Chemical Engineering of Transcription Factors UncoveredCell-Permeable μMax Modulators

Transcription factor engineering has emerged as a powerfulstrategyfor generating novel proteins for fundamental research and biomedicalapplications. Although various analogs have been developed, they remainlargely constrained to native sequences and structures. The generationof advanced analogs bearing noncanonical modifications with enhancedfunctional properties remains limited. Here we combined rational designwith total synthesis to engineer novel abiotic transcription factorswith enhanced stability and cell permeability. Using solid-phase synthesisand native chemical ligation, we created a library of 30 Max-derivedtranscription factor analogs incorporating novel modifications, suchas sequence mutations and aromatic staples at strategic sites. ThroughDNA-binding analysis and cellular uptake studies, we identified the μMax20 analog, which contains two mutations (Lys31 andLys57 to hArg) and exhibits potent DNA binding to the canonical enhancerbox (E-box) as well as intrinsic cell permeability. Notably, furthersite-specific modifications of μMax20 with aromaticstaples yielded improved analogs with enhanced stability and remarkablecellular delivery at nanomolar concentrations. Our lead μMax20 analog suppressed Myc-driven gene expression, as demonstrated byreporter gene assays and antiproliferative activity against Myc-dependentcancer cells. Altogether, these results highlight how combining chemicalprotein synthesis with late-stage modifications can be leveraged toenhance protein function and engineer novel bioactive modulators.