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Welcome to the CNP18-DENVRdrp-VirtualScreen wiki!
This repository outlines one of several projects related to our efforts to identify a small molecular compound that binds to the Dengue virus RNA-dependent RNA polymerase (RdRp). This specific repository describes our efforts at identifying a possible hit molecule from the Enamine REAL library.
Below is a summary of the project, and the sidebar to the the right allows you to explore each step if the process further; linking to additional data, tables, and other overviews we might only reference below.
To get an overview of the READDI-AViDD project please read the Rules of Participation and Origins of the Project.
You can also explore Other Repositories related to Dengue RdRp campaign.
The work done around the lead compound, RA-3301, has been summarized here
In mid-2023 we started a campaign to identify a new druggable pocket on the DENV RdRp and a subsequent virtual screen of the Enamine REAL space to helped us identify new starting points for developing a probe that binds to Dengue RdRp in this pocket. In this process, that took ~6 weeks, a new druggable pocket was identified computationally and confirmed through mutagenic studies, and then allowed a virtual screen using HIDDEN-GEM to generate a selection of 65 compounds that were sent for initial testing.
Of these, three hits showed antiviral activity and were used as starting point for further exploration. A first tentative exploration for the three hits was selected from commercially available sources. These should provide information about what parts are important for binding and will guide future SAR exploration.
Additional data for the three hits /RA-3272, RA-3301, RA-3318) show that RA-3301 is the top compound due to its favorable antiviral replication activity (EC90), best Kd of the tested compounds, as well as favorable aggregation profile. The main issue that needs to be addressed with this compound is:
- the very low solubility causing issues in some assays,
- that the CC50/EC50 < 5.
In Q1 of 2024, the first expansion was sent for testing and the results of these efforts are chelated on this page. In summary, initial single point data pointed at four compounds with potential activity. After dose-response determination, antiviral replication tests, and whole cell toxicity this was narrowed down to two analogues of RA-3301 that were of interest: RA-20636 and RA-20641.
Especially RA-20641 shows retained activity and CC50/EC50 > 5. Notably, in this compound the motif that presented as a possible PAIN in the parent compound has been removed. The lower part of the RA-3301 seems to play a minor role for activity this could allow us to use this part of the molecule overcome the issues with solubility. Notably, the retained activity may also be due the chloro-pyridine acting as a covalent SNAr warhead.
Synthetic chemistry efforts have commenced to do a conservative SAR analysis of RA-3301 and RA-20636. A combination of custom synthesis and in-house synthesis generated 31 compounds that were send for testing. The results are summarized below and a detailed description can be found here for RA-3301 and here for RA-20636.
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Resynthesizing RA-3301 was successful and yielded comparable EC50 to previous batches.
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Expanding around RA-20636 generated only 2 compounds of 6 with similar EC50 as the parent compound. Taking into account the aggregation issues and less than excellent therapeutic window of RA-20636. After further analysis, exploring this compound has been put on hold due to the limited avenues to move forward.
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Our assumption regarding RA-20641 showing we can change the lower part freely (that was backed up by computational modeling) of RA-3301 was too hasty - only 3 compounds of 23 showed comparable EC50 as RA-3301 with 2 only keeping activity towards DENV2.
The data from the third and fourth expansion show that the SAR around RA-3301 remains challenging to establish. Further, our current SAR is driven by antiviral activity as we are yet to confirm activity on RA-3301 or any of it's analogues using SPR, in the picogreen assay, or the gelbased assay.
In the third and fourth expansions we were able to conclude:
- That the PAIN motif is not causing toxicity, but instead the phenol is generating a significant part of the activity of the molecule.
- The hydroxy group can be removed replaced by a fluoro-group and the isoindole ring can be opened, which presents better synthetic variability.
- The lack of RdRp inhibition but in some cases binding to DENV2 NS5, has raised the question of where these compounds are binding. An MTase assay is being developed to check is the compounds are binding to that part of DENV2 NS5.
What we know of RA-3301 has been summarized here.
Dec 2024
The synthetic efforts in this project is slowing down until we can figure out:
- if the compounds are binding DENV2 MTase,
- if the compounds aggregation profiles are the cause of the antiviral activity (potential false positives).