Derivatives of imidazotriazine and pyrrolotriazine C-nucleosides as potential new anti-HCV agents.

Previous investigations identified 2'-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.

CD160 isoforms and regulation of CD4 and CD8 T-cell responses.

BACKGROUND: Coexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation. METHODS: Expression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1. RESULTS: We first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation. CONCLUSIONS: Antibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.

Discovery and Synthesis of C-Nucleosides as Potential New Anti-HCV Agents.

Nucleoside analogues have long been recognized as prospects for the discovery of direct acting antivirals (DAAs) to treat hepatitis C virus because they have generally exhibited cross-genotype activity and a high barrier to resistance. C-Nucleosides have the potential for improved metabolism and pharmacokinetic properties over their N-nucleoside counterparts due to the presence of a strong carbon-carbon glycosidic bond and a non-natural heterocyclic base. Three 2'CMe-C-adenosine analogues and two 2'CMe-guanosine analogues were synthesized and evaluated for their anti-HCV efficacy. The nucleotide triphosphates of four of these analogues were found to inhibit the NS5B polymerase, and adenosine analogue 1 was discovered to have excellent pharmacokinetic properties demonstrating the potential of this drug class.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

A simplified approach to predict CYP3A-mediated drug-drug interactions at early drug discovery: validation with clinical data.

1. The present study evaluates which factors should be incorporated into a simplified approach to reasonably predict CYP3A-mediated drug-drug interaction (DDI) at an early drug discovery stage. 2. CYP3A IC50 values were obtained using human liver microsomes (HLM) and hepatocytes. Plasma and microsomal protein binding and in vitro hepatocyte partition coefficient (Kp) were also determined for 10 drugs. Therapeutic human maximum plasma concentrations (Cmax) were retrieved from the literature. DDI predictions were performed using an equation incorporating the fraction of the substrate metabolized by CYP3A with the total or free plasma Cmax, with or without correction for hepatocyte Kp. 3. Based on the Ki data from HLM, the use of total Cmax provided a prediction of DDI within 2-fold of the observed clinical values for 9 out of 10 drugs. 4. In comparison, free drug corrections for both Cmax and Ki values from HLM led to an underprediction of DDI (>3-fold error for five drugs). 5. Data from hepatocytes showed, in general, lower prediction accuracy than data from HLM. 6. CYP3A-mediated DDIs can be predicted with a high level of accuracy based on Ki estimates from HLM data and the total therapeutic plasma Cmax of the inhibitors. This approach should be widely applicable to the assessment of clinically significant DDIs risk in early drug discovery programs.

The liver partition coefficient-corrected inhibitory quotient and the pharmacokinetic-pharmacodynamic relationship of directly acting anti-hepatitis C virus agents in humans.

Pharmacokinetic-pharmacodynamic (PK-PD) data analyses from early hepatitis C virus (HCV) clinical trials failed to show a good correlation between the plasma inhibitory quotient (IQ) and antiviral activity of different classes of directly acting antiviral agents (DAAs). The present study explored whether use of the liver partition coefficient-corrected IQ (LCIQ) could improve the PK-PD relationship. Animal liver partition coefficients (Kp(liver)) were calculated from liver to plasma exposure ratios. In vitro hepatocyte partition coefficients (Kp(hep)) were determined by the ratio of cellular to medium drug concentrations. Human Kp(liver) was predicted using an in vitro-in vivo proportionality method: the species-averaged animal Kp(liver) multiplied by the ratio of human Kp(hep) over those in animals. LCIQ was calculated using the IQ multiplied by the predicted human Kp(liver). Our results demonstrated that the in vitro-in vivo proportionality approach provided the best human Kp(liver) prediction, with prediction errors of <45% for all 5 benchmark drugs evaluated (doxorubicin, verapamil, digoxin, quinidine, and imipramine). Plasma IQ values correlated poorly (r(2) of 0.48) with maximum viral load reduction and led to a corresponding 50% effective dose (ED(50)) IQ of 42, with a 95% confidence interval (CI) of 0.1 to 148534. In contrast, the LCIQ-maximum VLR relationship fit into a typical sigmoidal curve with an r(2) value of 0.95 and an ED(50) LCIQ of 121, with a 95% CI of 83 to 177. The present study provides a novel human Kp(liver) prediction model, and the LCIQ correlated well with the viral load reductions observed in short-term HCV monotherapy of different DAAs and provides a valuable tool to guide HCV drug discovery.

