Safety and efficacy of AK0529 in respiratory syncytial virus-infected infant patients: A phase 2 proof-of-concept trial.

Background: Respiratory syncytial virus (RSV) infection is a cause of substantial morbidity and mortality in young children. There is currently no effective therapy available. Methods: This was a Phase 2 study of the oral RSV fusion protein inhibitor AK0529 in infants aged 1-24 months, hospitalized with RSV infection. In Part 1, patients (n = 24) were randomized 2:1 to receive a single dose of AK0529 up to 4 mg/kg or placebo. In Part 2, patients (n = 48) were randomized 2:1 to receive AK0529 at 0.5, 1, or 2 mg/kg bid or placebo for 5 days. Sparse pharmacokinetic samples were assessed using population pharmacokinetics modelling. Safety, tolerability, viral load, and respiratory signs and symptoms were assessed daily during treatment. Results: No safety or tolerability signals were detected for AK0529: grade ≥3 treatment-emergent adverse events occurring in 4.1% of patients in AK0529 and 4.2% in placebo groups, respectively, and none led to death or withdrawal from the study. In Part 2, targeted drug exposure was reached with 2 mg/kg bid. A numerically greater reduction in median viral load with 2 mg/kg bid AK0529 than with placebo at 96 h was observed. A -4.0 (95% CI: -4.51, -2.03) median reduction in Wang Respiratory Score from baseline to 96 h was observed in the 2 mg/kg group compared with -2.0 (95% CI: -3.42, -1.82) in the placebo group. Conclusions: AK0529 was well tolerated in hospitalized RSV-infected infant patients. Treatment with AK0529 2 mg/kg bid was observed to reduce viral load and Wang Respiratory Score. Clinical Trials Registration: NCT02654171.

Comparison of antiviral resistance across acute and chronic viral infections.

Antiviral therapy can lead to drug resistance, but multiple factors determine the frequency of drug resistance mutations and the clinical consequences. When chronic infections caused by Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV) are compared with acute infections such as influenza virus, respiratory syncytial virus (RSV), and other respiratory viruses, there are similarities in how and why antiviral resistance substitutions occur, but the clinical significance can be quite different. Emergence of resistant variants has implications for design of new therapeutics, treatment guidelines, clinical trial design, resistance monitoring, reporting, and interpretation. In this discussion paper, we consider the molecular factors contributing to antiviral drug resistance substitutions, and a comparison is made between chronic and acute infections. The implications of resistance are considered for clinical trial endpoints and public health, as well as the requirements for therapeutic monitoring in clinical practice with acute viral infections.

Design and Synthesis of Orally Bioavailable 4-Methyl Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors.

Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal capsid formation have been an attractive approach to block the replication of HBV viruses. We carried out multidimensional structural optimizations based on the heteroaryldihydropyrimidine (HAP) analogue Bay41-4109 (1) and identified a novel series of HBV capsid inhibitors that demonstrated promising cellular selectivity indexes, metabolic stabilities, and in vitro safety profiles. Herein we disclose the design, synthesis, structure-activity relationship (SAR), cocrystal structure in complex with HBV capsid proteins and in vivo pharmacological study of the 4-methyl HAP analogues. In particular, the (2S,4S)-4,4-difluoroproline substituted analogue 34a demonstrated high oral bioavailability and liver exposure and achieved over 2 log viral load reduction in a hydrodynamic injected (HDI) HBV mouse model.

Identification of a novel aminotetralin class of HDAC6 and HDAC8 selective inhibitors.

Herein we report the identification of a novel class of HDAC6 and HDAC8 selective inhibitors through a unique chemistry and phenotypic screening strategy. Tetrahydroisoquinoline 12 was identified as a potent HDAC6 and HDAC8 dual inhibitor from a focused library through cellular tubulin acetylation and p21 induction screening assays. Scaffold hopping from 12 led to the discovery of an aminotetralin class of HDAC inhibitors. In particular, the 3-R stereoisomer 32 showed highly potent inhibition against HDAC6 and HDAC8 with IC50 values of 50 and 80 nM, respectively. Treatment of neuroblastoma BE(2)C cells with 32 resulted in elevated levels of acetylated tubulin, TrkA, and neurite outgrowth with only marginal effects on p21 induction and histone H3 acetylation. Consistent with its weak enzymatic inhibition of HDAC1, 32 showed significantly less cytotoxicity than SAHA and moderately inhibited the growth of myeloma NCI-H929 and OPM-2 cells.

