25 January 2021

Fragments vs TNFα advanced with biophysics, linking, and growing

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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