Inhibitors of the Tick-Borne,Hemorrhagic Fever-Associated Flaviviruses

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2′-C-methylcytidine (2′-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2′-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2′-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2′-CMC, AHFV variants with reduced susceptibility to 2′-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2′-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2′-CMC.     

GRL-04810 and GRL-05010, Difluoride-Containing Nonpeptidic HIV-1 Protease Inhibitors (PIs) That Inhibit the Replication of Multi-PI-Resistant HIV-1 In Vitro and Possess Favorable Lipophilicity That May Allow Blood-Brain Barrier Penetration

We designed, synthesized, and identified two novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF), and a difluoride moiety, both of which are active against the laboratory strain HIV-1_LAI (50% effective concentrations [EC₅₀s], 0.0008 and 0.003 μM, respectively) with minimal cytotoxicity (50% cytotoxic concentrations [CC₅₀s], 17.5 and 37.0 μM, respectively, in CD4⁺ MT-2 cells). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1_NL4-3 variants selected by up to 5 μM lopinavir (EC₅₀s, 0.03 and 0.03 μM, respectively) and atazanavir (EC₅₀s, 0.02 and 0.04 μM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC₅₀ in 0.03 to 0.034 μM range for GRL-04810 and 0.026 to 0.043 μM for GRL-05010), while DRV had EC₅₀s between 0.02 and 0.174 μM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (log P) and distribution (log D) coefficients of −0.14 and −0.29, respectively. The in vitro blood-brain barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing the BBB than the currently available PIs, with apparent penetration indexes of 47.8 × 10⁻⁶ and 61.8 × 10⁻⁶ cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert efficient activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moieties may well enhance their penetration across the BBB.     

Nitro/Nitrosyl-Ruthenium Complexes Are Potent and Selective Anti-Trypanosoma cruzi Agents Causing Autophagy and Necrotic Parasite Death

cis-[RuCl(NO₂)(dppb)(5,5′-mebipy)] (complex 1), cis-[Ru(NO₂)₂(dppb)(5,5′-mebipy)] (complex 2), ct-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 3), and cc-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 4), where 5,5′-mebipy is 5,5′-dimethyl-2,2′-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruzi activity than benznidazole, the current antiparasitic drug. Complex 3 was the most potent, displaying a 50% effective concentration (EC₅₀) of 2.1 ± 0.6 μM against trypomastigotes and a 50% inhibitory concentration (IC₅₀) of 1.3 ± 0.2 μM against amastigotes, while it displayed a 50% cytotoxic concentration (CC₅₀) of 51.4 ± 0.2 μM in macrophages. It was observed that the nitrosyl complex 3, but not its analog lacking the nitrosyl group, releases nitric oxide into parasite cells. This release has a diminished effect on the trypanosomal protease cruzain but induces substantial parasite autophagy, which is followed by a series of irreversible morphological impairments to the parasites and finally results in cell death by necrosis. In infected mice, orally administered complex 3 (five times at a dose of 75 μmol/kg of body weight) reduced blood parasitemia and increased the survival rate of the mice. Combination index analysis of complex 3 indicated that its in vitro activity against trypomastigotes is synergic with benznidazole. In addition, drug combination enhanced efficacy in infected mice, suggesting that ruthenium-nitrosyl complexes are potential constituents for drug combinations.     

Structural modification of olibergin A,an isoflavonoid,from Dalbergia stipulacea Roxb. and its cytotoxicity

