To contribute to the global effort to combat COVID-19, Diamond has been able to solve a new structure of the SARS-CoV-2 main protease (MPro) at high resolution (PDB ID: 6YB7), and complete a large XChem crystallographic fragment screen against it (detailed below). Data have been deposited with the PDB, but we are making the results available immediately to the world on this page; additional work is ongoing, and updates will be continually posted here in coming days and weeks.
This work builds on the sensationally fast crystal structure of MPro at 2.16 Å in complex with a covalent inhibitor, released in January this year by Prof Zihe Rao (6LU7, published here, described here). We thus ordered the synthetic gene and cloned the full length protein as previously described for the SARS main protease (Xue et al 2007). This yielded crystals of the unliganded enzyme that diffracted to high resolution (1.25 Å) on beamline I04-1, in a different space group to the inhibtor complex, and the structure was determined and refined rapidly. Critically, this showed it had the active site empty and solvent accessible - perfect for fragment screening.
So it proved: the first 600-crystal experiment could be completed in 72 hours, through growing large numbers of crystals, optimising the soaking conditions, soaking and harvesting all 600 crystals and completing the data collection run on beamline I04-1. The hits from this initial run and other details were pre-released on March 6th.
On Tuesday March 17th we publicly released results from the full 1500-crystal experiment that yielded 58 non-covalent and covalent active-site fragments. Following data reprocessing and further analysis, an additional 13 structures were released on March 24th taking the final total to 66 active site fragments, 44 of which were covalently bound (full timeline here, download page here). This was an exceptionally large screen, and yielded an exceptionally rich readout, with vast opportunities for fragment growing and merging.
We have already triggered computationally-driven follow-up work internally, and externally joined forces to launch a fully-open crowdsourcing and crowdfunding iniative to establish urgently the shortest route possible to clinical impact by maximally exloiting the readout - you can help, read more here.
The screen encompassed three fragment libraries: the poised library; our collection of probing fragments (mainly FragLites, MiniFrags); and the electrophile library pre-screened by mass spec at the Weizmann.
Features of note include:
|S1 - the His163 - Glu166 motif||S2 - hits over catalytic Cys145||S3 - the aromatic wheel|
During analysis of the data, our new PanDDA-2 algorithm identified that crystals fall into 3 distinct clusters, separated mainly through subtle changes in unit cell parameters. These do not however appear to bear on the chemical opportunities in the binding site.
The London lab screened Mpro supplied by Diamond with 993 fragment electrophiles by their intact-protein mass-spectrometry platform (Resnick et al. JACS 2019). A preliminary screen (200 µM, 24 h, 4°C) revealed 130 compounds that irreversibly label the protein (>50%); a more stringent screen (5 µM, 1.5 h, RT) discriminated several compound series that still label robustly:
The full experiment is summarised in this spreadsheet: 74 compounds were screened at XChem by crystal soaking and co-crystallisation following Resnick et al, working in triplicate: resulting in 42 complex crystal structures. (Of the rest, 29 yielded crystals without bound ligand, and 3 could not be measured for experimental reasons.)
A particularly promising series are the chloroacetamides based on piperidine-amides: as they were not hits in many previous screens, they should be very selective to Mpro. Additionally, they reveal SAR (Structure activity relationships) even amongst the nine representatives in the library, suggesting binding can be optimised. Based on the several crystal structures of these hits, we have commenced synthesis of analogs and testing of optimized derivatives.
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