Fragment growing and merging: an inverse agonist of RORγt2

The nuclear receptor transcription factor RORγt2 is involved in the differentiation of Th17 cells, and is thus a target for inflammatory diseases. The protein contains a large, hydrophobic ligand binding site, and as a result most known inverse agonists have less than ideal physicochemical properties. In a paper recently published in J. Med. Chem., Samuel Hintermann and colleagues at Novartis have taken a fragment-based approach.

The researchers screened a library of 1408 fragments using a “differential static light scattering (DSLS)” assay, which is a type of thermal shift assay that measures denaturation and aggregation of the protein. A few dozen molecules that stabilized RORγt2 were tested in dose-response curves prior to crystallization trials, ultimately yielding 13 structures. Compound 1 was particularly interesting because it binds in the center of the cavity, providing growth vectors in two directions. It also makes a couple hydrogen bonds with the protein, as opposed to purely hydrophobic interactions.

Growing from the ethoxy position quickly led to improvements in affinity. To avoid the possibility of toxic iminoquinone metabolites, the researchers replaced the central phenyl ring with a pyridine, resulting in the low micromolar inverse agonist compound 8.

To further improve affinity, the researchers merged an element from a previously reported GlaxoSmithKline molecule (compound 2) onto compound 8, resulting in the potent compound 9, which was characterized in a battery of assays.

The crystal structure of compound 9 bound to the protein revealed that the core fragment moiety binds in the same manner as the original compound 1, though the added benzyl ether binds in a subpocket that had not previously been observed to bind ligands.

Kinetic studies using a reporter displacement assay revealed that compound 9 has both a slow on-rate as well as a slow off-rate, consistent with the fact that it is fully enclosed by the protein. The researchers performed molecular dynamics simulations to try to determine how the ligand could enter or leave, which suggested large conformational changes in a flexible region of the protein. Isothermal titration calorimetry (ITC) showed that the binding of compound 9 is enthalpically driven, with an unfavorable entropy. Although interpreting thermodynamics is fraught, this result makes intuitive sense given the hydrogen bonds formed and the fact that the molecule seems to rigidify the protein.

Biophysics is interesting, but of course biology is what was driving the program. Compound 9 is potent in a variety of cell assays and is also selective for RORγt2 over other nuclear hormone receptors. However, it is also mostly insoluble, and although it did show efficacy in a rodent inflammation model, plasma concentrations of compound 9 were highly variable between individual rats, which the authors attribute to poor physicochemical properties.

This is a nice application of fragment growing and merging that demonstrates how difficult it is to find useful leads for lipophilic sites: even with favorable biochemistry and biophysics, the pharmacokinetics are a slog. That said, others have made progress against similarly hydrophobic targets, so it will be fun to watch this story progress.