Discovery of a borylated aza-arene chemotype as a direct HSF1 inhibitor targeting the DNA-binding domain

Heat shock transcription factor 1 (HSF1) is a master regulator of proteostasis and a compelling yet challenging Anticancer target because of the scarcity of direct small-molecule inhibitors and the lack of canonical ligand-binding pockets. Here, we report the discovery of compound 1168, a borylated aza-arene identified by differential scanning fluorimetry (DSF)-based screening against the HSF1 DNA-binding domain (DBD). Orthogonal biophysical validation by microscale thermophoresis (MST) demonstrated direct binding of 1168 to HSF1-DBD with a dissociation constant (Kd) of 23.8 μM, while electrophoretic mobility shift assay (EMSA) showed that 1168 effectively disrupted HSF1-DBD/HSE complex formation and nearly completely abolished DNA binding at 12.5 μM under the assay conditions. Functionally, 1168 suppressed proliferation across multiple Cancer cell lines and showed enhanced sensitivity in androgen-dependent prostate Cancer cells. Mechanistically, 1168 inhibited HSF1 signaling, reduced the expression of downstream effectors including HSP70 and HSP90, and attenuated HSE-driven transcriptional activity. Furthermore, 1168 reduced the expression of HSF1 and impaired heat shock-induced HSF1 Ser326 phosphorylation and trimerization, thereby suppressing HSF1 activation. Importantly, the inhibitory effect of 1168 on HSP70 expression was markedly attenuated upon HSF1 knockdown, supporting HSF1-dependent activity in cells. Collectively, these findings identify 1168 as a direct HSF1-DBD-binding inhibitor and establish borylated aza-arenes as a promising chemotype for the development of HSF1-targeted Anticancer agents.