Design and Synthesis of Allosteric Inhibitors against Dengue Virus Protease

Although dengue fever is the most common arthropod-borne disease worldwide, with approximately 390 million infections per year, no specific therapeutic treatment is available to date. Therefore, this thesis addressed the design and synthesis of novel allosteric DENV protease inhibitors as potenti...

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1. Verfasser: Mark, Kerstin
Beteiligte: Diederich, Wibke E. (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2021
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Zusammenfassung:Although dengue fever is the most common arthropod-borne disease worldwide, with approximately 390 million infections per year, no specific therapeutic treatment is available to date. Therefore, this thesis addressed the design and synthesis of novel allosteric DENV protease inhibitors as potential anti-dengue drugs. As a starting point, an in silico High Throughput Screening (HTS) was performed targeting the closed conformation allosteric pocket of DENV3 protease using the ZINC15 lead-like library as input for docking. This resulted in a selection of 15 compounds that were purchased and tested in a fluorescence-based enzyme assay in vitro. The most potent compound from this set showed a promising IC50 value of 136 +/- 16 micromolar against DENV3 protease as well as a non-competitive binding mode and was successfully simplified to a core fragment, which also showed significant inhibition of the DENV protease. This core fragment served as starting point for an in silico fragment growing approach, which after docking and evaluation resulted in a selection of 17 compounds to be synthesized and tested in vitro. Four of these compounds showed significantly increased activity toward DENV3 protease compared to the initial hit in the fluorescence-based assay, with an IC50 value of 28 +/- 7.9 micromolar against DENV3 protease for the most potent one. A general structural motif common to the most effective inhibitors was identified and used as a template for a comprehensive SAR study that revealed important features of the protein-ligand interactions. The most potent compound resulting from this study (IC50(DENV3)=12.3 +/- 3.5 micromolar) also showed high potency against a binary DENV4 protease construct and against ZIKV protease, with an IC50 value against the latter in the low micromolar range. The non-competitive binding mode was also confirmed for the optimized ligand structures, and preliminary mutation experiments were conducted to clarify the actual binding site in the protease. Cell-based experiments revealed low cytotoxicity of the established ligand series at concentrations up to 25 micromolar and relative inhibition of ZIKV replication of more than 50% at concentrations of 5-10 micromolar. Overall, the results obtained in this thesis provide a good basis for further development of potent flaviviral protease inhibitors.
Umfang:199 Seiten
DOI:10.17192/z2022.0044