Chemical Engineering of Artificial Transcription Factors by Orthogonal Palladium(II)‐Mediated S‐Arylation Reactions

Abstract

Site‐selective functionalization strategies are in high demand to prepare well‐defined homogeneous proteins for basic research and biomedical applications. In this regard, cysteine‐based reactions have enabled a broad set of transformations to produce modified proteins for various applications. However, these approaches were mainly employed to modify a single reactive site with a specific transformation. Achieving site selectivity or multiple transformations, essential for preparing complex biomolecules, remains challenging. Herein we demonstrate the power of combining palladium(II)‐mediated C−S bond formation and C−S bond cleavage reactions to selectively edit desired cysteine sites in complex and uniquely modified proteins. We developed an orthogonal palladium(II) strategy for rapid and effective diversification of multiple cysteine sites (3–6 residues) with various transformations. Importantly, we employed our approach to prepare 10 complex analogues, including modified, stapled, and multimeric proteins on a milligram scale. Furthermore, we also synthesized a focused library of stabilized artificial transcription factors that displayed enhanced stability and potent DNA binding activity. Our approach enables rapid and effective protein editing and opens new avenues to engineer new biomolecules for fundamental research and therapeutic applications.

Site-Specific Acetylation of the Transcription Factor Protein Max Modulates Its DNA Binding Activity

“…The precise influence of altering the acetylation marks of TFs’ binding activity in these diseases is yet to be explored. We anticipate this demonstration to prepare homogeneous synthetic TFs with the desired PTM pattern to pave the way for further studies to dissect crosstalk between Max-PTMs and other essential bHLH-TFs as well as design new analogs with refined activity. − Importantly, given the different PTM levels and sites that are already identified in Max, − , a potential interplay between Max phosphorylation and acetylation can possibly represent an additional regulatory role in controlling the bHLH Myc/Max TF network, which requires further investigation. Finally, we envision that the synergy between total chemical synthesis and high-throughput technology will not only provide profound predictive insights into the impact of PTMs on the binding of additional oncogenic TFs to DNA but also lay the groundwork for comprehending other essential oligonucleotide-binding proteins of physiological significance.…”

Synthetic Peptides: Promising Modalities for the Targeting of Disease‐Related Nucleic Acids

“…[65] Several solid phase peptide synthesis (SPPS) approaches have been used to produce Omomyc [27][28][29]31] (as well as Myc and Max). [30,[66][67][68] Brown et al synthesized the whole protein using either microwave or infrared irradiation during SPPS and also explored native chemical ligation (NCL) to be able to make Omomyc from two fragments. Furthermore, they developed a high-throughput microbial expression workflow to introduce mutations and improve Omomyc binding affinity.…”

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC

“…15 An intriguing strategy to target MYC is based on the use of proteins that mimic the inhibitory activity of MAX and occupy E-Box DNA. [16][17][18][19][20][21][22] However, while protein based modalities generally hold immense potential in targeting 'undruggable' interactions, their usage is usually limited to extracellular targets, because they are unable to cross cell membranes. [23][24][25] Indeed, also the protein-based MYC inhibitors, of which Omomyc is the most studied representant, have mainly been explored as research tools and utilized via ectopic expression in cells or xenografts.…”