Fifteen derivatives were synthesized from olibergin A, a major isoflavonoid isolated from the stems of Dalbergia stipulacea Roxb. All compounds were evaluated for cytotoxicity against HCT-116, HT-29, MCF-7 and vero cell lines using MTT assay. Cytotoxicity results showed 5-hydroxy-7,2′,4′,5′-tetramethoxyisoflavone (5) was the most active with IC₅₀ values of 19.03 ± 0.70, 10.83 ± 1.65, 12.53 ± 0.70 and 13.53 ± 0.84 μM against HCT-116, HT-29, MCF-7 and vero cell lines, respectively. It should be noted that 5-hydroxy-7,2′,4′,5′-tetramethoxyisoflavone (5) showed two times less toxicity against vero cells than the cisplatin standard (IC₅₀ = 6.55 ± 0.81 μM) while 5 and cisplatin exhibited nearly equal cytotoxicity against the MCF-7 cell line. 5,7,2′,4′,5′-Pentamethoxyisoflavanone (10) showed an IC₅₀ value of 30.34 ± 1.15 μM against the HCT-116 cell line and exhibited weak cytotoxicity against normal cells, the vero cell line. In addition, 5,7,4′-trihydroxy-2′,5′-dimethoxyisoflavan oxime (13) demonstrated cytotoxicity against HT-29 cells with an IC₅₀ value of 31.41 ± 1.38 μM and displayed weak activity toward the vero cell line. The information revealed that these compounds were suitable for development to anticancer agents against HCT-116, HT-29 and MCF-7 cell lines.

Fifteen derivatives were synthesized from olibergin A, a major isoflavonoid isolated from the stems of Dalbergia stipulacea Roxb.     

In Vitro and In Vivo Inhibition of Murine Gamma Herpesvirus 68 Replication by Selected Antiviral Agents

We have evaluated the susceptibility of the murine gamma herpesvirus 68 (MHV-68) to a variety of antiviral agents. The acyclic nucleoside phosphonate analogs cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and adefovir [9-(2-phosphonylmethoxyethyl)adenine] efficiently inhibited the replication of the virus in Vero cells (50% effective concentrations [EC₅₀s], 0.008, 0.06, and 2.2 μg/ml, respectively). Acyclovir, ganciclovir, and brivudin [(E)-5-(2-bromovinyl)-2′-deoxyuridine] had equipotent activities (EC₅₀s, 1.5 to 8 μg/ml), whereas foscarnet and penciclovir were less effective (EC₅₀s, 23 and ≥30 μg/ml, respectively). The novel N-7-substituted nucleoside analog S2242 [7-(1,3-dihydroxy-2-propoxymethyl)purine] inhibited MHV-68 replication by 50% at 0.2 μg/ml. The susceptibilities of MHV-68 and Epstein-Barr virus (EBV) to cidofovir, HPMPA, adefovir, and acyclovir were found to be comparable. However, for penciclovir, ganciclovir, brivudin, and S2242, major differences in the sensitivity of MHV-68 and EBV were observed, suggesting that MHV-68 is not always an optimal surrogate for the study of antiviral strategies for EBV. When evaluated with a model for lethal MHV-68 infections in mice with severe combined immunodeficiency, cidofovir proved to be very efficient in protecting against virus-induced mortality (100% survival at 50 days postinfection), whereas acyclovir, brivudin, and adefovir had little or no effect.     

Biochemical Characterization of Recombinant Enterovirus 71 3C Protease with Fluorogenic Model Peptide Substrates and Development of a Biochemical Assay

Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant k_cat/K_m of 11.8 ± 0.82 mM⁻¹ min⁻¹. Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z′ factor of 0.87 ± 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC₅₀] = 1.89 ± 0.25 μM) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC₅₀] = 4.54 ± 0.51 μM; 50% cytotoxic concentration [CC₅₀] > 100 μM). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.     

A Conserved Hydrogen-Bonding Network of P2 bis-Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC₅₀s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIV_A02) compared to ritonavir (RTV; EC₅₀, >1.0 μM) and tipranavir (TPV; EC₅₀, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIV_DRV^R_P20), with a 2.6-fold increase in its EC₅₀ (0.097 μM) compared to its corresponding EC₅₀ (0.038 μM) against wild-type HIV-1_NL4-3 (HIV_WT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIV_A02 protease (PR_A02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PR_A02-RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PR_A02, supporting RTV''s compromised antiviral activity (EC₅₀, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIV_WT, HIV_A02, and HIV_DRV^R_P20.     

