Emerg Infect Dis 15:155C162. of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 2009 pandemic H1N1 (pH1N1) virus. Even though the efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug. IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 offers potential to be optimized as a new anti-influenza drug. (1). Like a human being respiratory pathogen, influenza disease causes recurrent seasonal epidemics, with global blood circulation of two subtypes (H1N1 and H3N2) of influenza A disease (IAV) and the two antigenically unique lineages (so-called Victoria and Yamagata) of influenza B disease (IBV), which leads to an estimated half million deaths each year (2). Sometimes, the disease also provokes a pandemic that affects global areas with incalculable socioeconomic damage (3, 4). These perpetuating events of influenza may result from antigenic drift or shift that cause strain and subtype changes of influenza viruses, respectively (5, 6). To deal with antigenic variance issues, universal treatment methods against numerous (sub)types of influenza viruses have been investigated (7,C10), with vaccine designs and platforms becoming the primary considerations to develop common vaccines inducing cross-reactive immunity in vaccinated individuals (11). While anti-neuraminidase (anti-NA) antibodies and additional immune repertoires have been also investigated for the development of broadly effective therapeutics (12,C16), several broadly neutralizing anti-hemagglutinin (anti-HA) antibodies have been found out: C05, which recognizes the receptor binding site (RBS) AF1 of HA (17); CR6261 and CR8020, which inhibit the fusion activity of the IDO-IN-12 organizations 1 and 2 IAV HAs, respectively (17); FI6, which neutralizes IAVs of both HA organizations (18); and CR9114, which has effectiveness against both IAVs and IBVs (19). These antibodies can be used as universally effective therapeutics (20), although there are still issues to be solved IDO-IN-12 in terms of their delivery routes and high developing costs (21). Antiviral medicines can be another option as universal treatment methods against numerous (sub)types of influenza viruses (22, 23). Most antivirals authorized by the Food and Drug Administration are small molecules (24), and several types of antiviral medicines have been used to treat influenza (25). However, due to the possibility of the emergence and dissemination of antiviral-resistant strains (26, 27), fresh types of antivirals are constantly needed. Nine antiviral drug candidates (baloxavir marboxil, a cap-dependent endonuclease inhibitor [28], which has been licensed recently in the United States; FluDase, a recombinant sialidase fusion protein [29]; JNJ-5806, a polymerase inhibitor; laninamivir octanoate, a neuraminidase inhibitor [30]; MEDI8852, an HA stem-binding monoclonal antibody [31]; NT-300, an HA intracellular trafficking inhibitor; pimodivir, a PB2 cap-snatching inhibitor [32]; radavirsen, a genetic transcription inhibitor of antisense oligonucleotide [33]; and VIS-410, an HA stem-binding monoclonal antibody [34]) are in medical tests (24), five of which are small molecules IDO-IN-12 that have advantages of standardization and manufacturing processes. Given the binding and fusion functions of the influenza disease HA protein, small molecules focusing on the HA may be of great importance in the prevention and treatment of influenza in humans. Several HA-targeted candidates are currently under investigation (35), of which umifenovir (Arbidol) is an orally active antiviral agent that inhibits HA fusion activity (36) and has been used to treat influenza in China and Russia. However, this inhibitor is not approved for use in other countries. In addition, the HA-targeting small-molecule inhibitors RO-5487624 and RO-5464466 and natural molecules SQ-02-S5 and pentacyclic triterpenoids are in preclinical studies (35). Recently, a small molecule (JNJ4796) was reported to inhibit HA-mediated membrane fusion by binding the residues that CR6261 interacts with in the HA stalk region (37). Despite.