ReCHEMbinant stapling enhances intracellular delivery and bioactivity of engineered protein inhibitors

SummaryProtein 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.Graphical abstractHighlights ReCHEMbinant engineering staples proteins for improved intracellular activity Stapled Omomyc variants, HeloMYCs, gain serum stability and cellular uptake HeloMYC-1421 shows enhanced MYC inhibition and blocks cancer cell proliferation The reCHEMbinant strategy broadens tools for engineering intracellular biologicsThe bigger pictureProteins are the body s natural machinery for carrying out highly specific biological functions, making them uniquely powerful as therapeutics. Unlike traditional small-molecule drugs, protein therapeutics can target large and complex biological surfaces that are otherwise undruggable. However, most protein drugs act only outside cells because they cannot efficiently cross cellular membranes. Many critical disease drivers, including oncogenic transcription factors (such as MYC), reside inside cells, making intracellular delivery of biologics one of the most important frontiers in modern drug discovery.In this study, we developed reCHEMbinant engineering, a broadly applicable strategy that integrates recombinant protein production with site-selective chemical stapling to create synthetically enhanced proteins. Using this approach, we generated HeloMYCs, stapled versions of the MYC inhibitor Omomyc that show potent DNA binding, serum resistance, and markedly enhanced cellular uptake, translating into potent inhibition of MYC-driven cancer programs. Beyond MYC, reCHEMbinant engineering might establish a versatile framework for designing next-generation intracellular biologics. In the long term, this strategy could enable the development of stable, cell-permeable protein therapeutics that modulate previously inaccessible targets involved in cancer, neurodegeneration, and infectious diseases, supporting innovation in safer and more effective treatments for human disease.ReCHEMbinant engineering enables chemical stapling of recombinant proteins to enhance their cellular uptake, serum stability, and general activity. Applying this strategy to the MYC inhibitor Omomyc produced HeloMYCs synthetically enhanced variants that display potent intracellular activity and better cell penetration and MYC inhibition than the natural parent protein.