Jyh-Haur Chern

Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay

ABSTRACT Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC50) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC50) of 0.17 ± 0.01 μM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.

Mutation in Enterovirus 71 Capsid Protein VP1 Confers Resistance to the Inhibitory Effects of Pyridyl Imidazolidinone

ABSTRACT Enterovirus 71 is one of the most important pathogens in the family of Picornaviridae that can cause severe complications in the postpoliovirus era, such as encephalitis, pulmonary edema, and even death. Pyridyl imidazolidinone is a novel class of potent and selective human enterovirus 71 inhibitor. Pyridyl imidazolidinone was identified by using computer-assisted drug design. This virologic investigation demonstrates that BPR0Z-194, one of the pyridyl imidazolidinones, targets enterovirus 71 capsid protein VP1. Time course experiments revealed that BPR0Z-194 effectively inhibited virus replication in the early stages, implying that the compound can inhibit viral adsorption and/or viral RNA uncoating. BPR0Z-194 was used to select and characterize the drug-resistant viruses. Sequence analysis of the VP1 region showed that the resistant variants differed consistently by seven amino acids in VP1 region from their parental drug-sensitive strains. Site-directed mutagenesis of enterovirus 71 infectious cDNA revealed that a single amino acid alteration at the position 192 of VP1 can confer resistance to the inhibitory effects of BPR0Z-194.

Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71.

ABSTRACT Enterovirus 71 (EV71) has emerged as an important virulent neurotropic enterovirus in young children. DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)-methyl]-piperazin-1-yl}-1-pheny-1H-pyrazolo[3,4-d]pyrimidine) was found to be a novel and potent inhibitor of EV71. The molecular target of this compound was identified by analyzing DTriP-22-resistant viruses. A substitution of lysine for Arg163 in EV71 3D polymerase rendered the virus drug resistant. DTriP-22 exhibited the ability to inhibit viral replication by reducing viral RNA accumulation. The compound suppressed the accumulated levels of both positive- and negative-stranded viral RNA during virus infection. An in vitro polymerase assay indicated that DTriP-22 inhibited the poly(U) elongation activity, but not the VPg uridylylation activity, of EV71 polymerase. These findings demonstrate that the nonnucleoside analogue DTriP-22 acts as a novel inhibitor of EV71 polymerase. DTriP-22 also exhibited a broad spectrum of antiviral activity against other picornaviruses, which highlights its potential in the development of antiviral agents.

Isatin-β-thiosemicarbazones as potent herpes simplex virus inhibitors

A series of isatin-β-thiosemicarbazones have been designed and evaluated for antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in a plaque reduction assay. Their cytotoxicity was examined using human rhabdomyosarcoma cells (RD cells). Several derivatives of isatin-β-thiosemicarbazone exhibited significant and selective antiviral activity with low cytotoxicity. It was found that the thiourea group at thiosemicarbazone and the NH functionality at isatin were essential for their antiherpetic activity. The synthesis and structure-activity relationship studies are presented.

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

ABSTRACT Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

Antiviral activity of pyridyl imidazolidinones against enterovirus 71 variants

Pyridyl imidazolidinone is a novel class of capsid binder which can inhibit enterovirus 71 (EV71). In this study, we tested the susceptibility of six recombinant viruses with different single-site mutations in VP1. Eleven modified pyridyl imidazolidinones were synthesized and used to probe the interaction between these compounds and the EV71 VP1 protein. We found that the D31N or E98K mutant viruses were susceptible to bulkier compounds, which suggested that mutations at these two sites in VP1 may widen the hydrophobic pocket of VP1, allowing bulkier compounds to enter and interfere VP1-receptor binding. Additionally, the Y116H mutant was more resistant to pyridyl imidazolidinone compounds containing a methyl group in the central position of the hydrophobic linker. When a trifluoromethyl group was substituted for the methyl group in the middle of the linker chain, the inhibitory effect was totally abolished in the Y116H mutant, suggesting that the interaction between Tyr (Y) 116 of VP1 and the central position of the linker chain of pyridyl imidazolodinone is very important for drug efficacy. A V192M mutant was resistant to most of the derivatives, indicating that residue 192 is a key mutation for resistance to pyridyl imidazolidinone.

Design, synthesis, and anti-HCV activity of thiourea compounds

A series of thiourea derivatives were synthesized and their antiviral activity was evaluated in a cell-based HCV subgenomic replicon assay. SAR studies revealed that the chain length and the position of the alkyl linker largely influenced the in vitro anti-HCV activity of this class of potent antiviral agents. Among this series of compounds synthesized, the thiourea derivative with a six-carbon alkyl linker at the meta-position of the central phenyl ring (10) was identified as the most potent anti-HCV inhibitor (EC(50) = 0.047 microM) with a selectivity index of 596.

Synthesis, activity, and pharmacokinetic properties of a series of conformationally-restricted thiourea analogs as novel hepatitis C virus inhibitors

A series of novel conformationally-restricted thiourea analogs were designed, synthesized, and evaluated for their anti-HCV activity. Herein we report the synthesis, structure-activity relationships (SARs), and pharmacokinetic properties of this new class of thiourea compounds that showed potent inhibitory activities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the fluorene compound 4b was found to possess the most potent activity (EC(50)=0.3 microM), lower cytotoxicity (CC(50)>50 microM), and significantly better pharmacokinetic properties compared to its corresponding fluorenone compound 4c.

Design and efficient synthesis of novel arylthiourea derivatives as potent hepatitis C virus inhibitors

A novel class of arylthiourea HCV inhibitors bearing various functionalities, such as cyclic urea, cyclic thiourea, urea, and thiourea, on the alkyl linker were designed and synthesized. Herein we report the synthesis and structure-activity relationships (SARs) of this novel class of arylthiourea derivatives that showed potent inhibitory activities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the new carbazole derivative 64, which has an eight-carbon linkage between the phenyl and carbazole rings and a tolyl group at the N-9 position of carbazole, was found to possess strong anti-HCV activity (EC50=0.031 microM), lower cytotoxicity (CC50 >50 microM), and higher selectivity index (SI >1612) compared to its derivatives.

Resistance Analysis and Characterization of a Thiazole Analogue, BP008, as a potent Hepatitis C Virus NS5A Inhibitor.

ABSTRACT Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the worlds population. The standard treatment for HCV infection is often poorly tolerated and ineffective. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In this report, BP008, a potent small-molecule inhibitor of HCV replication, was developed from a class of compounds with thiazol core structures by means of utilizing a cell-based HCV replicon system. The compound reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC50) and selective index value of 4.1 ± 0.7 nM and >12,195, respectively. Sequencing analyses of several individual clones derived from BP008-resistant RNAs purified from cells harboring HCV1b replicon revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. Q24L, P58S, and Y93H are the key substitutions for resistance selection; F149L and V153M play the compensatory role in the replication and drug resistance processes. Moreover, BP008 displayed synergistic effects with alpha interferon (IFN-α), NS3 protease inhibitor, and NS5B polymerase inhibitor, as well as good oral bioavailability in SD rats and favorable exposure in rat liver. In summary, our results pointed to an effective small-molecule inhibitor, BP008, that potentially targets HCV NS5A. BP008 can be considered a part of a more effective therapeutic strategy for HCV in the future.