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Welcome to the CNP17-DENVRdrp-EnamineCovalent wiki!
This repository outlines one of several projects related to our efforts to identify a small molecule 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 covalent library.
Below is a summary and the sidebar to the right will allow you to explore each step of the process further, linking to data and providing tables, data, and overviews that 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.
In August 2023 we started a high-throughput screening campaign of the Dengue Rdrp using a library of covalent ligands from Enamine. From the compounds showing more than 60% inhibition in the picogreen single-point assay we selected 62 compounds for which we determined the IC50. This generated 16 compounds with an IC50 below 30 μM. These compounds carried one of four warheads, with the acrylamides and chloroacetamides showing the most promising IC50's. We initially used the data from the IC50 screen and the HTS data to explore these two warheads for our first purchased expansion sets. Before this concluded we performed assay interference checks and found that a significant set of the original hits were false positives. This is not unexpected given the reactive nature of the warheads that can make them highly promiscuous. The compounds left as true hits are shown below.
Figure 1: Hits from the HTS of a library of covalent ligands
In this hit-set, two compounds (RA-2041 and -3109) were flagged in online filtering tools as possible PAINs (structures not related to the warheads) and were not explored further. Ligand RA-2478 and -3118 were hits in other screening campaigns using the same library. Acrylamide RA-2478 was of specific concerns as it showed activity against CHIKV nsp2, SARS CoV2 nsp13, and SARS CoV2 Rdrp. A small expansion set of three compounds was purchased and confirmed RA-2478 was not a suitable compound for further exploration; two of the compounds were inactive and the last was highly active but clearly a false positive. Additional tests on RA-3118 in a gel-based DENV2 RdRp interaction assay showed that RA-3118 is also a false positive, unable to block the function or the RdRp.
We thus focused our expansion efforts on the pyridine fluorosulfonamides RA-3145, -3150, and -3156 as this particular warhead seemed to be favored in DENV RdRp over CHIKV nsp2, SARS CoV2 nsp13, and SARS CoV2 Rdrp. As these compounds were more like fragments, the first expansion set (10 compounds) focused on a fairly wide exploration of the structure activity relationship around these hits.
May 31 2024
The 11 compounds from the first expansion have been tested by Jessica Smith in the nano-luciferase antiviral assay -> 4 compounds have been move to dose-response, antiviral replication, and cytotoxicity tests.