Engineering Bioactive Dimeric Transcription Factor Analogs via Flow Synthesis and Palladium Rebound Mediated Protein Cross-Coupling

Sebastian Pomplun

Sebastian Pomplun

Universiteit Leiden, Einsteinweg 55, 2333 CC Leiden

Dysregulation of the transcription factor MYC is involved in the majority of human cancers. The dimeric transcription factor complexes MYC/MAX and MAX/MAX bind to the same enhancer box (E-Box) DNA. MYC/MAX activates, and MAX/MAX inhibits gene transcription. Inspired by the MAX/MAX activity, we engineered covalently linked, synthetic homo- and heterodimeric analogs of MYC, MAX, and Omomyc, to inhibit MYC-dependent gene transcription. We prepared the dimers (167-231 residues) in a single shot via chemical flow-synthesis or via palladium mediated protein cross- coupling. All variants displayed correct folding, DNA binding activity, and enhanced structural stability compared to their non-covalent counterparts. The dimers are intrinsically cell-penetrating and inhibit cancer cell proliferation in low micromolar concentrations. Via RNA sequencing and RT-qPCR we showed that the synthetic dimers specifically downregulate MYC-dependent transcription. This work shows the intriguing potential synthetic protein based modalities for artificial gene regulation.

References:
[1] S. Pomplun et al., ACS Cent. Sci., 2021, 7, 8, 1408–1418. [2] M. Jbara,+ S. Pomplun+ et al., JACS, 2021, 143, 30, 11788–11798 (+ = equal contribution)

 

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