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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.

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The Story So Far

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 K_d 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.

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The first expansion

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 S_NAr warhead.

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SAR from second expansion

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.

• Resynthesizing RA-3301 and RA-20636 was successful and yielded comparable EC₅₀ to previous batches.

• Expanding around RA-20636 generated only 1 compound of 6 with similar EC₅₀ as the parent compound. Taking into account the aggregation issues and less than excellent therapeutic window, further expansion will depend on the results from aggregation and toxicity tests on RA-188638.

• 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.

• Concerns regarding the PAIN motif remains - data indicate a significant part of the activity is from this part of the molecule, some from the nitrile, and a small part for the benzene ring of the quinoline.

• Third expansion efforts will focus on confirming the possible Mannich Phenol activity from the PAIN motif.

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August 2024

• Second expansion evaluation at SGC Toronto (protein interaction and aggregation)
• Third expansion planing
• First and Second expansion proposed for testing in gelbased assay in Gotte Lab
• Co-crystal efforts ongoing in Arnold Lab