Targeting the GAS41 YEATS Domain with Novel Small-Molecule Inhibitors in Non-Small Cell Lung Cancer

Abstract

GAS41 (Glioma-amplified sequence 41) is a protein that is overexpressed in multiple indications of cancer. GAS41 contains a YEATS domain, and epigenetic protein-protein interaction domain that recognizes acylated lysine residues on histones, with high affinity for H3K27ac and H3K27cr. The GAS41 YEATS domain regulates gene expression by recruiting chromatin remodeling complexes to facilitate active expression of oncogenes as well as genetic silencing of tumor suppressor genes. GAS41 is amplified in non-small cell lung cancer, and loss of GAS41 or genetic perturbation of the residues involved in acylation binding results in a reduction in NSCLC cell growth and tumor proliferation. Therefore, the acylation binding pocket of the GAS41 YEATS domain is a potential therapeutic target for small-molecule inhibitor development. We report the discovery and development of first-in-class small-molecule inhibitors for the GAS41 YEATS domain. From a low-millimolar hit discovered in NMR-based fragment screening, extensive medicinal chemistry and inhibitor characterization efforts yielded low-micromolar inhibitors of the GAS41 YEATS domain. This enabled co-crystallization of the GAS41-YEATS and inhibitor, which guided further development. By leveraging the dimeric quaternary structure of full-length GAS41, we hypothesized that linking two inhibitors to engage two YEATS domains simultaneously in a bivalent manner could improve overall GAS41 binding. The resulting inhibitors had remarkably increased, mid-nanomolar inhibitory activity for GAS41 YEATS in biochemical assays and did engage two YEATS domains simultaneously in biochemical and cell-based assays. To improve drug-like potential, we returned to the monomeric inhibitor scaffold and set out to improve both binding affinity for GAS41 YEATS and cell permeability of these inhibitors. We developed a NanoBRET assay to assess intracellular inhibition of the GAS41 YEATS acylation binding. The lead compound of this series potently inhibits GAS41 YEATS in both biochemical and cellular inhibition assays and inhibits cellular proliferation in NSCLC, but not normal lung fibroblast, cell lines. This inhibitor has 8-fold selectivity for inhibiting cellular proliferation of wild-type A549 cells over an A549 GAS41-knockout cell line, demonstrating on-target inhibition of cancer cell growth. In transcriptomic experiments, we validated that the GAS41 inhibitor increases gene expression of CDKN1A, a tumor suppressor that is established as a target of GAS41 YEATS-mediated repression. Mechanistically, we discovered that the GAS41 YEATS inhibitor inhibits NSCLC cell migration, and the antiproliferative activity is caused through the generation of reactive oxygen species. This inhibitor is the first reported small-molecule inhibitor for GAS41 with demonstrated inhibition of NSCLC cellular proliferation. We also report the results of a high-throughput screen we initiated to discover additional scaffolds for GAS41 YEATS domain inhibitor development. Of an 82,000 compound library, we identified only one hit, which demonstrated low micromolar inhibition of the GAS41 YEATS domain. We developed this hit into a new series of inhibitors yielding a 2-fold improvement in binding affinity from the HTS hit, and this new scaffold represents a building block for further inhibitor development. Overall, the work in this dissertation facilitated the development of the first reported GAS41 YEATS domain inhibitors. The inhibitors reported will serve as valuable tools for interrogating the biological role of GAS41 YEATS domain binding in NSCLC tumorigenesis and proliferation. We demonstrate that inhibition of GAS41 YEATS by small molecules is a strategy for inhibiting NSCLC proliferation and may offer a new approach to developing NSCLC therapeutics.