Vol. 8 No. 3 September–December 2020 Available online at IJTID Website: https://e-journal.unair.ac.id/IJTID/ Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Case Report Eff ect of Zinc(II)-2,4,5-triphenyl-1H-imidazole Complex Against Replication DENV-2 in Vero Cell Aswandi Wibrianto1, Fatimah Martak2, Teguh Hari Sucipto3a, Siti Churrotin3, Ilham Harlan Amarullah3, Harsasi Setyawati4, Puspa Wardhani3, Aryati3, Soegeng Soegijanto3 1 Undergraduate Student, Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Indonesia 2 Department of Chemistry, Faculty of Natural Science, Institut Teknologi Sepuluh Nopember, Indonesia 3 Dengue Study Group, Institute of Tropical Disease, Universitas Airlangga, Indonesia 4 Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Indonesia Received: 8th April 2019; Revised: 29th January 2020; Accepted: 23rd April 2020 ABSTRACT Dengue virus (DENV) is a signifi cant pathogen emerging worldwide as a cause of infectious disease. DENVs are transmitted to humans through female mosquitoes from Aedes aegypti and Aedes albopictus species. Indonesia is one of the largest countries in the world in dengue endemic regions worldwide. Dengue fever was occurred for the fi rst time as an outbreak in Surabaya and Jakarta in 1968. Many eff orts have been made to prevent and treat DENV infections, and clinical trials of a number of vaccines are currently underway. Antiviral testing of DENV is an important alternative for drug characterization and development. Complex compounds are formed as a result of metal and organic complex reactions. Complex compounds can be used as an anti-infl ammatory, antimicrobial antifungal, antibacterial, antivirus. The Zn2+ ion can be used as an antiviral candidate. The purpose of this project was investigated Zinc(II)-2,4,5-triphenyl-1H-imidazole antiviral compound to be further tested for inhibitory eff ect on the replication of DENV-2 in cell culture. DENV replication was measured by antiviral activity assay and cytotoxicity assay. The inhibitory activity of Zinc(II)-2,4,5-triphenyl-1H-imidazole complex compound was determined by Viral ToxGloTM Assay. The cytotoxicity of Zinc(II)-2,4,5-triphenyl-1H-imidazole complex compound was determined by CellTiter96® AQuoeus assay. The inhibitory concentration (IC50) of Zinc(II)-2,4,5-triphenyl- 1H-imidazole against dengue virus type-2 was 34.42 μg/ml. The cytotoxic concentration (CC50) of compound against Vero cell was <100 μg/ml. The results of this study demonstrate the antidengue serotype 2 inhibitory activity of investigated Zinc(II)-2,4,5-triphenyl-1H-imidazole complex and its high toxicity in Vero cells. Further studies are not required before investigated Zinc(II)-2,4,5-triphenylimidazole can be applied in the treatment of DENV-2 infections. Keywords: Zinc (II), complex compound, cytotoxicity, inhibitory activity, DENV-2 ABSTRAK Virus Dengue (DENV) adalah patogen signifi kan yang muncul di seluruh dunia sebagai penyebab penyakit menular. DENV ditransmisikan ke manusia melalui nyamuk betina dari spesies Aedes aegypti dan Aedes albopictus. Indonesia adalah salah satu negara terbesar di dunia di daerah endemik demam berdarah di seluruh dunia. Demam berdarah terjadi untuk pertama kalinya sebagai wabah di Surabaya dan Jakarta pada tahun 1968. Banyak upaya telah dilakukan untuk mencegah dan mengobati infeksi DENV, dan uji klinis sejumlah vaksin saat ini sedang berlangsung. Pengujian antivirus DENV adalah alternatif penting untuk karakterisasi dan pengembangan obat. Senyawa kompleks terbentuk sebagai hasil dari reaksi kompleks logam dan organik. Senyawa kompleks dapat digunakan sebagai anti-infl amasi, antimikroba antijamur, antibakteri, antivirus. Ion Zn2+ dapat digunakan sebagai kandidat antivirus. Tujuan dalam proyek ini adalah menyelidiki senyawa antivirus Zink(II)-2,4,5-trifenil-1H-imidazol yang diuji lebih lanjut untuk efek penghambatan pada replikasi DENV-2 dalam kultur sel. Replikasi DENV diukur dengan uji aktivitas antivirus dan uji sitotoksisitas. Aktivitas penghambatan senyawa kompleks Zinc(II)-2,4,5-triphenyl- 1H-imidazol