants of BVDV that exhibited decreased susceptibility to this compound. When the non-structural genes of these variants were sequenced and compared with wild type, only one change was common to all the variants that also exhibited resistance to DPC-A69280–29 (>10-fold increase in IC50). This change was a T-to-A transversion at position 11198 of the BVDV genome, which would cause a predicted substitution of isoleucine for phenylalanine at amino acid 78 of the RNA-dependent RNA polymerase (RdRp). This substitution would occur in a region of the BVDV RdRp which has been proposed to be important for the formation of the RdRp homodimer that is essential for the activity of the enzyme. However, since DPC-69280-29 inhibits BVDV replication by interfering with the initiation of viral RNA synthesis, we discuss the possibility that this region of the BVDV RdRp also may play a role in the initiation process. Furthermore, since this region is located fairly close to the template RNA, we also propose that the role it plays may involve either template selection, stabilization or processivity.
The Quest for an Efficacious Antiviral for Respiratory Syncytial Virus
Authors: Torrence Paul F , Powell Linda D
Published: March 2015

Respiratory syncytial virus (RSV) continues as an emerging infectious disease not only among infants and children, but also for the immune-suppressed, hospitalized and the elderly. To date, ribavirin (Virazole) remains the only therapeutic agent approved for the treatment of RSV. The prophylactic administration of palivizumab is problematic and costly. The quest for an efficacious RSV antiviral has produced a greater understanding of the viral fusion process, a new hypothesis for the mechanism of action of ribavirin, and a promising antisense strategy combining the 2′-5′ oligoadenylate antisense (2–5′A-antisense) approach and RSV genomics.
A Cell-Based Model of HCV-Negative-Strand RNA Replication Utilizing a Chimeric Hepatitis C Virus/Reporter RNA Template
Authors: King Robert W , Zecher Marianne , Jeffries Matthew W , Carroll Denise R , Parisi Joseph M , Pasquinelli Claudio
Published: March 2015

The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric ‘HCV-like’ RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5′ and 3′ untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5′ HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4α that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.
Ribavirin Derivatives with a Hexitol Moiety: Synthesis and Antiviral Evaluation
Authors: Van Aerschot Arthur , Schepers Guy , Busson Roger , Rozenski Jef , Neyts Johan , De Clercq Erik , Herdewijn Piet
Published: March 2015

Current standard therapy for the treatment of chronic infections with hepatitis C virus consists of combination therapy with (pegylated) interferon-α and ribavirin. 1,5-Anhydrohexitol nucleoside analogues are constrained congeners known to mimic the ribonucleoside conformation. Within this series some analogues are endowed with strong antiviral properties, particularly against herpesviruses. The six-membered anhydrohexitol ring was, therefore, combined with the triazolyl carboxamide moiety of ribavirin, thus providing a new series of ribavirin analogues. None of the newly synthesized compounds elicited any substantial antiviral activity, neither against herpes simplex virus type 1 and 2, nor against bovine viral diarrhoea virus (a surrogate for hepatitis C virus) or the yellow fever virus.
HIV-1 Resistance Profile of the Novel Nucleoside Reverse Transcriptase Inhibitor β-D-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine (Reverset™)
Authors: Geleziunas Romas , Gallagher Karen , Zhang Hangchun , Bacheler Lee , Garber Sena , Wu Jing-Tao , Shi Guoen , Otto Michael J , Schinazi Raymond F , Erickson-Viitanen Susan
Published: March 2015

