Hu et al. Virology Journal (2017) 14:55 DOI 10.1186/s12985-017-0724-6 RESEARCH Open Access A novel benzo-heterocyclic amine derivative N30 inhibits influenza virus replication by depression of Inosine-5’- Monophospate Dehydrogenase activity Jin Hu†, Linlin Ma†, Huiqiang Wang, Haiyan Yan, Dajun Zhang, Zhuorong Li, Jiandong Jiang and Yuhuan Li* Abstract Backgroud: Influenza virus is still a huge threat to the world-wide public health. Host inosine-5’- monophosphate dehydrogenase (IMPDH) involved in the synthesis of guanine nucleotides, is known to be a potential target to inhibit the replication of viruses. Herein, we evaluated antiviral activity of a benzo-heterocyclic amine derivative N30, which was designed to inhibit IMPDH. Results: The results demonstrated that N30 inhibited the replication of H1N1, H3N2, influenza B viruses, including oseltamivir and amantadine resistant strains in vitro. Mechanistically, neuraminidase inhibition assay and hemagglutination inhibition assay suggested that N30 did not directly target the two envelope glycoproteins required for viral adsorption or release. Instead, the compound could depress the activity of IMPDH type II. Based on these findings, we further confirmed that N30 provided a strong inhibition on the replication of respiratory syncytial virus, coronavirus, enterovirus 71 and a diverse strains of coxsackie B virus. Conclusions: We identified the small molecule N30, as an inhibitor of IMPDH, might be a potential candidate to inhibit the replication of various viruses. Keywords: Benzo-heterocyclic amine derivative, IMPDH, Influenza A virus Background have limited efficacies as drug resistance occurrence Influenza A virus (IAV) is extremely prone to cause [7, 8], and they only worked at the early phase of periodic epidemics and pandemics in the world through virus infection. The inherent property of influenza evolution by point mutations or swapping of gene viruses to mutate, resulting in low efficacy of available segments, correspondingly [1–3]. drugs, has underscored the necessity of developing At present, vaccination and antiviral drugs are principle alternative strategies to provide protection against strategies to prevent influenza. Available anti-influenza pandemic influenza. drugs include inhibitors of neuraminidase (NA) (e.g. Widespread utilization of directly antiviral drugs oseltamivir and zanamivir) [4], M2 proton channel (aman- accelerates resistance problem, hence host cellular tadine and rimantadine) [5], and RNA-dependent RNA factors become attractive therapeutic targets to treat polymerase (RdRp) (favipiravir) [6]. However, influ- influenza virus infections. Recent studies were pio- enza vaccines must be reformulated each year due to neered in host purine metabolic pathway, a conserved the constant antigenic evolution of influenza. Add- process responsible for providing host cells with a itionally, NA inhibitors and M2 ion-channel inhibitors ready supply of guanosine triphosphate (GTP) for crit- ical cellular processes [9]. Host cells could produce GTP either in de novo pathway or salvage pathway. * Correspondence: [email protected] † While in the de novo synthesis of GTP, IMPDH cata- Equal contributors Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences lyzes the oxidation of inosine monophosphate (IMP) and Peking Union Medical College, Beijing, China © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hu et al. Virology Journal (2017) 14:55 Page 2 of 9 to xanthosine monophosphate (XMP) which is the F12 (1:1) (Invitrogen) supplemented with 1% antibiotics rate-limiting step. Growing evidences support that and 10% FBS. Cells were cultured at 37 °C in the inhibition of IMPDH decreases intracellular levels of presence of 5% CO2. guanosine nucleotides in DNA or RNA synthesis, thereby indirectly inhibits virus replication which Viruses and virus infection requires host guanine nucleotides as raw materials Influenza A/Fort Monmouth/1/1947 (H1N1) strain [10, 11]. As IMPDH inhibitors targeted on host cells, was perchased from ATCC. Clinical isolated strains A/ it would be less susceptible for selection of drug- TianjinJinnan/15/2009 (H1N1, oseltamivir-resistant), resistant strains. A/LiaoningZhenxing/1109/2010 (H1N1, oseltamivir-resis In early study, we established a series of novel benzo- tant), A/Wuhan/359/1995 (H3N2), A/FujianTongan/196/ heterocyclic amine derivatives and determined their in 2009 (H3N2, amantadine-resistant), A/HunanZhuhui/1222 vitro antiviral