Identification of small molecule inhibitors for influenza a virus using in silico and in vitro approaches

Juliann Nzembi Makau; Ken Watanabe; Takeshi Ishikawa; Satoshi Mizuta; Tsuyoshi Hamada; Nobuyuki Kobayashi; Noriyuki Nishida

doi:10.1371/journal.pone.0173582

Identification of small molecule inhibitors for influenza a virus using in silico and in vitro approaches

PLoS One. 2017 Mar 8;12(3):e0173582. doi: 10.1371/journal.pone.0173582. eCollection 2017.

Authors

Juliann Nzembi Makau^{1

2}, Ken Watanabe¹, Takeshi Ishikawa¹, Satoshi Mizuta¹, Tsuyoshi Hamada³, Nobuyuki Kobayashi¹, Noriyuki Nishida¹

Affiliations

¹ Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki, Japan.
² Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki, Japan.
³ Nagasaki Advanced Computing Center, Nagasaki University, Bunkyo-machi, Nagasaki, Japan.

Abstract

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.

MeSH terms

Amino Acid Sequence
Animals
Antiviral Agents / chemistry*
Antiviral Agents / pharmacology*
Cell Line
Computer Simulation*
Computers, Molecular
Drug Discovery* / methods
Humans
Influenza A Virus, H1N1 Subtype / drug effects
Influenza A virus / drug effects*
Models, Molecular
Protein Binding
Small Molecule Libraries*
Viral Core Proteins / antagonists & inhibitors
Viral Core Proteins / chemistry
Virus Replication

Substances

Antiviral Agents
Small Molecule Libraries
Viral Core Proteins

Grants and funding

This work was supported by a grant from the Takeda Science Foundation (NN), a grant from JSPS Next Generation World-Leading Researchers (NEXT Program), Grant Number GR082 (TH), and a grant from JSPS KAKENHI, Grant Numbers 25460125 and 16K00853 (KW). JNM was supported by the Nagasaki University Leading Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The funders had no role in study design, analysis, the decision to publish, or preparation of the manuscript.