Azadiradione regulates heat shock factor 1 function by interacting with its DNA ‐binding domain independent of the oligomerization domain

Heat shock factor 1 (HSF1) masters cellular proteostasis under stress by upregulating molecular chaperones that help refold or degrade misfolded proteins. HSF1 activation involves its monomer‐to‐oligomer transition and binding to its recognition sequence: the heat shock elements (HSEs) on its target gene promoters. HSF1 activity declines with age as well as in neurodegenerative disorders (NDs) such as Parkinson's disease, highlighting the need for therapeutic strategies to restore its function. Azadiradione (AZD), a limonoid isolated from Azadirachta indica seeds, directly activates HSF1 in cellular and preclinical ND models, unlike other small‐molecule activators reported elsewhere. In this study, the molecular basis of AZD‐mediated HSF1 activation was investigated using purified variants of HSF1, including those lacking its oligomerization and transactivation domains. Fluorescence polarization and dynamic light scattering assays revealed that AZD promotes the oligomerization of monomeric HSF1 to enhance its HSEs‐binding affinity by engaging its DNA‐binding domain (DBD). The oligomerization domain, which is required for stress‐induced HSF1 activation, appears redundant in AZD‐mediated activation. Furthermore, evidence suggests that AZD‐induced conformational alterations in the HSEs‐DNA facilitate its binding to the HSF1 monomer. Intriguingly, AZD was found to compromise the DNA‐binding ability of pre‐assembled trimeric/oligomeric HSF1. This finding also highlighted a potential anticancer effect of AZD, as cancer cells heavily depend on this HSF1 population for rapid proliferation and survival. Overall, these findings offer new insights into the functional regulation of HSF1 and suggest a framework for developing small‐molecule HSF1 activators with therapeutic potential for protein conformational disorders.