Identification and characterization of multiple TRIM proteins that inhibit hepatitis B virus transcription.

Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.

Characterization of nucleosome positioning in hepadnaviral covalently closed circular DNA minichromosomes.

Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.

Inhibition of influenza A virus (H1N1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin.

Hemagglutinin (HA) of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin.

Design and synthesis of benzenesulfonamide derivatives as potent anti-influenza hemagglutinin inhibitors.

Structural optimization of salicylamide-based hemagglutinin (HA) inhibitor 1 resulted in the identification of cis-3-(5-hydroxy-1,3,3-trimethylcyclohexylmethylamino)benzenesulfonamide 28 and its derivatives as potent anti-influenza agents. The lead compound 28 and its 2-chloro analogue 40 can effectively prevent cytopathic effects (CPE) caused by infection of influenza A/Weiss/43 strain (H1N1) with EC50 values of 210 and 86 nM, respectively. Mechanism of action studies indicate that 40 and its analogues inhibit the virus fusion with host endosome membrane by binding to HA and stabilizing the prefusion HA structure. With significantly improved metabolic stability, the reported series represents the first generation of orally bioavailable HA inhibitors that have a good selectivity window and potential for further development as novel anti-influenza agents.

Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats.

Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.

Discovery of novel 1-phenyl-cycloalkane carbamides as potent and selective influenza fusion inhibitors.

A novel class of HA inhibitors (4a) was identified based on ligand similarity search of known HA inhibitors. Parallel synthesis and further structural modifications resulted in 1-phenyl-cyclopentanecarboxylic acid (4-cyano-phenyl)-methyl-amide 4t as a potent and selective inhibitor to phylogenetic H1 influenza viruses with an EC(50) of 98 nM against H1N1 A/Weiss/43 strain and over 1000-fold selectivity against host MDCK cells.

Isobolographic characterization of interactions of retigabine with carbamazepine, lamotrigine, and valproate in the mouse maximal electroshock-induced seizure model.

The aim of this study was to characterize the pharmacodynamic, pharmacokinetic, and adverse-effect profiles of retigabine (RTG) in combination with carbamazepine (CBZ), lamotrigine (LTG), and valproate (VPA). The isobolographic analysis for parallel and nonparallel dose-response effects was used in the mouse maximal electroshock seizure (MES) model for evaluation of pharmacodynamic interaction. Potential adverse-effect profiles of interactions of RTG with CBZ, LTG, and VPA at the fixed ratio of 1:1 in the MES test were evaluated in the chimney (motor performance), passive avoidance (long-term memory), and grip strength (muscular strength) tests. Free plasma and total brain concentrations of CBZ, LTG, and VPA were determined by immunofluorescence and chromatography to assess pharmacokinetic interaction. In the MES model, RTG administered singly had its dose-response relationship curve (DRRC) parallel to that for VPA and nonparallel to that for CBZ and LTG. With isobolography for parallel DRRCs, the combination of RTG with VPA at fixed ratios of 1:3, 1:1, and 3:1 exerted supraadditive (synergistic) interaction. Isobolography for nonparallel DRRCs revealed that the combinations of RTG with CBZ and LTG at the fixed ratio of 1:1 produced additive interaction. In all combinations, neither motor coordination, long-term memory, nor muscular strength were affected. Only the combination of RTG with VPA at the fixed ratio of 3:1 was complicated by a pharmacokinetic increase in both free plasma and total brain VPA concentrations. All remaining combinations of RTG with VPA, CBZ, and LTG were pharmacodynamic in nature. RTG synergistically interacted with VPA and exerted additive interaction with CBZ and LTG in the mouse MES model.

Thiazolone-acylsulfonamides as novel HCV NS5B polymerase allosteric inhibitors: convergence of structure-based drug design and X-ray crystallographic study.

A novel series of thiazolone-acylsulfonamides were designed as HCV NS5B polymerase allosteric inhibitors. The structure based drug designs (SBDD) were guided by docking results that revealed the potential to explore an additional pocket in the allosteric site. In particular, the designed molecules contain moieties of previously described thiazolone and a newly designed acylsulfonamide linker that is in turn connected with a substituted aromatic ring. The selected compounds were synthesized and demonstrated low muM activity. The X-ray complex structure was determined at a 2.2A resolution and converged with the SBDD principle.

