GAS41 revisited: a chemical probe

The YEATS domain of the protein GAS41 is an epigenetic reader that modulates gene expression by binding to acetylated lysine residues in chromatin. Multiple lines of evidence suggest it could be a useful target for various cancers, in particular non-small cell lung cancer (NSCLC). Four years ago Practical Fragments highlighted a paper from Jolanta Grembecka, Tomasz Cierpicki, and colleagues at the University of Michigan describing a fragment screen and subsequent optimization of a hit to molecules with some cellular activity. In a new J. Med. Chem. paper, the same team now describes molecules with better cell potency, as well as a negative control.

 

Compound 1, the initial fragment hit, had weak affinity for GAS41, but replacing the t-butyl group with a proline led to compound 7, with low micromolar activity in a fluorescence polarization assay. On the other side of the molecule, modification and growth of the amide moiety led to compound 16, also with low micromolar activity. In the 2021 paper, molecules related to compound 16 were dimerized to bind to two YEATS domains in close proximity in the GAS41 dimer. This yielded mid-nanomolar inhibitors, but the molecules were also large, with limited cell permeability. In the new paper, the researchers instead combined medicinal chemistry learnings and used structure-based design to generate monomeric molecules, culminating in DLG-41.

 

The affinity of DLG-41 for GAS41 was measured as 1 µM using isothermal titration calorimetry (ITC). In accordance with best practices for chemical probes, the researchers also developed a negative control by replacing the thiophene moiety with a thiazole; this compound, DLG-41nc, shows negligible activity in two different biochemical assays.

 

DLG-41 showed high nanomolar activity in a NanoBRET assay, demonstrating that the molecule is both permeable and binds to the GAS41 protein in cells. Importantly, the IC₅₀ for the negative control DLG-41nc was > 25 µM in this assay. DLG-41 blocked proliferation in a panel of NSCLC cell lines, though DLG-41nc also showed some activity, albeit at higher concentrations. Gene expression studies in one cell line showed that DLG-41 caused changes in hundreds of genes, while DLG-41nc was inactive.

 

This is a nice example of fragment optimization in academia. With both biochemical and cell-based potency around one micromolar, DLG-41 is hovering on the edge of the 2015 suggestions for a chemical probe. But used alongside the negative control, the compound should be useful for further exploring the biology of GAS41.