Intracellular delivery is a bottleneck in the development of therapeutic peptides and proteins. Here, we demonstrate the efficient delivery of omoMYC, the first MYC inhibitor in clinical trials, using HBpep-SP, an engineered peptide forming liquid-liquid phase-separated coacervates. HBpep-SP coacervates facilitate efficient cellular uptake and intracellular delivery of the omoMYC peptide at concentrations lower than those required for spontaneous uptake. Strikingly, omoMYC coacervates result in reduced proliferation and apoptosis induction in the low c-MYC expressing cell lines HEK293 and SH-SY5Y cells, but not in HeLa and SK-N-BE(2) cells with high c-MYC/MYCN expression, respectively, suggesting that endogenous MYC/N levels may impact the effects of omoMYC. Importantly, our approach bypasses the need for cell penetration-enhancing chemical modifications, offering a novel strategy for the investigation of peptide drug mechanisms in therapeutic development.

Centre for Sustainable Materials School of Materials Science and Engineering Nanyang Technological University 50 Nanyang Avenue Singapore 637553 Singapore

Department of Cell and Molecular Biology Uppsala University 751 24 Uppsala Sweden

Department of Microbiology Tumor and Cell Biology Karolinska Institutet 171 65 Stockholm Sweden

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

School of Biological Sciences Nanyang Technological University 60 Nanyang Drive Singapore 637551 Singapore

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Lee T. I., Young R. A.. Transcriptional Regulation and Its Misregulation in Disease. Cell. 2013;152(6):1237–1251. doi: 10.1016/j.cell.2013.02.014. PubMed DOI PMC

Brodsky S., Jana T., Barkai N.. Order through Disorder: The Role of Intrinsically Disordered Regions in Transcription Factor Binding Specificity. Curr. Opin. Struct. Biol. 2021;71:110–115. doi: 10.1016/j.sbi.2021.06.011. PubMed DOI

Boija A., Klein I. A., Sabari B. R., Dall’Agnese A., Coffey E. L., Zamudio A. V., Li C. H., Shrinivas K., Manteiga J. C., Hannett N. M., Abraham B. J., Afeyan L. K., Guo Y. E., Rimel J. K., Fant C. B., Schuijers J., Lee T. I., Taatjes D. J., Young R. A.. Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell. 2018;175(7):1842–1855.e16. doi: 10.1016/j.cell.2018.10.042. PubMed DOI PMC

Karadkhelkar N. M., Lin M., Eubanks L. M., Janda K. D.. Demystifying the Druggability of the MYC Family of Oncogenes. J. Am. Chem. Soc. 2023;145(6):3259–3269. doi: 10.1021/jacs.2c12732. PubMed DOI PMC

Meyer N., Penn L. Z.. Reflecting on 25 Years with MYC. Nat. Rev. Cancer. 2008;8(12):976–990. doi: 10.1038/nrc2231. PubMed DOI

Ruiz-Pérez M. V., Henley A. B., Arsenian-Henriksson M.. The MYCN Protein in Health and Disease. Genes. 2017;8(4):113. doi: 10.3390/genes8040113. PubMed DOI PMC

Vita M., Henriksson M.. The Myc Oncoprotein as a Therapeutic Target for Human Cancer. Semin. Cancer Biol. 2006;16(4):318–330. doi: 10.1016/j.semcancer.2006.07.015. PubMed DOI

Nair S. K., Burley S. K.. X-Ray Structures of Myc-Max and Mad-Max Recognizing DNA. Cell. 2003;112(2):193–205. doi: 10.1016/S0092-8674(02)01284-9. PubMed DOI

Blackwood E. M., Eisenman R. N.. Max: A Helix-Loop-Helix Zipper Protein That Forms a Sequence-Specific DNA-Binding Complex with Myc. Science. 1991;251(4998):1211–1217. doi: 10.1126/science.2006410. PubMed DOI

Farina A., Faiola F., Martinez E.. Reconstitution of an E Box-Binding Myc:Max Complex with Recombinant Full-Length Proteins Expressed in Escherichia coli . Protein Expression Purif. 2004;34(2):215–222. doi: 10.1016/j.pep.2003.11.021. PubMed DOI PMC