Evaluation of phosphatidylinositol-4-kinase IIIα as a hepatitis C virus drug target.

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.

Cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV genotype 1 NS3/4A protease inhibitor.

The present study describes the cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV protease inhibitor currently in phase III clinical trials. BI 201335 showed a good Caco-II permeability (8.7 × 10(-6) cm/sec) and in vitro metabolic stability (predicted hepatic clearence (CL(hep)) <19% Q(h) in all species tested). Single dose PK revealed a clearance of 17, 3.0 and 2.6 mL/min/kg in rat, monkey and dog respectively, with a corresponding oral bioavailability of 29.1, 25.5 and 35.6%. Comparative plasma and liver PK profile in rodents showed a high liver Kp in the rat (42-fold), suggesting high target tissue distribution. Simple allometry based on animal PK predicted a human oral CL/F of 168 mL/min, within two-fold of the observed value (118 mL/min) at 240 mg in healthy volunteers. Allometry of volume of distribution generated a low exponent of 0.59, and a much lower predicted Vss/F (5-fold less than observed). Several different approaches of Vss/F prediction were evaluated and compared with the value observed in human. The averaged Vss/F from preclinical animals provides the best estimation of the observed human value (169 L vs. 175 L). Corresponding human "effective" t(1/2) values were also compared. The predicted human t(1/2) based on the CL from allometry with metabolic corrections and the averaged animal Vss represented the best estimation of the clinical data (12.1 vs. 17.2 hr). The present study demonstrated that the good preclinical ADMEPK profile of BI 201335 is consistent with that observed in the clinic. While preclinical data accurately predicted the human CL, the prediction of human Vss seems to be more challenging. The averaged Vss/F from all tested preclinical animals provided the best prediction of human Vss and the resulting "effective" t(1/2).

Preclinical characterization of BI 201335, a C-terminal carboxylic acid inhibitor of the hepatitis C virus NS3-NS4A protease.

BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K(i) values of 2.6 and 2.0 nM, respectively. K(i) values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC(50)s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection.

Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication.

A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.

Apricitabine: a novel deoxycytidine analogue nucleoside reverse transcriptase inhibitor for the treatment of nucleoside-resistant HIV infection.

Existing nucleoside reverse transcriptase inhibitors for HIV disease are limited by problems of resistance and, in some cases, long-term toxicity. Apricitabine (ATC; formerly BCH10618, SPD754 and AVX754) is a deoxycytidine analogue nucleoside reverse transcriptase inhibitor in clinical development. ATC retains substantial in vitro activity against HIV-1 containing many mutations associated with nucleoside reverse transcriptase inhibitor resistance, showing a less than twofold reduction in susceptibility in the presence of either up to five thymidine analogue mutations or the M184V mutation. ATC showed a low potential for cellular or mitochondrial toxicity in vitro. ATC is well absorbed orally, with a bioavailability of 65-80%. Its plasma elimination half-life (approximately 3 h), and the intracellular half-life of its triphosphate (TP) metabolite (6-7 h) support twice-daily dosing. Intracellular ATC-TP levels are markedly reduced in the presence of lamivudine or emtricitabine, indicating that clinical co-administration of ATC together with these agents will not be possible. The drug is renally eliminated, giving a low potential for hepatic drug interactions. In a double-blind, randomized, placebo-controlled Phase II monotherapy trial in antiretroviral-naive patients, ATC doses of 1,200 and 1,600 mg/day reduced plasma viral load levels by 1.65 and 1.58 log10 HIV RNA copies/ml, respectively, after 10 days of treatment (P<0.0001 versus placebo). ATC showed a low propensity to select for resistance mutants in vitro and during clinical monotherapy. ATC was well tolerated in volunteers and in HIV-infected patients. This promising profile suggests that ATC may be useful in treating patients who have failed previous lamivudine- or emtricitabine-containing regimens. Further studies to evaluate the long-term efficacy and tolerability of ATC are underway.

Efficacy and tolerability of 10-day monotherapy with apricitabine in antiretroviral-naive, HIV-infected patients.