activities. Notably, compounds 3d (N30) /2010 (H3N2, amantadine-resistant), BV/Shenzhen/155/ showed potent activity towards IAV at low micromolar 2005 and BY/FujianXinluo/54/2006 were kindly pro- concentrations [12]. In the present study, we developed vided by Professor Yuelong Shu at the Institute for the broad-spectrum antiviral activity of N30 in vitro, Viral Disease Control and Prevention, China Centers for including oseltamivir-resistant strains and amantadine- Disease Control and Prevention. These strains were all ob- resistant strains of influenza virus, coxsackie B virus, tained by propagating in 10-day-old embryonated chicken coronavirus, and respiratory syncytial virus. In addition, eggs for 48 or 72 h. we examined the inhibitory rate of the compound Coxsackie virus B (CVB) strains CVB1 (strain Conn-5) against two IAV envelope glycoproteins, hemagglutinin and CVB5 (strain Faulkner) were kindly provided by and neuraminidase, and investigated its effect on ex- Professor Zhaohua Zhong, Department of Microbiology, pression and the enzyme activity of IMPDH type II. Harbin Medical University. Enterovirus 71 (EV71) strain Our results indicated that N30 is a potential compound SZ98 was kindly provided by Dr. Qi Jin, Institute of with antiviral activities through suppressing the activity Pathogen Biology, Chinese Academy of Medical Science of IMPDH type II, these finding also proves that devel- and Peking Union Medical School, Beijing, China. EV71 opment of anti-influenza drugs directing at IMPDH is strain BrCr, CVB strains CVB2 (strain Ohio-1), CVB3 warranted. (strain Nancy), CVB4 (strain J.V.B.), CVB6 (strain Schmitt), respiratory syncytial virus (RSV)strain(longstrain)and Methods human coronavirus (229E) were all obtained from ATCC. Compounds EV71 virus and CVB virus were propagated in Vero cells, N30 (N-(4-nitrophenyl methyl) benzothiazole-6-amine, RSV, and coronavirus were propagated in Hep2 and MRC- Fig. 1a) was originally provided by Professor Zhuorong 5 cells respectively. Li at the Institute of Medicinal Bioechnology Chinese For influenza virus infections, MDCK cells were washed Academy of Medical Sciences, Beijing, China. Pirodavir with PBS and infected with influenza virus diluted in (Biochempartner), oseltamivir carboxylate (OC, Med- serum-free medium at 37 °C for 2 h. Then, the viral inocu- chem), amantadine hydrochloride (AH, Sigma-Aldrich) lum was replaced by maintenance medium supplemented and Ribavirin (RBV, Sigma-Aldrich) were used as refer- with 2 μg/ml TPCK-treated trypsin (Worthington) and ence drugs in vitro. N30, pirodavir and AH were dis- 0.08% BSA (Sigma-Aldrich). As for other virus infections, solved in dimethyl sulfoxide (DMSO, Sigm-Aldrich). OC virus was diluted in serum-free medium and incubated with and RBV were dissolved at phosphate buffered saline cells at 37 °C for 1 h. Then, the viral inoculum was replaced (PBS, Gbico). by maintenance medium supplemented with 2% FBS. Culture of cells Cytotoxicity test Cells in this study were all purchased from the America The cytotoxicity of N30 in MDCK, Vero, Hep2, and MRC- Type Culture Collection (ATCC). Madin-Darby canine 5 was evaluated by Cell Counting Kit (CCK, Transgen kidney (MDCK) cells were grown in Minimum Essen- Biotech). Briefly, cells were seeded in 96-well culture plates tial Medium (MEM, Invitrogen) containing 1% Non- (MDCK cells were 2.5 × 104 per well, Hep2 cells were 1 × Essential Amino Acids Solution (NEAA, Invitrogen), 104 per well, Vero and MRC-5 cells were 4 × 104 per well). 1% Penicillin-Streptomycin (10,000 U/mL) (Invitrogen) 16 h later, cells were incubated with serial two-fold dilu- and 10% fetal bovine serum (FBS, Gbico). African green tions of N30 for another 48 h. Then, 10 μLofCCKwas monkey kidney (Vero) cells and Human lung embryo- added to cells, the absorbance was read at 450 nm on nated cells (MRC-5) cells were cultured in MEM sup- Enspire (Perkin Elmer) after 4 hours incubation. The TC50 plemented with 1% antibiotics and 10% or 15% FBS (defined as the 50% toxicity concentration of drugs) were respectively. While Hep2 cells were grown in DMEM / determined by Reed and Muench method [13]. Hu et al. Virology Journal (2017) 14:55 Page 3 of 9 Fig. 1 Effects of N30 on IAV M2 protein and RNA expression in MDCK. a Chemical structure of N30. b The effect of N30 on viabilities of MRC-5, Vero, MDCK and Hep2 cells, which were measured by CCK. c The effect of N30 with different concentrations on IAV M2 protein and RNA expression. d The effect of N30 on