Soucek L., Helmer-Citterich M., Sacco A., Jucker R., Cesareni G., Nasi S.. Design and Properties of a Myc Derivative That Efficiently Homodimerizes. Oncogene. 1998;17(19):2463–2472. doi: 10.1038/sj.onc.1202199. PubMed DOI

Garralda E., Beaulieu M.-E., Moreno V., Casacuberta-Serra S., Martínez-Martín S., Foradada L., Alonso G., Massó-Vallés D., López-Estévez S., Jauset T., Corral De La Fuente E., Doger B., Hernández T., Perez-Lopez R., Arqués O., Castillo Cano V., Morales J., Whitfield J. R., Niewel M., Soucek L., Calvo E.. MYC Targeting by OMO-103 in Solid Tumors: A Phase 1 Trial. Nat. Med. 2024;30(3):762–771. doi: 10.1038/s41591-024-02805-1. PubMed DOI PMC

Atibalentja D. F., Deutzmann A., Felsher D. W.. A Big Step for MYC-Targeted Therapies. Trends Cancer. 2024;10(5):383–385. doi: 10.1016/j.trecan.2024.03.009. PubMed DOI PMC

Beaulieu M.-E., Jauset T., Massó-Vallés D., Martínez-Martín S., Rahl P., Maltais L., Zacarias-Fluck M. F., Casacuberta-Serra S., Serrano Del Pozo E., Fiore C., Foradada L., Cano V. C., Sánchez-Hervás M., Guenther M., Romero Sanz E., Oteo M., Tremblay C., Martín G., Letourneau D., Montagne M., Morcillo Alonso M. Á., Whitfield J. R., Lavigne P., Soucek L.. Intrinsic Cell-Penetrating Activity Propels Omomyc from Proof of Concept to Viable Anti-MYC Therapy. Sci. Transl. Med. 2019;11(484):eaar5012. doi: 10.1126/scitranslmed.aar5012. PubMed DOI PMC

Ellenbroek B. D., Kahler J. P., Arella D., Lin C., Jespers W., Züger E. A.-K., Drukker M., Pomplun S. J.. Development of DuoMYC: A Synthetic Cell Penetrant Miniprotein That Efficiently Inhibits the Oncogenic Transcription Factor MYC. Angew. Chem., Int. Ed. 2025;64:e202416082. doi: 10.1002/anie.202416082. PubMed DOI PMC

Wang E., Sorolla A., Cunningham P. T., Bogdawa H. M., Beck S., Golden E., Dewhurst R. E., Florez L., Cruickshank M. N., Hoffmann K., Hopkins R. M., Kim J., Woo A. J., Watt P. M., Blancafort P.. Tumor Penetrating Peptides Inhibiting MYC as a Potent Targeted Therapeutic Strategy for Triple-Negative Breast Cancers. Oncogene. 2019;38(1):140–150. doi: 10.1038/s41388-018-0421-y. PubMed DOI PMC

Lim J., Kumar A., Low K., Verma C. S., Mu Y., Miserez A., Pervushin K.. Liquid–Liquid Phase Separation of Short Histidine- and Tyrosine-Rich Peptides: Sequence Specificity and Molecular Topology. J. Phys. Chem. B. 2021;125(25):6776–6790. doi: 10.1021/acs.jpcb.0c11476. PubMed DOI

Gabryelczyk B., Cai H., Shi X., Sun Y., Swinkels P. J. M., Salentinig S., Pervushin K., Miserez A.. Hydrogen Bond Guidance and Aromatic Stacking Drive Liquid-Liquid Phase Separation of Intrinsically Disordered Histidine-Rich Peptides. Nat. Commun. 2019;10(1):5465. doi: 10.1038/s41467-019-13469-8. PubMed DOI PMC

Shebanova A., Perrin Q. M., Zhu K., Gudlur S., Chen Z., Sun Y., Huang C., Lim Z. W., Mondarte E. A., Sun R., Lim S., Yu J., Miao Y., Parikh A. N., Ludwig A., Miserez A.. Cellular Uptake of Phase-Separating Peptide Coacervates. Adv. Sci. 2024;11:2402652. doi: 10.1002/advs.202402652. PubMed DOI PMC

