インシリコ及びインビトロスクリーニングによるA型インフルエンザウイルスの増殖阻害活性をもつ低分子化合物の同定

Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. Nucleoprotein (NP) is an attractive target since it has an indispensable role in virus replication and its amino acid sequence is well conserved. In this study, we aimed to identify new inhibitors of the NP using a structure-based drug discovery algorithm, named Nagasaki University Docking Engine (NUDE), which has been established especially for the Destination for GPU Intensive Machine (DEGIMA) supercomputer. The hit compounds that showed high binding scores during in silico screening were subsequently evaluated for anti-influenza virus effects using a cell-based assay. A 4-hydroxyquinolinone compound, designated as NUD-1, was found to inhibit the replication of influenza virus in cultured cells. Analysis of binding between NUD-1 and NP using surface plasmon resonance assay and fragment molecular orbital calculations confirmed that NUD-1 binds to NP and could interfere with NP-NP interactions essential for virus replication. Time-of-addition experiments showed that the compound inhibited the mid-stage of infection, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 was also effective against various types of influenza A viruses including a clinical isolate of A(H1N1)pdm09 influenza with a 50% inhibitory concentration range of 1.8?2.1 μM. Our data demonstrate that the combined use of NUDE system followed by the cell-based assay is useful to obtain lead compounds for the development of novel anti-influenza drugs.長崎大学学位論文 学位記番号:博(医歯薬)甲第997号 学位授与年月日:平成29年9月20日Author: Juliann Nzembi Makau, Ken Watanabe, Takeshi Ishikawa, Satoshi Mizuta, Tsuyoshi Hamada, Nobuyuki Kobayashi, Noriyuki NishidaCitation: PLOS ONE, 12(3), e0173582; 2017Nagasaki University (長崎大学)課程博

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs

多剤耐性カンジダおよび細菌に対するカスポファンギンの新たな抗微生物活性

長崎大学学位論文 [学位記番号]博（医歯薬）甲第1326号 [学位授与年月日]令和3年3月22

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs

Susceptibility of influenza viruses to NUD-1 and neuraminidase inhibitors.

<p>Susceptibility of influenza viruses to NUD-1 and neuraminidase inhibitors.</p

Mode of action of NUD-1 against influenza virus replication.

<p>MDCK cells were infected with A/WSN/33 virus (MOI = 0.001) and the culture supernatant was collected at 12 h post-infection for TCID₅₀ assay. (A) Different cell and/or virus treatment protocols. (B) NUD-1 (2.5 μM) was added to cells before, during or after virus infection; or virus was incubated with NUD-1 before infection. C) Infected cells were exposed to NUD-1 (2.5 μM) or zanamivir (10 μM) at different time points after virus infection (0–3 h, 3–6 h, 6–9 h, 9–12 h). Virus yield for each treatment condition is represented as a percentage of the untreated control. The results are the mean ± SD obtained from at least two independent experiments. Student’s t-test was performed using Graphpad software. * indicates a <i>p</i>-value of less than 0.05.</p

Chemical structure and anti-influenza activity of NUDs 6–24.

<p>Commercially available analogs of NUDs 1–5 were acquired and evaluated for anti-influenza activity. The results are the average ± SD from two independent experiments.</p

Multiple alignment of IAV and IBV NP sequences.

<p>Sequences of A/WSN/33 (AAA43452.1), A/Puerto Rico/8/34 (NP_040982.1), A/Virginia/04/2009 (ACR08603.1), A/Aichi/2/68 (AFM71861.1) and B/Lee/40 (NP_056661.1) were obtained from the NCBI protein database and aligned using Clustal Omega software. The NP sequence for strain A/Virginia/ATCC2/2009 was not available, so strain A/Virginia/04/2009 was chosen as a representative sequence for A(H1N1)pdm09 influenza. Asterisks indicate conserved amino acid residues between IAV and IBV, while conserved residues among IAV strains are highlighted in grey. Amino acid residues important for NP-NP interactions in IAV and IBV are indicated in red and purple font, respectively. Tail loop regions are underlined.</p