Identification and structure-activity relationships of substituted pyridones as inhibitors of Pim-1 kinase.

A novel series of highly potent substituted pyridone Pim-1 kinase inhibitors is described. Structural requirements for in vitro activity are outlined as well as a complex crystal structure with the most potent Pim-1 inhibitor reported (IC(50)=50 nM). A hydrogen bond matrix involving the Pim-1 inhibitor, two water molecules, and the catalytic core, together with a potential weak hydrogen bond between an aromatic hydrogen on the R(1) phenyl ring and a main-chain carbonyl of Pim-1, accounts for the overall potency of this inhibitor.

Novel thiazolones as HCV NS5B polymerase allosteric inhibitors: Further designs, SAR, and X-ray complex structure.

Structure-activity relationships (SAR) of 1 against HCV NS5B polymerase were described. SAR explorations and further structure-based design led to the identifications of 2 and 3 as novel HCV NS5B inhibitors. X-ray structure of 3 in complex with NS5B polymerase was obtained at a resolution of 2.2A, and confirmed the design.

Isothiazoles as active-site inhibitors of HCV NS5B polymerase.

Isothiazole analogs were discovered as a novel class of active-site inhibitors of HCV NS5B polymerase. The best compound has an IC(50) of 200 nM and EC(50) of 100 nM, which is a significant improvement over the starting inhibitor (1). The X-ray complex structure of 1 with HCV NS5B was obtained at a resolution of 2.2A, revealing that the inhibitor is covalently linked with Cys 366 of the 'primer-grip'. Furthermore, it makes considerable contacts with the C-terminus, beta-loop, and more importantly, to the active-site of the enzyme. The uniqueness of this binding mode offers a new insight for the rational design of novel inhibitors for HCV NS5B polymerase.

Structure-based design of a novel thiazolone scaffold as HCV NS5B polymerase allosteric inhibitors.

A structure-based approach was performed to design a novel thiazolone scaffold as HCV NS5B inhibitors. A focused library was designed and docked by GOLD. One of the top-scored molecules was synthesized and shown to have similar potency to the initial hit. The X-ray complex structure was determined and validated our design rationale.

Identification of isothiazole-4-carboxamidines derivatives as a novel class of allosteric MEK1 inhibitors.

The development of potent, orally bioavailable, and selective series of 5-amino-3-hydroxy-N(1-hydroxypropane-2-yl)isothiazole-4-carboxamidine inhibitors of MEK1 and MEK-2 kinase is described. Optimization of the carboxamidine and the phenoxyaniline group led to the identification of 55 which gave good potency as in vitro MEK1 inhibitors, and good oral exposure in rat.

Discovery of 3-hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazoles as potent MEK1 inhibitors.

3-Hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazoles were discovered as potent in vitro MEK1 inhibitors.

Parallel synthesis of 5-cyano-6-aryl-2-thiouracil derivatives as inhibitors for hepatitis C viral NS5B RNA-dependent RNA polymerase.

From random screening of our compound libraries, we identified a hit compound with an IC50 of 27 microM against hepatitis C viral NS5B RNA-dependent RNA polymerase. By using a parallel synthetic strategy, a series of its derivatives were synthesized. From their anti-HCV activity screening, compounds with single digital 3.8 micromolar activity were obtained.

Recent patents on nucleoside and nucleotide inhibitors for HCV.

Hepatitis C virus (HCV) infection is a leading cause of liver diseases such as cirrhosis and hepatocellular carcinoma. There are estimated 170 million people worldwide chronically infected with the virus. The lack of highly effective and safe therapeutics for HCV infection has spurred intensive efforts to develop anti-HCV drugs as evidenced by the large number of new patent applications filed each year. Nucleoside and nucleotide inhibitors are the analogues of DNA or RNA substrates, and they inhibit viral polymerases by acting as chain terminators, viral mutagens, or simple competitive inhibitors. The successful development of various nucleoside and nucleotide inhibitors for the treatment of HIV and HBV infections has prompted the drug industry to seek similar strategies for HCV. This review summarizes recently issued or published patents covering nucleoside and nucleotide inhibitors for HCV. The claimed chemical structures and available biological activities, mechanism of action, and drug resistance profiles are discussed. The development status of several promising nucleoside inhibitors is also described.