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

bDengue 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 highthroughput 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 (EC 50 ) of 1.03 ؎ 0.09 M. 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. D engue virus (DENV) (serotypes 1 to 4) belongs to the familyFlaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents such as yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and DENV (1). More than 70 members of the Flavivirus genus are important human pathogens that cause significant morbidity and mortality (2). DENV is a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic, leading to 50 to 100 million human infections each year (3, 4). DENV infection frequently leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (5-7). Approximately 500,000 cases of DHF and DSS have been reported among more than 100 countries, causing approximately 12,500 deaths per year (3). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapeutic agents are available for humans, and disease treatment is limited to supportive care (4,8,9). Considering the spread of this epidemic and the severity of DENV, an effective anti-DENV drug is urgently needed.DENV is an enveloped RNA virus consisting of a 10.7-kb single-stranded, positive-polarity RNA genome associated with multiple copies of capsid proteins. DENV RNA is translated as a single polyprotein upon entering the host cell and is cleaved by host proteases and the virus-encoded two-component protease (NS...

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

“…We used mycophenolic acid (MPA) as a positive control of inhibition of viral replication. The data show that DMB220 was able to inhibit all four serotypes of DENV, with EC 50 s ranging between 2.2 and 2.8 M (Table 3), whereas the anti-DENV potency of MPA ranged between 0.4 and 0.6 M across the four serotypes, in agreement with previous reports (52,53). The 50% cytotoxic concentration (CC 50 ) of DMB220 was 18 M, as assayed in BHK-21 cells using the Viral ToxGlo Assay (Table 3).…”

Inhibitors compounds of the flavivirus replication process

“…Greater preclinical development is warranted.NS3- ProteasEBowman-Birk inhibitorDENVBollati et al, 2010 [2]A starting point is provided for the design of specific inhibitors. The current situation of this compounds is unknown.BP13944DENVYang et al,2014 [48]BP13944 inhibited viral replication or RNA synthesis in all four DENV serotypes. Further work is required to determine the interaction mechanism.Compound 32 (keto amides)DENVSteuer et al, 2011 [49]An inhibitory effect on DENV replication was determined in a dose-dependent manner.…”

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

“…Various strategies for Dengue NS2B/NS3 inhibitor development were recently reviewed (Luo et al, 2015), including design of both peptidic (Prusis et al, 2013; Xu et al, 2012) and non-peptidic small molecule inhibitors (Cabarcas-Montalvo et al, 2016; Chu et al, 2015; Deng et al, 2012; Lai et al, 2013a; Lai et al, 2013b; Li et al, 2015; Liu et al, 2014; Tomlinson and Watowich, 2011; Viswanathan et al, 2014; Wu et al, 2015; Zhou et al, 2013). There are already indications that resistance against protease inhibitors develops readily (Yang et al, 2011; Yang et al, 2014), consistent with the HCV protease inhibitor experience (Romano et al, 2010). The high error rate of the Zika polymerase is likely to produce a diversity of viral variants, facilitating resistance development, similar to the HCV protease inhibitor experience (Halfon and Locarnini, 2011).…”