Nucleoside reverse transcriptase inhibitors (NRTIs) represent the cornerstone of highly active antiretroviral therapy when combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) or HIV-1 protease inhibitors (PIs). Unlike the NNRTIs and PIs, NRTIs must be successively phosphorylated by cellular kinases to a triphosphate form, which represents the active metabolite possessing antiviral activity. Emergence of viral resistance to NRTIs has severely hampered treatment options for persons infected with HIV-1. As such, there is an urgent need to develop NRTIs capable of suppressing NRTI-resistant strains of HIV-1. We have recently reported that the cytidine analogue D-d4FC (DPC817, Reverset™) effectively inhibits clinically prevalent resistant strains of HIV-1. In this report, we have extended these findings and now describe a detailed resistance profile for this novel NRTI. By examining a panel of 50 viruses carrying RTs derived from HIV-1 clinical isolates displaying a wide range of NRTI resistance mutations, we report that the median fold increase in effective antiviral concentration for such a panel of viruses is 3.2, which is comparable to tenofovir (2.8-fold) and didanosine (2.4-fold). D-d4FC is highly effective at inhibiting subsets of lamivudine-and zidovudine-resistant variants but, like other NRTIs, seems less potent against multi-NRTI-resistant viruses, particularly those carrying the Q151M complex of mutations. Finally, in vitro selections for HIV-1 mutants capable of replicating in the presence of D-d4FC yielded a mutant carrying the RT K65R mutation. This mutation confers 5.3- to 8.7-fold resistance to D-d4FC in vitro. These findings suggest that D-d4FC may represent an alternative NRTI for the treatment of individuals infected with lamivudine- and zidovudine-resistant strains of HIV-1.
RNA as a Target for Developing Antivirals
Authors: McKnight Kevin L , Heinz Beverly A
Published: March 2015

The base of knowledge concerning RNA structure and function has been expanding rapidly in recent years. Simultaneously, an increasing awareness of the pivotal role RNA plays in viral diseases has prompted many researchers to apply new technologies in high-throughput screening and molecular modelling to the design of antiviral drugs that target RNA. While the two RNA viruses with the greatest unmet medical need, HIV and HCV, have been most actively pursued, the approaches discussed in this review are relevant to all virus infections. Both traditional small-molecule and large-molecule therapeutics, such as antisense, ribozymes and interfering dsRNAs have been described, and several molecules are under development for commercialization. The purpose of this review is to summarize the current state of the art in this field and to postulate new directions in the future.
N4-Acyl-Modified D-2′,3′-Dideoxy-5-Fluorocytidine Nucleoside Analogues with Improved Antiviral Activity
Authors: Shi Junxing , Mathew Judy S , Tharnish Phillip M , Rachakonda Suguna , Pai S Balakrishna , Adams Marjorie , Grier Jason P , Gallagher Karen , Zhang Hangchun , Wu Jing-Tao , Shi Guoen , Geleziunas Romas , Erickson-Viitanen Susan , Stuyver Lieven , Otto Michael J , Watanabe Kyoichi A , Schinazi Raymond F
Published: March 2015

A series of 2,3-dideoxy (D2) and 2,3-didehydro-2,3-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4- p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4- p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4- p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5′-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5′-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.
Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase
Authors: Ray Adrian S , Schinazi Raymond F , Murakami Eisuke , Basavapathruni Aravind , Shi Junxing , Zorca Suzana M , Chu Chung K , Anderson Karen S
Published: March 2015

β-D and β-L-enantiomers of 2′,3′-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2′-fluorine substitution on the pre-steady state incorporation of 2′-deoxycytidine-5′-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-β-L-2′,3′-dideoxy-3′-thia-5-fluoro-cytidine (L-FTC, Emtriva™), and (+)-β-D-2′,3′-dide-hydro-2′,3′-dideoxy-5-fluorocytidine (D-D4FC, Reverset™), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2′ position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.
Synthesis and Antiviral Activity of Some 5′-N-Phthaloyl-3′-Azido-2′,3′-Dideoxythymidine Analogues
Authors: Balzarini Jan , De Clercq Erik , Kaminska Beata , Orzeszko Andrzej
Published: March 2015

