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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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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2021
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| Online Access: | PDF Full Text |
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| Summary: | 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. |
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| Physical Description: | 199 Pages |
| DOI: | 10.17192/z2022.0044 |