First-in-class dual inhibitors of MASTL and Aurora A kinase: Discovery of selective cyclohexa[b]thiophenes with potent anticancer activity

Abstract

The dysregulation of mitotic kinases, particularly Microtubule Associated Serine/Threonine Kinase Like (MASTL) and Aurora A kinase, play pivotal roles in tumor progression and resistance to therapy. Herein, we report cyclohexa[b]thiophenes as first-in-class dual inhibitors of MASTL and Aurora A kinase. The lead compound, MA4, demonstrated potent inhibition of both kinases with IC₅₀ values of 0.16 ± 0.01 μM for Aurora A and 0.56 ± 0.16 μM for MASTL. Kinase selectivity profiling against a panel of 277 kinases revealed a high degree of specificity against both targets. In vitro antiproliferative screening using the NCI-60 human cancer cell line panel revealed broad-spectrum cytotoxicity, with MA4 exhibiting submicromolar GI₅₀ values across multiple malignancies, outperforming previously reported cyclohexa[b]thiophenes in the multidose screening. Mechanistic studies, including microscale thermophoresis (MST) and NanoBRET target engagement assays, confirmed direct binding to both kinases. Computational studies, including molecular docking and molecular dynamics simulations, revealed key interactions stabilizing MA4 within the ATP-binding sites of both kinases. We demonstrated the potent anticancer activity of MA4 in 3D tumor spheroids, along with its favorable pharmacokinetic profile. Additionally, MA4 exhibited no inhibitory activity against hERG and demonstrated selectivity toward cancer cells over normal cell lines, further supporting its potential for in vivo applications. These findings establish cyclohexa[b]thiophenes as promising dual kinase inhibitors with high selectivity, offering a compelling strategy for targeting mitotic dysregulation in cancer therapy.