David Standring

Synthesis and biological evaluation of aryl-phospho-indole as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

A novel series of 3-aryl-phospho-indole (API) non-nucleoside reverse transcriptase inhibitors of HIV-1 was developed. Chemical variation in the phosphorus linker led to the discovery of 3-phenyl-methyl-phosphinate-2-carboxamide 14, which possessed excellent potency against wild-type HIV-1 as well as viruses bearing K103N and Y181C single mutants in the reverse transcriptase gene. Chiral separation of the enantiomers showed that only R enantiomer retained the activity. The pharmacokinetic, solubility, and metabolic properties of 14 were assessed.

Telbivudine, a nucleoside analog inhibitor of HBV polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir

Telbivudine, a nucleoside analog inhibitor of the viral polymerase of hepatitis B virus (HBV), has been approved for the treatment of chronic HBV infection, along with the nucleoside inhibitors lamivudine and entecavir, and the nucleotide inhibitors adefovir and tenofovir. The resistance profiles of these agents were investigated via drug treatment of HepG2 cells stably transfected with wild-type or mutant HBV genomes bearing known resistance mutations. Telbivudine was not active against HBV strains bearing lamivudine mutations L180M/M204V/I but remained active against the M204V single mutant in vitro, potentially explaining the difference in resistance profiles between telbivudine and lamivudine. Against HBV genomes with known telbivudine-resistance mutations, M204I and L80I/M204I, telbivudine, lamivudine and entecavir lost 353- to >1000-fold activity whereas adefovir and tenofovir exhibited no more than 3-5-fold change. Conversely, against HBV cell lines expressing adefovir resistance mutations N236T and A181V, or the A194T mutant associated with resistance to tenofovir, telbivudine remained active as shown by respective fold-changes of 0.5 (N236T) and 1.0 (A181V and A194T). These in vitro results indicate that nucleoside and nucleotide drugs have different cross-resistance profiles. The addition of telbivudine to ongoing adefovir therapy could provide effective antiviral therapy to patients who develop adefovir resistance.

Assembly and antigenicity of hepatitis B virus core particles.

Recent studies in Xenopus oocytes and other systems have led to an understanding of the HBV capsid, or core particle, assembly process. Nascent HBV core polypeptides rapidly dimerize. Accumulation of free dimers to a signature concentration (approximately 0.8 microM) then triggers a highly cooperative capsid assembly reaction. This dimer-to-capsid transition is accompanied by a switch from HBe to HBc antigenicity and appears to be nucleated by interaction between core protein and RNA: deletion of a protamine-like RNA binding domain at the C-terminus of the core protein markedly increases the concentration of dimers needed to drive capsid assembly. The simple assembly pathway seen for HBV capsids mirrors that of R17 bacteriophage.

2'-C-Methyl branched pyrimidine ribonucleoside analogues : potent inhibitors of RNA virus replication

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain ‘natural’ pyrimidine bases, but possess a β-methyl substituent at the 2′-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA), single-stranded negative (ssRNA−) or double-stranded (dsRNA), revealed potent activities for D-2′-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5′-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2′-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.

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

ABSTRACT 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.

91 ANTIVIRAL ACTIVITY OF THE LIVER-TARGETED NUCLEOTIDE HCV POLYMERASE INHIBITOR IDX184 CORRELATES WITH TROUGH SERUM LEVELS OF THE NUCLEOSIDE METABOLITE IN HCV-INFECTED CHIMPANZEES

