Tadahisa Teramoto

Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives.

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j-n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface.

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases.

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one-1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC(50) values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure-activity relationship studies, in vitro screening and X-ray crystallography.

Structural complexity of Dengue virus untranslated regions: cis -acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb2+ ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region.

Requirements for West Nile Virus Minus- and Plus-Strand Subgenomic RNA Synthesis in vitro by the viral RNA- dependent RNA Polymerase Expressed in E. coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Inhibitors of Dengue Virus and West Nile Virus Proteases Based on the Aminobenzamide Scaffold

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.

Effects of detergents on the West Nile virus protease activity

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at 0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC(50) value of 5.7+/-2.5 microM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.

Antiviral activities of selected antimalarials against dengue virus type 2 and Zika virus

&NA; In a previous study, twelve antimalarial compounds, amodiaquine (AQ) and derivatives, were shown to have potent anti‐dengue viral (DENV) activity by using the stable DENV2 Renilla luciferase reporter replicon expressing BHK‐21 cells, infectivity (plaque), and the qRT‐PCR assays. In this study, we performed molecular modeling on these compounds to determine their stereo‐electronic properties required for optimal antiviral activity. Based on the similarity of calculated stereo‐electronic profiles, specifically the electrostatic potential profiles of the compounds, and in silico screening of related compounds from literature, we identified three additional compounds, Quinacrine (QC), Mefloquine (MQ), and GSK369796. Analysis of their antiviral activities indicated that all three compounds have high anti‐DENV activity in the DENV2 replicon expressing cells with EC50 values of 5.30 ± 1.31 &mgr;M (QC), 3.22 ± 0.37 &mgr;M (MQ), and 5.06 ± 0.86 &mgr;M (GSK369796). The infectivity assays revealed the EC50 values of 7.09 ± 1.67 &mgr;M (QC), 4.36 ± 0.31 &mgr;M (MQ) and 3.03 ± 0.35 &mgr;M (GSK369796). The mode of action of these compounds is through inhibition of autophagy, thereby affecting DENV2 replication. Moreover, these compounds also showed antiviral activity against the rapidly emerging Zika virus (ZIKV) with EC50 values of 2.27 ± 0.14 &mgr;M (QC), 3.95 ± 0.21 &mgr;M (MQ), and 2.57 ± 0.09 &mgr;M (GSK369796). HighlightsFrom the antiviral activity of twelve amodiaquine derivatives, an interaction pharmacophore model was developed.Based on the model three new antimalarials were identified and verified as potent inhibitors of dengue and Zika viruses.Mode of action of these compounds is through inhibition of autophagy, especially the lysosomal proteases.This current study may lead to repurposing of antimalarial drugs for treatment of flaviviral infections.

Substitution of NS5 N-terminal domain of dengue virus type 2 RNA with type 4 domain caused impaired replication and emergence of adaptive mutants with enhanced fitness

Background: The four dengue virus serotypes, evolved from a common ancestor, are global human pathogens. Results: Interserotypic substitution of the functional domain essential for 5′-capping was detrimental to RNA replication. Conclusion: The chimeric RNA gradually evolves replication fitness through adaptive mutations in genes encoding two replication proteins. Significance: This study provides a possible pathway for generating attenuated dengue virus vaccine. Flavivirus NS3 and NS5 are required in viral replication and 5′-capping. NS3 has NS2B-dependent protease, RNA helicase, and 5′-RNA triphosphatase activities. NS5 has 5′-RNA methyltransferase (MT)/guanylyltransferase (GT) activities within the N-terminal 270 amino acids and the RNA-dependent RNA polymerase (POL) activity within amino acids 271–900. A chimeric NS5 containing the D4MT/D4GT and the D2POL domains in the context of wild-type (WT) D2 RNA was constructed. RNAs synthesized in vitro were transfected into baby hamster kidney cells. The viral replication was analyzed by an indirect immunofluorescence assay to monitor NS1 expression and by quantitative real-time PCR. WT D2 RNA-transfected cells were NS1- positive by day 5, whereas the chimeric RNA-transfected cells became NS1-positive ∼30 days post-transfection in three independent experiments. Sequence analysis covering the entire genome revealed the appearance of a single K74I mutation within the D4MT domain ∼16 days post-transfection in two experiments. In the third, D290N mutation in the conserved NS3 Walker B motif appeared ≥16 days post-transfection. A time course study of serial passages revealed that the 30-day supernatant had gradually evolved to gain replication fitness. Trans-complementation by co-expression of WT D2 NS5 accelerated viral replication of chimeric RNA without changing the K74I mutation. However, the MT and POL activities of NS5 WT D2 and the chimeric NS5 proteins with or without the K74I mutation are similar. Taken together, our results suggest that evolution of the functional interactions involving the chimeric NS5 protein encoded by the viral genome species is essential for gain of viral replication fitness.