With crystal structures of
protein-ligand interactions becoming increasingly accessible, it is easy to
forget that proteins do not exist as the static structures seen on page or screen.
Indeed, back in 2018 we
quoted Karplus quoting Feynman that “everything that
living things do can be understood in terms of the jiggling and wiggling of
atoms,” and even the smallest proteins have lots of atoms. In an open-access
paper published in
Proc. Nat. Acad. Sci. USA earlier this year, Heike
Schönherr, David Shaw, and collaborators at Relay Therapeutics, D.E Shaw Research,
Pharmaron, and Columbia University take advantage of these movements.
The researchers were interested
in finding selective inhibitors of fibroblast growth factor receptor 2 (FGFR2),
which is activated in many cancers. The four members of the FGFR family are so
closely related that finding selective inhibitors is difficult. Inhibiting FGFR1
can lead to hyperphosphatemia, while inhibiting FGFR4 can cause diarrhea, side
effects seen with the approved fragment-derived drug
erdafitinib.
Although the structures of FGFR1
and FGFR2 are very similar, extended (25 µs) molecular dynamics simulations revealed
that the so-called P-loop of the proteins behaved differently: in FGFR1 it became
disordered, while in FGFR2 it remained more rigid. The researchers sought to
take advantage of these differences with a
covalent inhibitor.
The researchers started with a
non-selective
hinge-binding fragment, compound 1. Adding an acrylamide warhead
led to a nanomolar inhibitor with modest selectivity for FGFR2. (All IC
50
values are measured after 30 minute incubations.) Growing the molecule into the
so-called back pocket of the kinase led to compound 5, with nearly 100-fold
selectivity for FGFR2 over FGFR1.
The path from compound 5 to lirafugratinib
(also called
RLY-4008) looks straightforward but was anything but. First, the aryl
acrylamide was a metabolic liability, so the researchers attenuated the reactivity
by adding a methyl group. Mechanistic studies with this molecule revealed that while
it had only a slightly better affinity (K
I) for FGFR2 than FGFR1, it
had a k
inact value about 15-fold higher for FGFR2. Molecular dynamics
studies suggested that the relevant cysteine in FGFR1 is locked in a position
too far from the acrylamide to react, while the corresponding cysteine in FGFR2
may be able to more closely approach the acrylamide warhead.
Further optimization, guided by
extended molecular dynamics simulations, led eventually to lirafugratinib with
~250-fold selectivity for FGFR2 over FGFR1 and >5000-fold selectivity over
FGFR4. Remarkably, the noncovalent version of lirafugratinib, compound 11, shows
dramatically lower affinity for both FGFR1 and FGFR2 and very little
selectivity between them. The ligand seems to assume a different binding mode
after covalent bond formation, which could explain these differences in selectivity.
Mouse studies of lirafugratinib
showed tumor stasis or regression without increased serum phosphate levels. More
importantly, early clinical data has shown “minimal hyperphosphatemia and
diarrhea.”
This is a lovely example of structure
and
dynamics-based design (SDBD?). Commonly cited advantages of covalent
drugs include improved potency and extended pharmacological effects, but this
work shows that they can also achieve remarkable selectivity between closely
related proteins, even when both proteins contain cysteine residues in the same
location. Moreover, an open-access
paper in
Cancer Discov. that dives
more deeply into the biology shows that lirafugratinib is selective across the
kinome, inhibiting just two of 468 kinases other than FGFR2 by >75% at 500
nM.
The next time you’re trying to
find a selective inhibitor for one member of a protein family, it
may be worth taking a covalent approach, and paying close attention to dynamics
along the way.