Background: ANA598 is a novel HCV non-nucleoside polymerase inhibitor currently in clinical development for the treatment of hepatitis C. Due to the high potential for developing resistance to any single direct antiviral used as monotherapy in hepatitis C, future therapies for HCV are expected to involve combinations of direct antivirals to increase antiviral potency and suppress viral resistance. The results from in vitro combination studies provide support for future clinical exploration of combination regimens that include ANA598. Methods: Combination studies were conducted using wt Huh7-Luc or M414T-containing 1b dicistronic replicons. Cells were cultured in the presence of compounds for 72 hours using both fixed ratios and checkerboard concentration matrices of test agents. Cultured media from PBMCs treated with the active metabolite of ANA773 were used to evaluate the combination effects of this agent with ANA598. The observed inhibitory activity of two agents in combination was compared to the combined action predicted from the dose responses for individual agents assuming Loewe additivity or Bliss independence. Results using CalcuSyn and internally developed analytical procedures were compared. Results: ANA598 was combined pairwise in vitro with IFN-a, the HCV NS3/4 protease inhibitor Telaprevir, the NS5B nucleoside polymerase inhibitor PSI-6130, or the TLR7 agonist ANA773 (currently in clinical development by Anadys). We have previously demonstrated that there is no overlap in viral mutations conferring resistance to these agents in vitro. Combinations were evaluated in both wt and mutant replicons containing the M414T mutation that confers resistance to palm site non-nucleoside NS5B inhibitors. No cytotoxicity was detected for any of the combinations tested. For each combination evaluated, the antiviral interaction between the compounds was determined to be additive to synergistic. Conclusions: The in vitro combination studies demonstrate that additive to synergistic antiviral effects are observed when ANA598 is combined with other anti-HCV agents having distinct mechanisms of action and nonoverlapping resistance profiles. Such combinations may produce a greater viral load reduction and potentially delay the emergence of drug resistance in vivo. Consequently, appropriately constructed combination regimens may provide clinical benefit by improving response rates and allowing non-responders to current therapies to be treated more effectively.

Discovery of the Aryl-phospho-indole IDX899, a Highly Potent Anti-HIV Non-nucleoside Reverse Transcriptase Inhibitor.

Here, we describe the design, synthesis, biological evaluation, and identification of a clinical candidate non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a novel aryl-phospho-indole (APhI) scaffold. NNRTIs are recommended components of highly active antiretroviral therapy (HAART) for the treatment of HIV-1. Since a major problem associated with NNRTI treatment is the emergence of drug resistant virus, this work focused on optimization of the APhI against clinically relevant HIV-1 Y181C and K103N mutants and the Y181C/K103N double mutant. Optimization of the phosphinate aryl substituent led to the discovery of the 3-Me,5-acrylonitrile-phenyl analogue RP-13s (IDX899) having an EC50 of 11 nM against the Y181C/K103N double mutant.

Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study.

Ribonucleoprotein Complex Formation by the Human Hepatitis B Virus Polymerase

Human hepatitis B virus (HBV) polymerase (pol or RT), when expressed in Xenopus oocytes upon injection of synthetic minimal pol RNA (RT RNA), assembles into a higher molecular weight complex with the characteristics of a ribonuclear protein (RNP) complex. In vitro RNA competition binding data suggest that RT RNA is preferentially packaged into this complex even though it lacks the authentic viral encapsidation signal, epsilon, and viral capsid protein sequences. Consistent with this finding, the in vitro polymerase reaction performed in pol-expressing oocyte extracts generates primarily HBV-specific DNAs even when the pol template is challenged with a coinjected non-HBV competitor RNA. These results suggest that interaction between pol and its cognate RNA can be mediated by sequences other than the known packaging elements. We speculate that HBV RNP complexes containing at least polymerase and viral RNA may play a role in viral nucleocapsid assembly and may help to segregate HBV reverse transcription from the cellular milieu in vivo.

Discovery of benzophosphadiazine drug candidate IDX375: A novel hepatitis C allosteric NS5B RdRp inhibitor

Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells, and as a consequence is an attractive target for selective inhibition. This paper describes the discovery of a novel family of HCV NS5B non-nucleoside inhibitors inspired by the bioisosterism between sulfonamide and phosphonamide. Systematic structural optimization in this new series led to the identification of IDX375, a potent non-nucleoside inhibitor that is selective for genotypes 1a and 1b. The structure and binding domain of IDX375 were confirmed by X-ray co-crystalisation study.