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as β-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 μM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 μM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC₅₀) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC₅₀] of 50 to >100 μM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.     

Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib by Zinc: Reversal of Amikacin Resistance in Acinetobacter baumannii and Escherichia coli by a Zinc Ionophore

In vitro activity of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by ZnCl₂ with a 50% inhibitory concentration (IC₅₀) of 15 μM. Growth of Acinetobacter baumannii or Escherichia coli harboring aac(6′)-Ib in cultures containing 8 μg/ml amikacin was significantly inhibited by the addition of 2 μM Zn²⁺ in complex with the ionophore pyrithione (ZnPT).     

Effects of Antiviral Drugs on Organic Anion Transport in Human Placental BeWo Cells

Placental drug transfer is important for achieving better pharmacotherapy in pregnant women and in fetuses. In the present study, we examined the effects of anti-hepatitis C virus (HCV) and anti-HIV drugs on organic anion transport in human placental BeWo cells. The cellular uptake of two fluorescence organic anions, 8-(2-[fluoresceinyl]aminoethylthio)adenosine-3′,5′-cyclic monophosphate (8-FcAMP) and fluorescein, was temperature and concentration dependent. The Michaelis constant (K_m) and the maximum uptake rate (V_max) for 8-FcAMP transport in BeWo cells were estimated to be 6.45 ± 0.75 μM and 25.55 ± 5.93 pmol/mg protein/10 min, respectively. The K_m and V_max values for fluorescein uptake were estimated to be 31.2 ± 11.8 μM and 510.9 ± 90.6 pmol/mg protein/10 min, respectively. Several known substrates of organic anion transporters in human placenta, including atorvastatin, glibenclamide, estrone-3-sulfate, and rifampin, inhibited cellular uptake of 8-FcAMP and fluorescein in BeWo cells. Transport of 8-FcAMP and fluorescein was inhibited by the antiviral drugs boceprevir, telaprevir, elvitegravir, and maraviroc. These findings suggest that some antiviral drugs are sufficiently potent to influence placental drug transfer and cause drug-drug interactions.     

Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication by Blocking Viral Ribonuclease H Activity

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC₅₀) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC₅₀) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC₅₀] = 25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.     

Investigation of some benzoquinazoline and quinazoline derivatives as novel inhibitors of HCV-NS3/4A protease: biological,molecular docking and QSAR studies

Morbidity and mortality due to hepatitis C virus (HCV) is a globe health concern. Hence, there is a persistent demand to design and optimize current HCV therapy and develop novel agents. HCV NS3/A4 protease plays an essential role in HCV life cycle and replication. Thus, HCV NS3/A4 protease inhibitors are one of the best therapeutic targets for the identification of novel candidate drugs. Recent studies have shown some benzoquinazolines as potent antiviral agents and promising HAV-3C protease inhibitors. In the present study, a series of benzo[g]quinazolines (1–13) and their quinazoline analogues (14–17) were evaluated for their HCV-NS3/4A inhibitory activities using in vitro assay. Our results revealed that the target compounds inhibited the activity of the NS3/4A enzyme, (IC₅₀ = 6.41 ± 0.12 to 78.80 ± 1.70 μM) in comparison to telaprevir (IC₅₀ = 1.72 ± 0.03 μM) as a reference drug. Compounds 1, 2, 3, 9, 10 and 13 showed the highest activity (IC₅₀ = 11.02 ± 0.25, 6.41 ± 0.12, 9.35 ± 0.19, 9.08 ± 0.20, 16.03 ± 0.34 and 7.21 ± 0.15 μM, respectively). Molecular docking was performed to study the binding modes of the docked-chosen benzo[g]quinazolines, hydrogen bonding, and amino acid residues at the catalytic triad of the NS3/4A enzyme of HCV. The QSAR was determined to explore the relationships between the molecular structures of the targets and their biological activities by developing prediction models among the known HCV NS3/A4 inhibitors and then to predict the inhibitory activity of the target molecules synthesized.