ditentukan dengan Viral ToxGloTM Assay. Sitotoksisitas senyawa kompleks Zinc(II)-2,4,5-triphenyl- * Corresponding Author: teguhharisucipto@staf.unair.ac.id 184 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 3 September–December 2020: 183–188 1H-imidazol ditentukan dengan uji CellTiter96® AQuoeus. Konsentrasi penghambatan (IC50) Zinc(II)-2,4,5-trifenil- 1H-imidazol terhadap virus dengue tipe-2 adalah 34,42 μg/ml. Konsentrasi sitotoksik (CC50) senyawa terhadap sel Vero adalah <100 μg/ml. Hasil penelitian ini menunjukkan aktivitas penghambatan serotipe 2 antidengue dari Zinc(II)-2,4,5- trifenil-1H-imidazol yang diteliti dan toksisitasnya yang tinggi dalam sel Vero. Studi lebih lanjut tidak diperlukan sebelum investigasi Zinc(II)-2,4,5-trifenil-1H-imidazol dapat diterapkan dalam pengobatan infeksi DENV-2. Kata kunci: Seng (II), senyawa kompleks, sitotoksisitas, aktivitas penghambatan, DENV-2 How to Cite: Effect of Zinc(II)-2,4,5-triphenyl-1H-imidazole Complex Against Replication DENV-2 in Vero Cell. Wibrianto, A. Martak, F. Setyawati, H. Sucipto, TH. Churrotin, S. Amarullah, IH. Wardhani, P. Aryati, A. Soegijanto, S. Indonesian Journal of Tropical and Infectious Disease, 8(3), 183–188. imidazole-1-β-ᴅ-ribofuranoside is examined for four diff erent types of viruses from the fl aviridae family in vitro, including hepatitis C virus (HCV), Japanese viral encephalitis (JEV), West Nile virus (WNV), and dengue virus (DENV) in vitro against NTPases/helicases. The compound showed activity highly active against WNV with IC50 was 23 μM.7 Complex compounds are formed as a result of metal and organic compound reactions. Complex compounds can be used as an anti-infl ammatory8, antimicrobial9, antifungal, antibacterial10, and antivirus11. Based on previous research, Copper(II)-imidazole derivatives can be used as antiDENV-2, can be used as low toxicity and potential as drug candidates. The compound exhibited adsorption inhibitory activity against DENV-2 at IC50 = 2.3 μg/ml.12 The Zn2+ ion can be used as an antiviral candidate.13 Zn2+ ions can change the activity of various transcription factors and thus, patterns of cellular and viral gene expression.14 Thus, the antiviral test of the compound Zinc(II)-2,4,5- triphenyl-1H-imidazole was investigated. MATERIALS AND METHODS Chemicals and Media Chemical reagents used in this research were Zinc(II)-2,4,5-triphenyl-1H-imidazole complex compound, Minimum Essential Eagle Medium (Sigma-Aldrich, Germany), dengue virus serotype 2 Surabaya isolate (KT012509), Vero cells (African Green Monkey Kidney), Viral ToxGloTM assay (Promega, USA), CellTiter96® INTRODUCTION Dengue virus (DENV) is a virus carried by the fl avivirus genus of the family Flaviviridae. Dengue virus (DENV) consists of four serotypes which is dengue virus type 1, dengue virus type 2, dengue virus type 3, and dengue virus type 4. Dengue virus is transmitted to humans through female mosquitoes from Aedes aegypti and Aedes albopictus species. World Health Organization (WHO) reported 390 million dengue infections per year.1 Indonesia is one of the largest countries in the world with dengue endemic areas. Surabaya and Jakarta were the cities where dengue disease was fi rst reported in Indonesia in 1968.2 Many studies have been conducted to overcome the threat of dengue virus infections, and clinical trials of a number of vaccines are currently on the way.3 Antiviral testing on DENV is a very important method in the development and characterization of drugs. Supplementary to vaccines, inhibitors in each natural cycle of viral replication have the potential to cure dengue virus infection and indeed compounds such as RNA replication inhibitors have been tested as such.4 However, there is no commercially available drug with antiviral activity for DENV.5 L i g a n d 2 , 3 , 5 - t r i p h e n y l - 1 H - i m i d a z o l e compound is a derivate of imidazole. Imidazole- containing drugs that have strong therapeutic properties have encouraged scientists to synthesize many novel chemotherapeutic agents consisting of these