Abstract A functional screen of an adenovirus-delivered shRNA library that targets ∼ 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.
BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This ...3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow CL, 0.7%; bioavailability F, 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.
Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected ...with influenza B virus. A hemagglutinin mutation (198 Thr→Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg→Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.
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.
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.
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.
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.
The discovery of a 1,5-dihydrobenzob1,4diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzob1,4diazepine-2,4-dione hit ...established structure–activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.
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.
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