A variety of substituted 5′- N-phthaloyl-3′-azido-2′,3′-dideoxythymidine derivatives has been evaluated for their activity against HIV-1, HIV-2 and Moloney murine sarcoma virus (MSV) in cell culture. Most of the 3′-azido-2′,3′-dideoxythymidine (AZT, zidovudine) derivatives showed antiviral activity in the lower micromolar concentration range and there was a close correlation between their anti-HIV and anti-MSV activity (r=0.99). The adamantyl phthaloyl derivative was active at sub-micromolar concentrations. None of the compounds showed marked cytostatic activity. They did not inhibit recombinant HIV-1 reverse transcriptase. All compounds were inactive against HIV in thymidine kinase-deficient cells, pointing to the compounds' requirement to release free AZT to afford antiviral efficacy.
Viramidine, a Prodrug of Ribavirin, Shows Better Liver-Targeting Properties and Safety Profiles Than Ribavirin in Animals
Authors: Lin Chin-Chung , Yeh Li-Tain , Vitarella Domenico , Hong Zhi
Published: March 2015

Ribavirin, part of the current first line combination therapy for the treatment of chronic hepatitis C, may cause haemolytic anaemia and poses a significant challenge to the clinical management of the disease. Viramidine, a prodrug of ribavirin, is currently under development. In-vitro partition demonstrated that viramidine had less association with RBCs than ribavirin in rat, monkey and man, and thus has less liability for haemolytic anaemia than ribavirin. In a whole body autoradiography study in rats following oral dosing (30 mg/kg) of [14C]ribavirin or [14C]viramidine to monkeys, viramidine produced 32% higher radioactivity in the liver than ribavirin, indicating a better liver-targeting properties. In portal vein-cannulat-ed cynomolgus monkeys following single oral dosing (30 mg/kg) of [3H]viramidine or [3H]rib-avirin, viramidine retained 3X higher radioactivity in the liver than ribavirin. Viramidine dosing also produced a higher viramidine to ribavirin ratio in portal plasma than in systemic plasma, indicating that the liver was the main site for the viramidine conversion to ribavirin and subsequent trapping of the drug. After multiple oral dosing (10 mg/kg) of [14C]ribavirin or [14C]viramidine to monkey, viramidine yielded three times the drug level in the liver but only half in RBCs compared to rib-avirin. Viramidine and ribavirin had comparable toxicity profiles in a 28-day toxicity study in rats. In contrast, viramidine had much better safety profiles than ribavirin in a 28-day toxicity study in monkeys. In conclusion, viramidine has better liver-targeting properties and safety profiles than ribavirin in animals.
Synthesis and Evaluation of Antirhinovirus Activity of 3-Hydroxy and 3-Methoxy 2-Styrylchromones
Authors: Desideri Nicoletta , Mastromarino Paola , Conti Cinzia
Published: March 2015

Recently, we identified 2-styrylchromones as a new class of antirhinovirus flavonoids with moderate activity against both rhinovirus groups A and B. In order to improve the antiviral effect of the first series of tested 2-styrylchromones, a hydroxy or methoxy group was introduced in position 3 of the chromone ring. Cytotoxicity and antiviral activity of the new synthesized compounds were evaluated in HeLa cell cultures infected with rhinoviruses 1B and 14, selected as representative serotypes for viral groups B and A of human rhinoviruses (HRVs), respectively. These antiviral results compared to those obtained for 3-unsubstituted 2-styrylchromones indicate the greater potency of 3-hydroxy and 3-methoxy derivatives against both serotypes.
Antiviral Activity of CTC-8 against Herpes Simplex Virus (HSV-1) in Cell Culture: Evidence for a Selective Antiviral Effect via a Cellular Mechanism
Authors: Fitzmaurice Tim , Harper David R , Field Hugh J
Published: March 2015

