The cytokine tumor necrosis
factor α (TNFα) is a key mediator of inflammation and has long been a target
for rheumatoid arthritis, Crohn’s disease, psoriasis, and a host of other
inflammatory diseases. Several biologic drugs, such as adalimumab, are approved
but these monoclonal antibodies and fusion proteins can be immunogenic or
induce neutralizing antibodies. Small molecules could avoid these pitfalls and also reach organs, such as the brain, less accessible to biologic agents. Impressive efforts towards this goal have just been reported in J.
Med. Chem. by Justin Dietrich, Chaohong Sun, and colleagues at AbbVie.
(Andrew Petros presented this work at the CHI DoT meeting last September.)
The researchers began by
screening 18,000 fragments using two-dimensional (13C-HSQC) NMR against
TNFα in which the methyl groups of isoleucine, valine, leucine, and methionine
were isotopically labeled. Only 11 fragments caused significant perturbations, an
0.06% hit rate reflecting the difficulty of finding hits against this target. All
the fragments were characterized by SPR, and compound 1 turned out to have reasonable
affinity and ligand efficiency. Synthesis of a few dozen analogs led to
compound 2, with improved activity.
TNFα forms a homotrimer, and a
crystal structure of compound 2 bound to TNFα revealed that two copies of the fragment
bind within a large hydrophobic cavity at the interface of the three protein
monomers. Not present in the apo-form of the protein, this central pocket is
formed by the movement of tyrosine side chains, causing desymmetrization of the
protein trimer. The researchers linked the two nearby fragments to produce
compound 3 with improved affinity but decreased ligand efficiency. Further
optimization led to compound 4, which was active in cells. But perhaps not
surprisingly given its size and lipophilicity, this molecule had high clearance
and poor oral bioavailability in mice.
A second fragment, compound 6, had
lower affinity than fragment 1, and parallel chemistry efforts generated only
flat SAR. A crystal structure revealed that compound 6 bound in a similar
manner as compound 1, with two copies in the central cavity. Surprisingly, a crystal
structure of compound 8, which differs from compound 6 only by a single methyl group
and actually has slightly lower affinity, revealed a singly copy bound in the
central void. Scaffold hopping led to compound 9, which was ultimately optimized
through structure-based design and careful attention to drug-like properties to
compound 12. This molecule is orally bioavailable and showed activity in a
mouse arthritis model.
This is a lovely paper that illustrates
several important lessons. First, as the researchers note, “we have learned
from multiple programs, including this one, aimed at developing small-molecule inhibitors
of protein-protein interactions, that biophysical methods, when used to drive a
fragment-based approach, offer the greatest chance of success.” NMR was
essential for finding the initial fragments, SPR provided necessary
thermodynamic and kinetic information, and crystallography led to the
breakthrough discovery of the binding mode of compound 8.
Second, although the initial
series generated by fragment linking ultimately did not advance, it proved
critical for developing chemical tools, validating assays, and providing structural
insights.
And finally, this paper is a paean
to persistence for difficult targets. As the researchers note, scientists have
been seeking small molecule inhibitors of TNFα for decades, and although compounds
were reported as early as 2005, most of these have had poor physicochemical properties.
Seemingly undruggable targets can sometimes be unlocked. But it usually takes
time.
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