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Abstract
Transcription factors (TFs) are crucial proteins involed in regulation of gene expression and cellular behavior. Their dysregulation can precipitate diseases, notably cancer, positioning TFs as potential targets for treatment. However, the complexity of targeting protein-protein and protein-DNA interactions presents significant challenges, hindering the development of TF-targeting drugs within the current pharmacological arsenal. In particular, bZIP protein family, consisting of 53 members drives numerous types of cancer, representing a family of attractive yet “undruggable” pharmacological targets. We pioneered the development of a modular platform - a synthetic DNA-binding domain (sDBD) - which mimicked the FOS/JUN heterodimer, both of which are members of the bZIP protein family. Through iterative optimization of the sDBD structure, we provided valuable insights into the structure-activity relationship involving the DNA-binding domain of bZIP proteins interacting with DNA and explored diverse chemistries in protein engineering. In subsequent research, we highlighted the significant role the Leucine zipper domain of bZIP proteins assumes in DNA recognition. Leveraging these attributes, we designed a new modular platform: synthetic transcription repressors (STRs). Systematic fine-tuning of the Leucine zipper segment within STRs has elucidated the underlying mechanisms of dimerization amongst bZIP proteins. Notably, a lead STR derivative, derived from the FOS/JUN heterodimer, demonstrated potent and specific binding to its target AP-1 DNA. We broadened the scope of STRs to target other bZIP protein family members, notably the unfolded protein response protein, XBP1. STRs derived from XBP1 exhibited strong binding affinity and specificity to their UPRE DNA targets. Through vigilant stabilization of secondary, tertiary, and quaternary structural elements, STRs exhibited superior DNA binding affinity, cellular permeability, and proteolytic stability while retaining DNA recognition specificity. Under hypoxic conditions, STR globally suppressed HIF1 binding to gene promoters and enhancers, thereby curtailing gene expression. We demonstrated that STRs impeded the aggressive phenotypes of triple negative breast cancer both in vitro. We further showed that systemic administration of STR globally blocked the HIF1-mediated hypoxia response, mitigating aggressive growth and metastasis. Finally, we extended the STR approach to a distinct synthetic scaffold derived from the MAX homodimer, a bHLH family member. MAX STR exhibited potent EBox DNA binding affinity, specificity, and efficient cell permeability, while counteracting MYC binding to EBox in cells. Crystal structure analysis of the STR: E-box DNA complex confirmed near identitcal DNA recognition to full-length bHLH transcription factors. We also demonstrated the potential for reprogramming MAX STR to target other bHLH family proteins. Collectively, these findings underscored the flexibility and utility of the synthetic transcription repressor (STR) platform, thereby establishing it as a robust pharmacological tool to target transcription factors.