Over the past sixteen months SARS-CoV-2 has infected more than 146 million people worldwide and killed over 3 million. Highly effective vaccines are now available, but not everywhere, and how long the vaccinations will last as new variants arise remains unknown. COVID-19 will likely be with us indefinitely, necessitating drugs as well as vaccines.
More than a year ago we highlighted the COVID Moonshot effort, which began with a crystallographic screen against the essential main protease (Mpro) to find starting points for drug discovery. In a new open-access J. Biomol. NMR paper, Ioannis Vakonakis and collaborators at University of Oxford and University of Patras have attempted to characterize some of the hits using saturation transfer difference NMR (STD NMR, see here for a brief description).
The researchers had access to a 950 MHz NMR (jealous much?). Samples were screened at 10 µM protein using irradiation of a Mpro methyl group with a chemical shift at 0.5 ppm. To try to minimize differences in relaxation parameters among different ligands, only the strongest STD signals in aromatic moieties were examined.
Of 39 non-covalent ligands discovered in the crystallographic screen, five either did not produce an NMR signal or the spectra were inconsistent with the expected structures, suggesting the ligands may be insoluble or unstable in aqueous buffer. The remaining 34 compounds were nominally screened at 0.8 mM each, but the reference spectra differed in intensity by as much as 15-fold, suggesting dramatic differences in concentration. Since the strength of an STD signal is related to both affinity and concentration, this could obviously complicate interpretation of results. As we’ve written previously, careful curation of your library is essential.
Of thirteen active site ligands, only four showed strong STD signals. Dose-response titrations between 0.05 and 4 mM revealed dissociation constants of 1.6-1.7 mM for two of them, with the other two being too weak to accurately measure. Molecular dynamics simulations starting with the known structures were consistent with these results, with the tighter binders tending to maintain their positions more than the weaker binders.
The researchers also characterized 650 elaborated molecules from the COVID Moonshot, some of which had been reported to be nanomolar inhibitors. Disturbingly, 35 gave no NMR signal and another 86 yielded weak signals. Among those remaining there was a weak correlation (R2=30%) between IC50 in an enzymatic assay and the STDratio (integrated signal intensity of peaks in the STD spectrum over reference spectrum). STD NMR is not appropriate for molecules with Kd < 10 µM, so the researchers also used a competition experiment in which four putative high-affinity molecules would compete a weaker “spy” fragment. This exercise confirmed two ligands but not two others, calling into question their mechanism.
STD NMR is often used as part of an assay cascade prior to attempting crystallography, but as crystallography throughput increases there is a case for starting with crystallography, as we argued five years ago. Results from the 39 crystallographic hits perhaps gives pause to that notion, or at least emphasize the need for confirmatory assays. It is easy to be seduced by a high-resolution structure, but because of its sensitivity crystallography may identify ligands so weak as to be unadvanceable. As for the 650 elaborated molecules, it’s too early to draw conclusions, though it’s good to always be on the lookout for false positives.
Hopefully the COVID Moonshot will ultimately lead to drugs against SARS-CoV-2. But even if it doesn’t, the intensive focus of multiple techniques on a few proteins is providing useful guidance and best practices that will be applicable to other targets.