Research Article: Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling

Date Published: January 24, 2019

Publisher: Public Library of Science

Author(s): Maywan Hariono, Sy Bing Choi, Ros Fatihah Roslim, Mohamed Sufian Nawi, Mei Lan Tan, Ezatul Ezleen Kamarulzaman, Nornisah Mohamed, Rohana Yusof, Shatrah Othman, Noorsaadah Abd Rahman, Rozana Othman, Habibah A. Wahab, Alexandre G. de Brevern.


Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 μM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 μM and 16 μM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.

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Dengue, caused by Dengue Virus (DENV), is the most important mosquito-borne viral disease affecting the tropics and subtropics [1]. Endemic in more than 100 countries [2,3], the virus is estimated to cause 390 million infections each year [4]. DENV infections can result in serious diseases including dengue fever, dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and even death [5]. There are no approved antiviral drugs for these diseases and currently, patients are treated with supportive care to relieve fever, pain, and dehydration [6]. A tetravalent dengue vaccine (CYD-TDENV or Dengvaxia), the first dengue vaccine has recently been registered in several countries [7]. Despite being a leading cause of hospitalisation and death among children in some Asian and Latin American countries [8], this vaccine is not recommended for use in children under 9 years of age due to safety concerns [7]. Therefore, there exists an urgent need for antiviral therapies to treat dengue.

Thioguanine was identified as a potential scaffold for DENV-2 NS2B/NS3pro inhibitor based on virtual screening, in vitro assay and molecular modelling. The thioguanine scaffold is composed of pyramidine and imidazole rings attached to an amine and a thiol group which may contribute to the compound’s activity. As far as we are aware, no report of pyrimidine inhibition to DENV-2 NS2B/NS3pro, however, 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine has been reported to demonstrate an activity against SARS-CoV-3C like protease [61]. In addition, pyrimidine has also been shown to actively stop the growth of DENV-2 but it relies on the capability of pyrimidine to inhibit dihydroorotate dehydrogenase (DHODH), an enzyme required for viral pyrimidine biosynthesis [62]. Imidazoles have also been investigated against dengue virus [63] however the exact molecular mechanism or which protein they target is still unknown. The 6-thioguanine scaffold has been reported to non-competitively inhibit ubiquitin specific protease in various cancers [64]. With this background, we postulated that thioguanine derivatives might potentially be good inhibitors against DENV-2 NS2B/NS3pro.

We presented here the computational design of potential DENV-2 NS2B/NS3pro inhibitors. From the virtual screening of National Cancer Institute Database, four compounds including D0713 were observed to have moderate inhibition activity on the protease. D0713 has a thioguanine scaffold in its structure prompting us to consider the scaffold for designing the new thioguanine derivatives as potential DENV-2 NS2B/NS3pro inhibitors. Fifteen compounds were synthesised and the bioactivity against dengue protease showed variation in inhibition activity from inactive to moderately active (1000>IC50>18 μM). Based on this information, a further design of six new compounds was conducted by concentrating on the attachment of amide group. All the compounds showed inhibition activity against the dengue protease with compound 18 being the most potent (IC50 of 0.38 μM). This result agrees well with the MM/PBSA calculation which showed that the interactions are mainly contributed by polar and non-polar interactions. Hydrogen bonding analysis demonstrates the importance of amino acid residues Asn174 (occupancy 60%), Asp75 (8.22%), Tyr183 and Ser157 (2.85 and 2.73%). This is further supported by the experimental results which showed that 18 could be further developed as DENV-2 NS2B/NS3pro inhibitor. It is hoped that the results obtained from this study could be used in designing more active compounds as potential dengue protease inhibitors.




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