Fragments vs TIM-3

In order to thrive, cancer cells need to evade the immune system. Preventing them from doing so is the goal of cancer immunotherapy. Although it has not entirely lived up to its initial hopes, this promising approach has generated multiple new targets, such as T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), whose upregulation correlates with tumor progression. Several antibodies targeting this protein are working their way through the clinic, but small molecules may have advantages in terms of oral dosing and improved tumor penetration. The discovery of one small molecule binder is reported in a new J. Med. Chem. paper by Stephen Fesik and colleagues at Vanderbilt University.

 

As is customary for this group, the project began with a two-dimensional (¹H/¹⁵N HMQC) NMR screen of 13,824 fragments, each at 0.8 mM in pools of 12. This yielded 101 hits, a respectable 0.7% hit rate, and higher than might be expected for this immunoglobulin-like protein. The hits belonged to 11 chemotypes, and 18 had dissociation constants better than 1 mM and ligand efficiencies (LE) better than 0.25 kcal mol^-1 per heavy atom. All of the fragments caused similar resonance perturbations, suggesting a common binding pocket, though as specific backbone resonance assignments were not known the exact location was unclear. Compound 1 was pursued due to its (relatively) high affinity, LE, and chemical tractability.

 

Substitutions off two vectors of the molecule improved affinity, and combining these substituents led to compound 22. This molecule bound sufficiently tightly that NMR could no longer be used to measure the dissociation constant. At this point the researchers were able to solve a crystal structure of the compound bound to TIM-3, revealing that it binds to a protein loop with the tricyclic core sandwiched between two tryptophan residues. The structure also revealed a portion of the molecule that extended toward solvent, and this insight was used to construct a fluorescent probe for use in a fluorescence polarization anisotropy (FPA) competition assay to accurately measure binding of more potent molecules.

 

 

With the probe results and crystal structure in hand, the researchers continued to optimize the molecule by growing towards a couple arginine and aspartic acid residues. This led to compound 34, which again started bottoming out the FPA assay and necessitated constructing yet another fluorescent probe. Further optimization using structure-based design ultimately led to compound 38, the most potent molecule in the series. NMR experiments revealed that compound 38 causes a rigidification of the TIM-3 loop where it binds.

 

And that’s where things stand. Unfortunately no data are presented as to whether compound 38 blocks binding of TIM-3 to its biological partners. The binding site is actually somewhat distant from where natural ligands bind, suggesting that the compounds would likely need to act allosterically. Moreover, the researchers note that many of the compounds are not particularly soluble. Still, whether the compounds move forward or not, this is a nice example of finding fragments that bind to a novel target and using diverse insights to improve them by several orders of magnitude.