HCV NS3/A4 protease inhibitors are one of the best therapeutic targets for the identification of novel candidate drugs. A series of benzo[g]quinazolines and their quinazoline analogues were evaluated for their HCV-NS3/4A inhibitory activities.     

Evaluation of PD 404,182 as an Anti-HIV and Anti-Herpes Simplex Virus Microbicide

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672–681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD''s virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC₅₀) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC₅₀ determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC₅₀], >300 μM), freshly activated human peripheral blood mononuclear cells (CC₅₀, ∼200 μM), and primary CD4⁺ T cells, macrophages, and dendritic cells (CC₅₀, >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco''s phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD''s microbicidal candidacy in animals and humans.     

A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor,GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration Property In Vitro

We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC₅₀], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC₅₀], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1_NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC₅₀, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2_ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles.     

In Vitro Activity and Resistance Profile of Samatasvir,a Novel NS5A Replication Inhibitor of Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC₅₀s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC₅₀ shift in the presence of 40% human serum in the genotype 1b replicon. The EC₉₀/EC₅₀ ratio was low (2.6). A 50% cytotoxic concentration (CC₅₀) of >100 μM provided a selectivity index of >5 × 10⁷. Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.     

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-l-2′,3′-Didehydro-2′,3′-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells

ß-l-2′,3′-Didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM; 50% cytotoxic dose [CD₅₀] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[³H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5′-mono-, 5′-di-, and 5′-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß-d-N⁴-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μM l-[³H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/10⁶ cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/10⁶ cells. The intracellular half-life of the 5′-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5′-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5′-diphosphate during a drug-free incubation. The high 5′-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.A large number of nucleoside analogues have been described as inhibitors of hepatitis B virus (HBV) and HIV replication. Recently l-nucleoside analogues in particular have gained increasing interest. They are characterized by an opposite configuration from that of the natural d-nucleoside analogues and represent one of the most attractive groups of antiretroviral compounds, including ß-l-2′,3′-dideoxy-3-thiacytidine (3TC) and its 5-fluoro derivative (FTC), ß-l-2′,3′-didehydro-2′,3′-dideoxy-cytidine (l-d4C) and its 5-fluoro derivative (l-d4FC), ß-l-thymidine, ß-l-fluoroarabinosylyluracil (l-FMAU), and ß-l-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-cytidine (l-2′Fd4C) (3, 5, 22).Some of them not only have been found to be more potent than their corresponding d-nucleosides but seem to exhibit lower cytotoxicity and have been proved to be effective and selective agents for the treatment of chronic hepatitis B virus infections (4). However, only long-term therapy with a single nucleoside for several years was shown to be able to completely suppress HBV DNA in serum of patients and to reverse the progression of the disease. The disadvantage connected with such therapy regimens is the development of drug-resistant HBV strains (22). Therefore, the challenge will be to develop more-efficient drugs for shorter treatment regimens and to combine them to reach synergistic or at least additive drug action. This approach has been described not only as being highly efficient for the treatment of HIV infections but also as preventing the development of resistant mutants. Therefore, AIDS therapy is considered a model for future therapy of chronic HBV infections (17).Recently we described a series of new ß-l-N⁴-hydroxydeoxycytidine and ß-l-5-methyl-deoxycytidine derivatives as inhibitors of HBV replication. Between them, ß-l-2′,3′-didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) (Fig. ​(Fig.1)1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD₅₀] for HepG2 cells = 2,500 μM) (12).Open in a separate windowFIG. 1.Structure of l-Hyd4C and possible metabolites formed by reduction (l-d4C) or by deamination (l-d4U).These encouraging features have prompted us to investigate the cellular pharmacology of l-Hyd4C in a hepatic cell line. This included the activation of this unnatural l-deoxycytidine nucleoside to its 5′-mono-, 5′-di-, and 5′-triphosphate, the search for other metabolites, and the estimation of the intracellular half-lives (t_1/2) of the 5′-di- and 5′-triphosphate of l-Hyd4C.(This work was presented in part at BIT''s 5th Anniversary Congress of International Drug Discovery Science and Technology, 7 to 13 November 2007, Xi''an and Beijing, China.)     

