Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses.

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations.

OBJECTIVES: Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. METHODS: In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. RESULTS: Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were >10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P < 0.001). CONCLUSIONS: Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Comparable In Vitro Activities of Second-Generation HIV-1 Integrase Strand Transfer Inhibitors (INSTIs) on HIV-1 Clinical Isolates with INSTI Resistance Mutations.

Second-generation HIV-1 integrase strand transfer inhibitors (INSTIs) dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) showed a high genetic barrier to resistance and limited cross-resistance with first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG). In this study, DTG, BIC, and CAB demonstrated a comparable activity on a panel of INSTI-resistant strains isolated from patients exposed to RAL, EVG, and/or DTG, with a significantly reduced susceptibility only with the pathway Q148H/K/R plus one to two additional INSTI mutations.

The HIV-1 reverse transcriptase E138A natural polymorphism decreases the genetic barrier to resistance to etravirine in vitro.

OBJECTIVES: The HIV-1 reverse transcriptase (RT) natural polymorphism E138A is included among the mutations with a minor impact on response to etravirine. However, the interpretation of E138A on etravirine susceptibility is not consistent across different genotypic resistance algorithms. The aim of the study was to investigate the effect of E138A on the genetic barrier to resistance to etravirine in vitro. METHODS: A panel of 20 clinically derived recombinant viruses (10 with WT 138E and 10 with 138A, all without any other resistance mutation) were cultured in the presence of increasing etravirine concentrations and analysed for genotypic changes at virus breakthrough. Parallel experiments were conducted with 138E/A/G/K/Q NL4-3-based clones. RESULTS: In the NL4-3 background, codon 138 changes increased etravirine resistance in the following order: Q > K > A > G > E. The 138A viruses were less susceptible to etravirine compared with the 138E viruses [median (IQR) fold change, 1.8 (1.5-2.8) versus 1.3 (0.8-1.8); P = 0.026], overcame etravirine pressure earlier [HR (95% CI) for viral outgrowth with 138A, 5.48 (2.95-28.24); P < 0.001] and grew at higher drug concentrations [median (IQR), 1350 (1350-1350) versus 0 (0-1350) nM; P = 0.005]. A variety of etravirine resistance-related mutations and changes in the RT connection and RNase H domains accumulated without any consistent pattern depending on baseline codon 138. CONCLUSIONS: E138A can contribute to reduced response to etravirine through a decreased genetic barrier to resistance. In vitro drug resistance selection is a valuable complement to define the full potential of low-level resistance mutations.

Neutralizing activity of African lineage Zika virus immune sera towards Asian lineage.

Genetic and phylogenetic studies indicated that Zika virus (ZIKV) has evolved into 2 major lineages, the African and Asian. However, ZIKV has been described as a single serotype. This study aimed at assessing the cross-neutralization between ZIKV African and Asian lineages strains. Sixthy-five samples collected in 2007 and 30 samples collected from the same subjects in 2011/2012 in West Africa and positive to neutralizing antibody against ZIKV MR-766 strain (African lineage) were tested against ZIKV H/PF/2013 strain (Asian lineage) by microneutralization assay. All samples showing neutralizing antibodies against MR-766 strain showed also neutralizing activity against H/PF/2013 strain, although with lower titers. This is consistent with about 120 amino acid differences between the two strains. Despite differences in the magnitude of neutralizing activity against different ZIKV strains, all samples showed neutralizing antibody titers considered to be protective.

Proviral location affects cognate peptide-induced virus production and immune recognition of HIV-1-infected T cell clones.

BACKGROUNDHIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (ECs) and people on long-term therapy suggest that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing.METHODSIn this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells.RESULTSThe proviral landscape was dominated by 2 large clones with replication-competent proviruses integrated into zinc finger (ZNF) genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-fold less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene.CONCLUSIONSWe provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence.FUNDINGOffice of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.