Last week we highlighted NMR
screens against RNA from SARS-CoV-2. As we noted then, much of the drug discovery
action has focused on the virus’s main protease, called Mpro or 3CLp.
These efforts have included two separate screens by crystallography and/or mass
spectrometry. A new (open-access) paper in Angew. Chem. Int. Ed. by Benoit
Deprez, Xavier Hanoulle, and collaborators at CNRS and University Lille describes
an NMR screen against this same protein.
The main protease is 306 amino
acid residues long and forms a 67.6 kDa dimer in solution. Proteins this large
are challenging for protein-detected NMR, both because the number of potentially
overlapping resonances increases and because of line broadening. Nonetheless,
the researchers used several sophisticated NMR techniques to assign more than
60% of backbone resonances as well as quite a few main-chain and side-chain hydrogens
to gain information on binding locations.
A library of 960 fragments was purchased
from Life Chemicals and Maybridge. These were pooled in groups of five, with
each fragment at 377 µM, and screened by Water-LOGSY and, for 427 fluorinated
fragments, 19F line broadening and chemical shift perturbation. This
exercise yielded 159 hits.
These hits were validated in a
protein-detected 1H, 15N TROSY-HSQC experiment, which confirmed
38 fragments, giving an overall hit rate of around 4%, comparable to that seen in
the crystallographic fragment screen against Mpro. Also in common
with the previous screen is the fact that most of the fragments (32) bind
somewhere in the active site, while a few (5) bind at the dimerization
interface. Fragment hits tended to be both larger and more lipophilic than
those in the overall library.
Earlier this year we highlighted
an NMR study of non-covalent fragment hits from the crystallographic fragment screen, which
found that only two of them had measurable affinities, and both were weak (KD
= 1.6 mM at best). In contrast, one of the new fragments, F01, has a dissociation
constant of 73 µM. With a molecular weight of 287 Da and 20 non-hydrogen atoms
this is a somewhat portly fragment, but it does have a ligand efficiency of 0.3
kcal mol-1 per heavy atom. It also shows functional activity with an
IC50 = 54 µM in a biochemical assay and EC50 = 150 µM in an
antiviral assay. The researchers further characterized their molecule crystallographically,
which confirmed that it binds to the active site; it makes three hydrogen bond
interactions and multiple hydrophobic contacts with the protein.
Although crystallography has been
receiving increasing attention among fragment-screening techniques, this paper
is a reminder than NMR remains highly relevant, even for larger proteins that
crystallize easily. And at the end of the day, it’s not how you screen but what
you find and what you do next. Hopefully folks will follow up on F01. While PF-07321332
is making rapid progress in the clinic against this enzyme and the COVID Moonshot effort is moving molecules into animal studies, HIV has taught us
that we’ll need multiple small molecule drugs.
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