Fragments in the clinic: Lirafugratinib

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 IC₅₀ 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.