As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.high-throughput screening | antiinfluenza | influenza NP | influenza PB1 | chemical genetics

The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism-disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozinresistant strains. This work provides a new approach to design antiinfluenza drugs.T he RNA-dependent RNA polymerase (RDRP) of the influenza A virus is composed of polymerase basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA) (1). The function of RDRP for viral replication requires association with the nucleoprotein (NP) (2) to form the ribonucleoprotein (RNP) complex. Only low resolution structures of the RNP complex are available from cryo-EM studies (2-9), whereas high resolution structures have been reported for some individual components or fragments (10-12). Crystal structures of NP indicate that it exists in trimers (13,14), with the tail-loop (residues 402-428) region playing an important role in the trimerization (Fig. 1A). Based on the structural information, it was suggested that the tail-loop binding pocket could be a target for antiinfluenza therapeutics (13,14).Disruption of the NP-NP interaction as a strategy for designing antiinfluenza drugs has been further reported. Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers. These results support the importance of NP in the RDRP activity and viral replication, and the possibility of NP as a drug target. However, it remains to be shown that molecules capable of disrupting the NP-NP interaction would inhibit viral replication.Recently Kao et al. (19) and our group (20) reported the use of high throughput screening to identify nucleozin and its analogs as inhibitors that halt viral replication by binding to NP and causing it...

T he forkhead-associated (FHA) domain, discovered in 1995 (10) and first suggested to bind phosphoproteins in 1998 (24), is known to specifically recognize phosphothreonine (pT) to exert its function (5,20). Although the sequence homology among different FHA-containing proteins is relatively low, the structural architecture of FHA domains is highly conserved. It contains a six-stranded ␤-sheet and another five-stranded ␤-sheet, forming a ␤-sandwich. The FHA-pT binding has been shown to regulate diverse biological functions, ranging from DNA damage repair to cell cycle checkpoints to signal transduction (18). Furthermore, the mechanism of FHA-phosphoprotein binding varies greatly among different FHA-containing proteins. The structure, specificity, mechanism, and biological functions of FHA domains have been summarized in recent reviews (16,18).TRAF-interacting protein with an FHA domain (TIFA) was first identified as a tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) binding protein. Consisting of 184 amino acids, TIFA is the smallest FHA domain-containing protein in humans (Fig. 1A). In the absence of TNF-␣ stimulation, TIFA overexpression in HEK 293T cells can activate NF-B and AP-1 (14), suggesting a direct involvement of TIFA in TNF-mediated immune responses. This involvement of TIFA was further attributed to the binding of TRAF2, which requires the TRAF domain of TRAF2 and almost the entire TIFA protein (residues 1 to 162) (14). TIFA was also reported to bind to TNF-associated factor 6 (TRAF6) (25). The consensus binding site of TIFA for TRAF6 was mapped to be glutamic acid 178 (E178) (11, 25), indicating different binding mechanisms in TIFA-TRAF2 and TIFA-TARF6 interactions. In addition, TIFA overexpression, even in the absence of interleukin-1 (IL-1), was shown to activate NF-B and c-Jun aminoterminal kinase (JNK), possibly through its enhancement of TRAF6 binding to IL-1 receptor-associated kinase 1 (IRAK-1). On the other hand, mutation of E178 abolished the binding of TIFA to TRAF6 and the ensuing activation of NF-B (25). In a follow-up report, TIFA was shown to promote oligomerization and ubiquitination of TRAF6, leading to activation of IB kinase (IKK), based on in vitro studies (6).Although the studies of Takatsuna et al. (25) and Ea et al. (6) have previously established the key function of TIFA in its interaction with TRAF6, several issues still remain inconclusive. For example, TIFA has been suggested to be phosphorylated, and the integrity of the FHA domain of TIFA is essential for its function (6, 25), but little information has been unveiled about the molecular basis of TIFA phosphorylation and its functional consequences.In this work, we report that threonine 9 (T9) is a newly identified phosphorylation site of TIFA and that the phosphorylation level of T9 increases upon TNF-␣ treatment. Based on data collected here, we concluded that TIFA-FHA binds to this pT9 site. Such a TIFA-FHA/pT9 binding directs TIFA self-association and promotes NF-B activation through the oligomerizatio...