Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Abstract

Influenza A virus infection is a major global health concern causing significant mortality, morbidity, and economic loss. Antiviral chemotherapeutics that target influenza A virus are available; however, rapid emergence of drug-resistant strains has been reported. Consequently, there is a burgeoning need to identify novel anti-influenza A drugs, particularly those that target host gene products required for virus replication, to reduce the likelihood of drug resistance. In this study, a small interfering RNA (siRNA) screen was performed to identify host druggable gene targets for anti-influenza A virus therapy. The host organic anion transporter-3 gene (OAT3), a member of the SLC22 family of transporters, was validated as being required to support influenza A virus replication. Probenecid, a prototypical uricosuric agent and chemical inhibitor of organic anion transporters known to target OAT3, was shown to be effective in limiting influenza A virus infection in vitro (50% inhibitory concentration [IC 50 ] of 5.0 ؋ 10 ؊5 to 5.0 ؋ 10 ؊4 M; P < 0.005) and in vivo (P < 0.05). Probenecid is widely used for treatment of gout and related hyperuricemic disorders, has been extensively studied for pharmacokinetics and safety, and represents an excellent candidate for drug repositioning as a novel anti-influenza A chemotherapeutic.

Differences in Purinergic Amplification of Osmotic Cell Lysis by the Pore-Forming RTX Toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the Role of Pore Size

“…Indeed, we also found here that two nonselective pannexin inhibitors, probenecid and carbenoxolone, antagonized ApxIA-induced hemolysis in a concentration-dependent manner (data not shown). However, such data need to be interpreted with caution, as the regulation of pannexin 1 channel activity is poorly understood and both inhibitors are known to affect also other membrane transporter functions (61)(62)(63)(64). Lysis of erythrocytes by ApxIA was further found to be antagonized by P2X inhibitors (200 M PPADS or 2 M BBG) that did not interfere with the activity and properties of toxin pores formed in planar bilayer membranes.…”

Targeting Cell Division Cycle 25 Homolog B To Regulate Influenza Virus Replication

“…The RNAi screening that was performed identified numerous host cell genes that are required for IAV replication (16,19,20). One of the genes identified was CDC25B, a member of the dual-specificity CDC25 phosphatases that has been shown to dephosphorylate CDK1 and ERK1/2 (21,47).…”

“…To assess CDC25B and IFN-␤ gene expression, cDNA were synthesized using random hexamers as primers (Thermo Scientific). cDNAs were subsequently used for quantitative PCR amplifications using CDC25B, IFN-␤, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene-specific primers and RT 2 SYBR green qPCR Master Mix (SABioscience) in an MX3005P thermocycler as previously described (16). The levels of viral RNA, CDC25B, and IFN-␤ gene expression were normalized to that of GAPDH, and their expression levels relative to mock-treated samples were calculated using the 2 (Ϫ⌬⌬Ct) formula.…”

“…For measurement of influenza A viral copy number, total RNAs collected from infected A549 cells, BEAS2B cells, or lungs of infected mice were used for a quantitative real-time PCR (qRT-PCR) assay using a OneStep RT-PCR kit (Qiagen). A universal influenza primer-probe set (InfA forward primer, InfA reverse primer, and InfA probe; Bioresearch Technologies, Inc.) was used for amplification and detection of influenza A virus RNA as previously described (16).…”

“…Several host factors have been previously identified to promote IAV replication in different stages of the virus life cycle. Among these, organic anion transporter 3 (OAT3) and vacuolar ATPase (vATPase) have recently been shown to facilitate IAV entry into host cells, while other host factors such as importin-␣ and calcium/calmodulin-dependent protein kinase II ␤ (CAMK2B) have postentry roles (15)(16)(17). IAV also utilizes host factors, such as cellular P58IPK, which has been implicated in inhibition of the host double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) response, to modulate antiviral responses (18).…”