A synthetic programme produced a series of compounds related to natural prostaglandins, which are known to affect the growth of a number of viruses. Several of the compounds showed potent biological activity including antiviral effects. The compound CTC-8 [(S)-4-tert-butyldimethylsilyloxy-2-cyclopenten-1-one] contains the cyclopentenone ring of prostaglandin A1, but the extended side chains common to the prostaglandin family are truncated. The present study demonstrates that CTC-8 inhibits HSV-1 replication in cell culture at sub-toxic concentrations. The antiviral effect was evidenced by reduction in infectious virus yield, although the compound was not effective in the standard plaque-reduction assay. Time-of-addition studies and other experiments provide a possible explanation for these results by suggesting that the antiviral activity is confined to a single cycle. Under the standard conditions of high-multiplicity infection in BHK cells it was notable that CTC-8 is most effective when added for a short period 6–8 h post-infection. Furthermore, multiple passage of HSV-1 in the presence of CTC-8 did not result in the selection of resistant mutants. The results of these and other experiments are consistent with the hypothesis that the mechanism by which CTC-8 inhibits virus replication involves a cellular target. These results encourage further research into the therapeutic potential of this series of compounds.
Development of New Antivirals for Herpesviruses
Authors: Eizuru Yoshito
Published: March 2015

The long-term treatment of herpesvirus infections with current antivirals in immunocompromised hosts leads to the development of drug-resistant viruses. Because nearly all currently available antivirals finally target viral DNA polymerase, virus resistant to one drug often shows cross-resistance to other drugs. In addition, nearly all the antivirals show various kinds of side effects or poor bioavailability. This evidence highlights the need for developing new antivirals for herpesviruses that have the different viral targets. Recently, high-throughput screening of large compound collections for inhibiting specific viral enzymes, or in vitro cell culture assay, has identified several new antivirals that target different viral proteins. These include the inhibitors of helicase/primase complex, terminase complex, portal protein and UL97 protein kinase. In addition, non-nucleoside inhibitors for viral DNA polymerase have been also developed. This review will focus on these new compounds that directly inhibit viral replication.
Inhibition of Severe Acute Respiratory Syndrome-Associated Coronavirus (SARSCoV) by Calpain Inhibitors and β-D-N4-Hydroxycytidine
Authors: Barnard Dale L , Hubbard Valerie D , Burton Jared , Smee Donald F , Morrey John D , Otto Michael J , Sidwell Robert W
Published: March 2015

We evaluated two types of compounds for efficacy in inhibiting SARSCoV replication in vitro: calpain inhibitors (a class of cellular cysteine proteinases) and a number of nucleoside analogues. Cytopathic effect reduction assays visually determined with spectrophotometric verification by neutral red (NR) uptake assay were used to evaluate cytotoxicity and antiviral potency of the compounds. Significantly inhibitory compounds were then evaluated in virus yield reduction assays. Two calpain inhibitors, Val-Leu-CHO (calpain inhibitor VI) and Z-Val-Phe-Ala-CHO (calpain inhibitor III), were the most potent inhibitors of SARSCoV. By virus yield reduction assay, calpain inhibitor VI had a 90% effective concentration (EC90) of 3 μM and calpain inhibitor III had an EC90 of 15 μM. β-D-N4-hydroxycytidine was the most selective nucleoside analogue inhibitor with an EC90 of 6 μM by virus yield reduction assay. These compounds or analogues warrant further evaluation as potential therapies for treating SARS or could be used as lead compounds for discovery of more potent SARSCoV inhibitors.
In Vitro and in Vivo Transport and Delivery of Phosphorothioate Oligonucleotides with Cationic Liposomes
Authors: Miyano-Kurosaki Naoko , Barnor Jacob Samson , Takeuchi Hiroaki , Owada Takashi , Nakashima Hideki , Yamamoto Naoki , Matsuzaki Tetsuo , Shimada Fumiyuki , Takaku Hiroshi
Published: March 2015

A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of anti-sense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1NL432 at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 μM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post-infection, we confirmed that p24 antigen production was inhibited by the anti-sense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/ cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.
Inhibitors of Measles Virus
Authors: Barnard Dale L
Published: March 2015