OBJECTIVE: Apricitabine (formerly AVX754 and SPD754) is a deoxycytidine analogue nucleoside reverse transcriptase inhibitor in clinical development for patients with HIV disease. This study evaluated the antiretroviral efficacy, tolerability and safety of apricitabine monotherapy, administered for 10 days in antiretroviral-naive, HIV-1 infected adults. METHODS: Adult patients (> or = 18 years) with HIV infection (CD4 count > or = 250 cells/microl; plasma HIV-1 RNA level 5000-100 000 copies/ml) were randomized to 10 days' double-blind oral therapy with placebo or apricitabine 400 mg/day, 800 mg/day, 1200 mg/day, or 1600 mg/day. RESULTS: At 7 days, all apricitabine doses produced statistically significant log10 reductions in plasma HIV RNA levels from baseline relative to placebo (n = 13; P < 0.0001), as follows: -1.16 (400 mg; n = 11), -1.28 (800 mg; n = 12), -1.44 (1200 mg; n = 14), -1.30 (1600 mg; n = 13). After 10 days, the log10 viral load reductions with apricitabine 1200 mg (-1.65; P = 0.01) and 1600 mg/day (-1.58; P = 0.04) were significantly greater than that with the 400-mg dose (-1.18). No clinically relevant changes were observed in CD4 or CD8 cell indices. Apricitabine was well tolerated and showed no tendency to select any particular resistance mutation. CONCLUSION: Apricitabine monotherapy showed promising antiretroviral efficacy, good tolerability and a low propensity for resistance selection in antiretroviral-naive HIV-infected patients treated for 10 days. These results warrant further evaluation of the long-term clinical efficacy and tolerability of apricitabine.

In vitro activity of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor (NRTI), against 215 HIV-1 isolates resistant to other NRTIs.

SPD754 (also known as AVX-754) is a deoxycytidine analogue nucleoside reverse transcriptase inhibitor (NRTI) with antiretroviral activity against HIV-1 and HIV-2 in vitro and against recombinant viruses containing thymidine analogue mutations (TAMs). In order to better establish the activity of SPD754 against HIV-1 containing TAMs, twelve panels of up to twenty clinical isolates with defined TAM combinations were selected from the ViroLogic database. Phenotypic viral susceptibility to SPD754 and five other NRTIs was tested using the PhenoSense HIV assay and expressed as median fold-change compared with a reference strain. In total, 215 isolates were selected, representing four TAM patterns in both pathways by which TAMs accumulate clinically. The presence of five TAMs in the 41, 215 pathway, at codons 41, 67, 210, 215, and 219 of reverse transcriptase (RT), produced a median 1.8-fold reduction in SPD754 susceptibility, compared with fold reductions to zidovudine, lamivudine, abacavir, didanosine and tenofovir of 438, 4.8, 4.5, 1.4 and 3.6, respectively. Five TAMs in the 67, 70, 219 pathway (at codons 41, 67, 70, 215 and 219) reduced SPD754 susceptibility by a median 1.3-fold, compared with fold reductions for the aforementioned NRTIs of 108, 3.2, 3.0, 1.3 and 2.5, respectively. M184V addition reduced SPD754 susceptibility by 1.8-fold in the presence or absence of TAMs. SPD754 retains a substantial proportion of its antiviral activity against HIV-1 containing multiple TAMs, with or without the M184V mutation. These data suggest that SPD754 is a promising new NRTI for the treatment of NRTI-experienced HIV-infected patients.

Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. Crystal structures and mechanism of inhibition.

X-ray crystal structures of two non-nucleoside analogue inhibitors bound to hepatitis C virus NS5B RNA-dependent RNA polymerase have been determined to 2.0 and 2.9 A resolution. These noncompetitive inhibitors bind to the same site on the protein, approximately 35 A from the active site. The common features of binding include a large hydrophobic region and two hydrogen bonds between both oxygen atoms of a carboxylate group on the inhibitor and two main chain amide nitrogen atoms of Ser(476) and Tyr(477) on NS5B. The inhibitor-binding site lies at the base of the thumb domain, near its interface with the C-terminal extension of NS5B. The location of this inhibitor-binding site suggests that the binding of these inhibitors interferes with a conformational change essential for the activity of the polymerase.

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy.

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.