Pharmacokinetics and Distribution over the Blood-Brain Barrier of Zalcitabine (2′,3′-Dideoxycytidine) and BEA005 (2′,3′-Dideoxy-3′-Hydroxymethylcytidine) in Rats, Studied by Microdialysis

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2′,3′-dideoxycytidine; n = 4) or BEA005 (2′,3′-dideoxy-3′-hydroxymethylcytidine; n = 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (C_max) was 31.4 ± 5.1 μM (mean ± standard error of the mean) for the brain and 238.3 ± 48.1 μM for muscle. The time to C_max was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 ± 0.64 and 0.85 ± 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 ± 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with C_maxs of 5.7 ± 1.4 μM in the brain and 61.3 ± 12.0 μM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 ± 1.45 and 0.64 ± 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 ± 0.026. The log octanol/water partition coefficients were found to be −1.19 ± 0.04 and −1.47 ± 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% ± 4%) and BEA005 (18% ± 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (P = 0.481), half-life in muscle (P = 0.279), and level of protein binding (P = 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (P = 0.032), clearance (P = 0.046), volume of distribution (P = 0.027) in muscle, and octanol/water partition coefficient (P = 0.019).     

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2′F-2′C-Me-UTP was efficiently incorporated by HCV polymerase with apparent K_d (equilibrium constant) and k_pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s⁻¹, respectively, giving an overall substrate efficiency (k_pol/K_d) of 0.0059 ± 0.0015 μM⁻¹ s⁻¹. We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2′ position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH₂ > F-C-Me > C-Me > N₃ > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2′F-2′C-Me-, 2′C-Me-, and 2′ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2′-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2′-modified NTP analogs against HCV polymerase.     

4-Amino Bis-Pyridinium Derivatives as Novel Antileishmanial Agents

The antileishmanial activity of a series of bis-pyridinium derivatives that are analogues of pentamidine have been investigated, and all compounds assayed were found to display activity against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major, with 50% effective concentrations (EC₅₀s) lower than 1 μM in most cases. The majority of compounds showed similar behavior in both Leishmania species, being slightly more active against L. major amastigotes. However, compound VGP-106 {1,1′-(biphenyl-4,4′-diylmethylene)bis[4-(4-bromo-N-methylanilino)pyridinium] dibromide} exhibited significantly higher activity against L. donovani amastigotes (EC₅₀, 0.86 ± 0.46 μM) with a lower toxicity in THP-1 cells (EC₅₀, 206.54 ± 9.89 μM). As such, VGP-106 was chosen as a representative compound to further elucidate the mode of action of this family of inhibitors in promastigote forms of L. donovani. We have determined that uptake of VGP-106 in Leishmania is a temperature-independent process, suggesting that the compound crosses the parasite membrane by diffusion. Transmission electron microscopy analysis showed a severe mitochondrial swelling in parasites treated with compound VGP-106, which induces hyperpolarization of the mitochondrial membrane potential and a significant decrease of intracellular free ATP levels due to the inhibition of ATP synthesis. Additionally, we have confirmed that VGP-106 induces mitochondrial ROS production and an increase in intracellular Ca²⁺ levels. All these molecular events can activate the apoptotic process in Leishmania; however, propidium iodide assays gave no indication of DNA fragmentation. These results underline the potency of compound VGP-106, which may represent a new avenue for the development of novel antileishmanial compounds.     

In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen,a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122

Miravirsen is a β-d-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC₅₀) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC₅₀ concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5′UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01200420","term_id":"NCT01200420"}}NCT01200420).