Escape Mutations in NS4B Render Dengue Virus Insensitive to the Antiviral Activity of the Paracetamol Metabolite AM404

Abstract

Despite the enormous disease burden associated with dengue virus infections, a licensed antiviral drug is lacking. Here, we show that the paracetamol (acetaminophen) metabolite AM404 inhibits dengue virus replication. Moreover, we find that mutations in NS4B that were previously found to confer resistance to the antiviral compounds NITD-618 and SDM25N also render dengue virus insensitive to AM404. Our work provides further support for NS4B as a direct or indirect target for antiviral drug development. Dengue virus (DENV) is a major health concern. The virus was initially estimated to cause 50 million to 100 million infections each year (1, 2), but more recent estimates suggest even greater numbers (390 million infections, of which 96 million manifest clinically) (3). Dengue is a mosquito-transmitted infection that was once considered a tropical disease, but the virus is spreading rapidly across the globe and is now endemic in 128 countries, with up to 4 billion people at risk of infection (1, 4-7). No specific drug or licensed vaccine is available for DENV infection, leaving vector control the only option to prevent transmission, although this approach is threatened by the emergence of insecticide resistance (8-10). A specific antiviral therapeutic agent would be an important tool to inhibit virus replication and transmission and to reduce the global burden of DENV.To identify new inhibitors of DENV replication, we screened the NIH Clinical Collection, a library of small molecules with a history of use in humans, using a replicon-based assay in HeLa cells (11). In this DENV serotype 2 (DENV2) (strain New Guinea C [NGC])-based replicon (RepDVPacLuc), the structural genes are replaced by a puromycin resistance gene and a firefly luciferase (FLuc) reporter gene, which can be assessed as a readout for virus replication (11,12). AM404 (PubChem identification no. 6604822) was one of the compounds that, at a concentration of 10 M, reduced FLuc activity in HeLa DENV2 replicon cells by Ͼ50%, relative to the dimethyl sulfoxide (DMSO) control, without affecting cell viability by Ͼ20%. AM404, also known as Narachidonoylphenolamine, is an active metabolite of paracetamol (acetaminophen) (Fig. 1A) and is suggested to be responsible for all or part of its analgesic activity (13,14).To confirm the results from our screen, we analyzed an independent batch of AM404 (Tocris Bioscience; purity, 99.5% by HPLC) for antiviral activity on HeLa DENV2 replicon cells and found that AM404, but not paracetamol, reduced FLuc activity in a dose-dependent manner (50% effective concentration [EC 50 ], 3.6 M [95% confidence interval [CI], 3.0 to 4.2 M]) (Fig. 1B). As expected (15-17), the nucleoside analogue ribavirin (SigmaAldrich), which was used as a positive control, also inhibited virus replication in a dose-dependent manner (EC 50 , 2.2 M [95% CI, 1.8 to 2.6 M]) (Fig. 1B). Importantly, none of the compounds affected cell viability, as assessed by a colorimetric assay for cell metabolic activity (Fig. 1B). We next analyzed whether AM404 al...

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein

“…Intriguingly, as summarized in Fig. 10, except for F164L, which confers DENV resistance to SDM25N and AM404 and localizes at the cytosolic loop between TMD3 and TMD4 (65,67), all other drug resistance mutations are mapped in the transmembrane domains. Furthermore, except for DENV V63 and YFV P219 mutations, which specifically confer resistance to spiropyrazolopyridone compound-14a and BDAA, respectively, all other DENV and YFV drug resistance mutations identified thus far confer resistance to multiple drugs (25).…”

“…Interestingly, several structurally distinct compounds have been identified that inhibit DENV or YFV replication through targeting NS4B (40,(63)(64)(65)(66)(67). In addition, multiple inhibitors of the NS4B protein of hepatitis C virus, a distantly related member of the Flaviviridae family, are currently under preclinical and clinical development for treatment of hepatitis C infection (68).…”

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection

“…Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25). Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26)(27)(28)(29)(30)(31)(32). It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.…”

Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis

“…Several biological activities on eukaryotic cells have previously been reported for AM404, including activating the capsaicin receptor TRPV1, inhibiting COX, activating cannabinoid receptors, inhibiting cellular anandamide reuptake, and inhibiting NFAT activity and IκB kinase beta phosphorylation and activation (Bertolini et al, ; Caballero et al, ; Högestätt et al, ; Mallet et al, ; Ottani, Leone, Sandrini, Ferrari, & Bertolini et al, ). A recent study also identified AM404 as an antiviral agent that effectively inhibits replication of the dengue virus by binding with the NS4B protein (van Cleef, Overheul, Thomassen, Marjakangas, & van Rij, ). To our knowledge, no studies exist on the antibacterial activities of AM404 and its mode of action.…”