The Story so Far - StructuralGenomicsConsortium/CNP16-iminoquinone-SarsRdRp-mechanstic-study GitHub Wiki

We are wanting to understand how the iminoquinone reacts with the cysteine SH in the protein. To study this (and to understand the observed reactivity with GSH) we need a proxy for the reactive SH groups. The question of which reagent to use was discussed in Issue 1.

The initial hit RA-0001351 has been synthesized (details in Issue 2). Two others (RA-0001663 and RA-0001664) (shown in Issue 3) were bought.

Efforts have been made to try to synthesize cysteine adducts (first attempt also described in Issue 2). An attempt was made to follow the reaction of N-acetyl cysteine with the initial hit RA-0001351 in buffer (conditions mimicking the relevant biochemical assays are described in Issue 3) using NMR, but this proved not possible due to the poor solubility of RA-0001351 under the aqueous conditions used (see Issue 4). A more detailed description of an attempt to carry out the reaction (using N-acetyl cysteine methyl ester) and isolate the adducts (as well as monitor by NMR spectroscopy) was described in Issue 5: A protected-cysteine-RA-0001351 adduct was synthesized and characterized using LCMS, 1H NMR; but it's C13 NMR missing signals.

The reaction of RA-0001351 with protected cysteine was conducted using a buffer identical to that used in the gel-based assay (excluding 0.01% Triton X-100) to confirm the formation of the synthesized adduct under these conditions. Initially, the reaction yielded only a trace signal of the adduct. However, after multiple attempts and troubleshooting, it was determined that the solubility of RA-0001351 in the buffer was the issue. Subsequently, increasing the amount of DMSO to solubilize RA-0001351 resulted in a significant signal of the adduct being successfully observed using LC-MS (see issue 7).

To provide additional evidence demonstrating that sulfur binds to nitrogen in the adduct, two proxies, 4-nitrophenol and 4-bromophenol, were used as substrates to react with RA-0001351, resulting in the synthesis of corresponding adducts. Efforts have been made to grow crystals from these two adducts, and their crystal samples have been sent to the National Crystallography Service (NCS) for structure determination.

Current Step:

Trying reactions using nitrogen and oxygen nucleophiles.

Next Steps:

Seek Theoretical Support: Reach out to computational chemists for theoretical support, such as Density Functional Theory (DFT) calculations, to determine the reaction mechanisms of RA-0001351 with cysteine. This will help validate and further understand the binding interactions between sulfur and nitrogen in the adduct.

Test Additional Hits: Test the other two purchased hits, RA-0001663 and RA-0001664, with protected cysteine to observe if signals of corresponding adducts can be detected using LC-MS. This experimentation will expand the scope of understanding for these compounds' interactions with cysteine residues.