Abstract
Transcription factor engineering has emerged as a powerful strategy for generating novel proteins for fundamental research and biomedical applications. Although various analogs have been developed, they remain largely constrained to native sequences and structures. The generation of advanced analogs bearing noncanonical modifications with enhanced functional properties remains limited. Here we combined rational design with total synthesis to engineer novel abiotic transcription factors with enhanced stability and cell permeability. Using solid-phase synthesis and native chemical ligation, we created a library of 30 Max-derived transcription factor analogs incorporating novel modifications, such as sequence mutations and aromatic staples at strategic sites. Through DNA-binding analysis and cellular uptake studies, we identified the μMax20 analog, which contains two mutations (Lys31 and Lys57 to hArg) and exhibits potent DNA binding to the canonical enhancer box (E-box) as well as intrinsic cell permeability. Notably, further site-specific modifications of μMax20 with aromatic staples yielded improved analogs with enhanced stability and remarkable cellular delivery at nanomolar concentrations. Our lead μMax20 analog suppressed Myc-driven gene expression, as demonstrated by reporter gene assays and antiproliferative activity against Myc-dependent cancer cells. Altogether, these results highlight how combining chemical protein synthesis with late-stage modifications can be leveraged to enhance protein function and engineer novel bioactive modulators.