ReCHEMbinant stapling enhances intracellular delivery and bioactivity of engineered protein inhibitors

Protein therapeutics have transformed drug discovery by enabling modulation of challenging targets inaccessible to small molecules. However, most proteins lack the ability to penetrate cells, where many critical drug targets reside. Here, we present reCHEMbinant protein engineering, a strategy designed to generate synthetically enhanced proteins with improved structural stability, serum resistance, and cellular uptake. Applying this approach to Omomyc, a protein-based MYC inhibitor, we developed several reCHEMbinant stapled variants (HeloMYCs) exhibiting low-nanomolar DNA-binding affinity. Notably, the i, i + 7 biphenyl-stapled construct HeloMYC-1421 outperformed Omomyc across several functional assays, including potent inhibition of MYC-driven gene expression in luciferase reporter assays and selective antiproliferative effects in MYC-dependent cells. Live-cell imaging showed that these enhanced effects result from significantly improved cellular uptake. Transcriptional reprogramming was further confirmed by RNA sequencing (RNA-seq). Together, our findings establish reCHEMbinant engineering as a chemically defined strategy for stapling entire recombinant proteins to enhance their intracellular bioactivity.