entities. N5-(4-fluorophenyl)-N4- (2-(pyridin-4-yl)benzyl)-1H-imidazole-4,5- dicarboxami-de, a derivate of imidazole, was reported anti-DENV activity.6 4-carbamoyl-5- (4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl) 185Aswandi Wibrianto, et al.: Effect of Zinc(II)-2,4,5-triphenyl-1H-imidazole Complex Against Replication Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 AQuoeus One Solution Cell Proliferation Assay (Promega, USA). Antiviral Activity Assay Confluent monolayers of Vero cells were prepared on a 96-well plate (1 × 106 cells/10 ml) and counted using a hemocytometer, and the titer of DENV-2 (2 × 104 FFU/well) was expressed in Foci-Forming Units (FFU) after incubating at 37°C for 2 days. The concentrations of Zinc(II)- 2,4,5-triphenyl-1H-imidazole were 50 μg/mL; 25 μg/mL; 12.5 μg/mL; 6.25 μg/mL; 3.13 μg/ mL; 1.57 μg/mL; 0.78 μg/mL; and 0.39 μg/mL with addition 100 μL Viral ToxGloTM Assay per well. The 50% inhibitory concentration (IC50) of DENV-2 replication by each compound was further investigated by using GloMax® Discover System. Cytotoxicity Assay A cytotoxicity assay was performed using CellTiter96® AQ uoeus One Solution Cell Proliferation reagent. The CellTiter96® Assay is a modifi cation of the MTT assay method portrayed by Akter.15 The concentrations of Zinc(II)-2,4,5- triphenyl-1H-imidazole were 100 μg/mL; 200 μg/mL; 400 μg/mL; 600 μg/mL; 800 μg/mL; and 1000 μg/mL. The medium was allowed to equilibrate for 1 hour; then 20μl/well of CellTiter 96® AQueous One Solution Reagent was added. After 1 hour at 37°C in a humidifi ed, 5% CO2 atmosphere, the absorbance at 490nm was recorded using GloMax® Discover System. Viral Detection by Reverse Transcriptase- Polymerase Chain Reaction RNA replication was estimated using the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The purpose of this assay was to known RNA replication after treatment. Briefl y, DENV-2 RNA was extracted from the DENV-2 infected cells and cell culture supernatant using RNA extraction kit by Qiagen, Germany. The two-step kit (Toyobo, Japan) was used for cDNA synthesis and Polymerase Chain Reaction (PCR) following the manufacturer’s instructions. Primer oligonucleotide sequences were as follows by Bhatnagar et. al. 2012.16 Amplifi cation condition was 54 °C for one minute (annealing temperature) and the amplifi ed product was the analyzed on 1.5% agarose gel. RESULTS AND DISCUSSION The cytotoxicity of Zinc(II)-2,4,5-triphenyl- 1H-imidazole complex compound was determined by CellTiter96® AQuoeus assay and the recorded CC50 value is <100 μg/ml to Vero cells. When compared with a previous study, Copper(II) was found to be nontoxic to human erythrocyte cells to concentrations of 500 μg/ml.17 CC50 is the cytotoxicity level of [Cu(2,4,5-triphenyl-1H- imidazole)2]n (compound) to cause death to 50% of Vero cells.12 The toxicity value of Cobalt(II) complex with 2,4,5-triphenyl-1H-imidazole ligand was 362.24 mg/L, which was not toxic.18 The toxicity value of 2-methyl-4,5-diphenyl- 1H-Immidazole ligand compound was 192,3 μg/ ml.19 The toxicity of [Mn(2-(4-chlorophenyl)- 4,5-diphenyl-1H-imidazole)2(H2O)2]·2H2O was >200 μg/ml which had less toxicity.20 Zinc(II)–2- (2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen- 4-one complex compound defi ned cytotoxicity with CC50 at 3.59 μg/ml.21 But, the metal-free imidazole more toxic for Vero cells (CC50 = 5.03 μg/ml).22 Activity against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells (CC50) by zinc(II) complexes with hexyl-Me2-cyclam (HMC; 3,14- dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.07,12)- docosane) were >372 μM and >372 μM with selectivity index >35 and >3. Activity against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells (CC50) by Zn(II)–HMC diacetate were 110.67 ± 12.67 μM and 110.67 ± 12.67 μM with selectivity index 32 and <1.23 The complex stability is highly dependent on both the metallic ion and the ligands. As for the central ion (M2+), Zn(II) more unstable than Cu(II), Mn(II), and Co(II). The Zn(II) complex has grater polarizability that that