Measles virus (MV) infections have been almost eradicated in some industrialized nations. However, MV continues to cause severe disease and mortality in the world and is responsible for clusters of exogenous-borne disease in essentially disease-free countries. Because of the ebb and flow of immunization campaigns, especially in the poverty-stricken and war-torn Third World, and the ominous potential for severe disease and mortality, it is vital that research for discovery of therapeutic countermeasures should continue. To that end, a number of compounds have been evaluated for efficacy in vitro and in animal models, and several therapeutic modalities have been tested in the clinic. The only current therapies used in the clinic include ribavirin administered orally or intravenously, alone or in combination with immune serum globulin; these therapies have demonstrated variable efficacy. Therefore, drug discovery efforts have been launched to supplement the existing treatments for MV infections. Antisense molecules, adenosine and guanosine nucleosides, including ring-expanded ‘fat’ nucleoside analogues, brassinosteroids, coumarins, peptide inhibitors, modulators of cholesterol synthesis and a variety of natural products have been screened for efficacy and toxicity both in vitro and in animals. However, none of these agents has gone into human clinical trials and most will not merit further development due to toxicity concerns and/or low potency. Thus, further research is needed to develop more potent and less toxic drugs that could be used for treating MV infections to supplement the existing MV vaccine campaigns.
Clinical Utility of Current NNRTIs and Perspectives of New Agents in This Class under Development
Authors: Zhang Zhijun , Hamatake Robert , Hong Zhi
Published: March 2015

Highly active antiretroviral therapy (HAART) has significantly reduced the number of deaths caused by AIDS. However, the antiviral efficacy of HAART comprising protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is frequently accompanied by a decrease in patients' quality of life. PI-based therapies often fail due to poor adherence caused by heavy pill burden, complex dosing schedules and undesirable side effects. The current trend is to switch from PI-based to PI-sparing regimens consisting of non-nucleoside reverse transcriptase inhibitors (NNRTIs) and NRTIs. Despite some encouraging results from NNRTI-containing therapies, two major concerns in using the currently available NNRTIs remain: 1) low genetic barrier to the emergence of resistance and 2) cross-resistance due to single mutations that often render the whole class of NNRTIs ineffective. Clearly, new and improved NNRTIs are needed to address these concerns.
Attempt to Reduce Cytotoxicity by Synthesizing the L-Enantiomer of 4′-C-Ethynyl-2′-Deoxypurine Nucleosides as Antiviral Agents against HIV and HBV
Authors: Kitano Kenji , Kohgo Satoru , Yamada Kohei , Sakata Shinji , Ashida Noriyuki , Hayakawa Hiroyuki , Nameki Daisuke , Kodama Eiichi , Matsuoka Masao , Mitsuya Hiroaki , Ohrui Hiroshi
Published: March 2015

We investigated the potential of 4′- C-substituted nucleosides for the treatment of HIV-1 and HBV. Of the nucleosides we prepared, several 4′- C-ethynyl-2′-deoxypurine nucleosides showed the most potent anti-HIV activity. However, two candidates, 4′-C-ethynyl-2′-deoxyguanosine and 9-(2-deoxy-4-C-ethynyl-β-D- ribo-pentofuranosyl)-2,6-diaminopurine, were very toxic during in vivo study. On the other hand, lamivudine (3TC) is known to show remarkable activity against HIV and HBV with lower cytotoxicity. Therefore, we attempted to synthesize the L-enantiomer of 4′- C-ethynyl-2′-deoxypurine nucleosides in 20–21 steps. These methods consisted of preparing 4-C-ethynyl-L-sugar, starting from D-arabinose and then condensing the L-sugar derivative with 2,6-diaminopurine. 4′- C-Ethynyl-2′-deoxyguanosine was also prepared by enzymatic deamination from the 2,6-diaminopurine derivative. The compounds' antiviral activity against HIV and HBV was then evaluated. Unfortunately, they demonstrated no activity and no cytotoxicity.