Cu(II), Mn(II), and Co(II) because it contains more d-electrons, and the Zn(II) complex produced more product ions soluble in water.24 This eff ect causes Zn(II) to be more toxic, because Zn2+ in the medium are 186 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 3 September–December 2020: 183–188 more numerous, so it damages the cell wall faster than complex compound that have high stability such as Cu(II), Mn(II), and Co(II). The percentage inhibition of the development of dengue virus type-2 by the test sample of Zinc(II)-2,4,5-triphenyl-1H-imidazole complex compound was shown on fi gure 1. The IC50 value was determined from the concentration–response curve (Figure 1); the IC50 value was 34.42 μg/ml, R2 was 0.9196. Based on the value of the IC50 Zinc(II)-2,4,5-triphenyl-1H-imidazole complex compound was a medium toxic compound. Antiviral activity was also shown in Figure 2, these fi ndings were corroborated by results obtained from RT-PCR which indicated signifi cant reduction in the amount of DENV-2 genomic RNA levels. The highest percentage of viral inhibition was observed after treating the infected cells with 50 μg/ml. Based on the previous study, [Cu(2,4,5- triphenyl-1H-imidazole)2]n complex compound exhibited adsorption inhibitory activity against DENV-2 at IC50 = 2.3 μg/ml. The inhibition at IC50 was not signifi cantly high (p<0.005) compared to that of the metal-free imidazole (IC50 = 0.13 μg/ ml).12 The maximal inhibitory concentration (IC50) of Copper(II)chloride Dihydrate against DENV-2 was 0.13 μg/ml.22 Activity against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells (IC50) by zinc(II) complexes with hexyl-Me2-cyclam (HMC; 3,14- dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.07,12) docosane) were 10.51 ± 0.23 μM and 133.78 ± 14.10 μM. Activity against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells (IC50) by Zn(II)– HMC diacetate were 3.50 ± 0.33 μM and >110.67 μM.23 Anti-HIV-1 activity (IC50) in C8166/IIIB, MT-4/GUN1 and PBLs/IIIB were 8.0 μg/ml, 3.5 μg/ml, and 9.3 μg/ml, respectively. The IC50 value of the Cobalt(II)–Morin complex for DENV-2 was 3.08 μg/ml.25 MB21, a benzimidazole derivative, was found to be the most potential inhibitor of cloned proteases (IC50 = 5.95 μM).26 This study suggest that of Zinc(II)-2,4,5- triphenyl-1H-imidazole complex compound can’t be an attractive antiviral option. It would be interesting to further investigate whether 2,4,5-triphenyl-1H-imidazole complex with other metal. The result of this study, Zinc(II)-2,4,5- triphenyl-1H-imidazole complex compound more toxic than Cu(II)-2,4,5-triphenyl-1H-imidazole, this is caused by the Zn (II) complex being unstable compared to the Cu (II) complex. CONCLUSION Further studies are not required before Zinc(II)- 2,4,5-triphenyl-1H-imidazole can be applied in the medication of DENV-2 infections. This study did not show the potential of the Zinc(II)-2,4,5- triphenyl-1H-imidazole complex as a candidate for antiviral agents against DENV-2 because it was shown to be toxic to Vero cells. Figure 1. Inhibition of DENV-2, at variation concentrations of Zinc(II)-2,4,5-triphenyl- 1H-imidazole complex compound 1000 bp 500 bp 100 bp (a) (b) (c) (d) (e) (f) (g) (h) Figure 2. Electrophoresis on 1.5% agarose of RT- PCR after treatment, molecular weight marker (100 bp), (a) treatment with 50 μg/ml compound, (b) 25 μg/ml, (c) 12.5 μg/ml, (d) 6.25 μg/ml, (e) 3.13 μg/ml, (f) 1.57 μg/mL, (g) 0.78 μg/mL, and (h) 0.39 μg/mL 187Aswandi Wibrianto, et al.: Effect of Zinc(II)-2,4,5-triphenyl-1H-imidazole Complex Against Replication Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 CONFLICT OF INTEREST There is no confl ict of interest of this paper. ACKNOWLEDGEMENT This research was supported by a Research Grant from Mandat Universitas Airlangga (HRMUA) 2019; the Institute of Tropical Disease (ITD); the Center of Excellence (COE) program by the Ministry of Research and Technology (RISTEK) Indonesia; and the Chemistry Department of Universitas Airlangga. REFERENCES 1. 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