Sun Y., Lau S. Y., Lim Z. W., Chang S. C., Ghadessy F., Partridge A., Miserez A.. Phase-Separating Peptides for Direct Cytosolic Delivery and Redox-Activated Release of Macromolecular Therapeutics. Nat. Chem. 2022;14(3):274–283. doi: 10.1038/s41557-021-00854-4. PubMed DOI

Sun Y., Wu X., Li J., Radiom M., Mezzenga R., Verma C. S., Yu J., Miserez A.. Phase-Separating Peptide Coacervates with Programmable Material Properties for Universal Intracellular Delivery of Macromolecules. Nat. Commun. 2024;15(1):10094. doi: 10.1038/s41467-024-54463-z. PubMed DOI PMC

Sun Y., Xu X., Chen L., Chew W. L., Ping Y., Miserez A.. Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery. ACS Nano. 2023;17(17):16597–16606. doi: 10.1021/acsnano.3c02669. PubMed DOI

Tang H., Su Z.-D., Wei H.-H., Chen W., Lin H.. Prediction of Cell-Penetrating Peptides with Feature Selection Techniques. Biochem. Biophys. Res. Commun. 2016;477(1):150–154. doi: 10.1016/j.bbrc.2016.06.035. PubMed DOI

Liu H.-S., Jan M.-S., Chou C.-K., Chen P.-H., Ke N.-J.. Is Green Fluorescent Protein Toxic to the Living Cells? Biochem. Biophys. Res. Commun. 1999;260(3):712–717. doi: 10.1006/bbrc.1999.0954. PubMed DOI

Demma M. J., Mapelli C., Sun A., Bodea S., Ruprecht B., Javaid S., Wiswell D., Muise E., Chen S., Zelina J., Orvieto F., Santoprete A., Altezza S., Tucci F., Escandon E., Hall B., Ray K., Walji A., O’Neil J.. Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene. Mol. Cell. Biol. 2019;39(22):e00248-19. doi: 10.1128/MCB.00248-19. PubMed DOI PMC

Kumar P., Nagarajan A., Uchil P. D.. Analysis of Cell Viability by the Lactate Dehydrogenase Assay. Cold Spring Harbor Protoc. 2018;2018(6):pdb.prot095497. doi: 10.1101/pdb.prot095497. PubMed DOI

Shen S., Shao Y., Li C.. Different Types of Cell Death and Their Shift in Shaping Disease. Cell Death Discovery. 2023;9(1):284. doi: 10.1038/s41420-023-01581-0. PubMed DOI PMC

Rogakou E. P., Nieves-Neira W., Boon C., Pommier Y., Bonner W. M.. Initiation of DNA Fragmentation during Apoptosis Induces Phosphorylation of H2AX Histone at Serine 139. J. Biol. Chem. 2000;275(13):9390–9395. doi: 10.1074/jbc.275.13.9390. PubMed DOI

Purhonen J., Banerjee R., Wanne V., Sipari N., Mörgelin M., Fellman V., Kallijärvi J.. Mitochondrial Complex III Deficiency Drives C-MYC Overexpression and Illicit Cell Cycle Entry Leading to Senescence and Segmental Progeria. Nat. Commun. 2023;14(1):2356. doi: 10.1038/s41467-023-38027-1. PubMed DOI PMC

Annibali D., Whitfield J. R., Favuzzi E., Jauset T., Serrano E., Cuartas I., Redondo-Campos S., Folch G., Gonzàlez-Juncà A., Sodir N. M., Massó-Vallés D., Beaulieu M.-E., Swigart L. B., Mc Gee M. M., Somma M. P., Nasi S., Seoane J., Evan G. I., Soucek L.. Myc Inhibition Is Effective against Glioma and Reveals a Role for Myc in Proficient Mitosis. Nat. Commun. 2014;5(1):4632. doi: 10.1038/ncomms5632. PubMed DOI PMC

Soucek L., Jucker R., Panacchia L., Ricordy R., Tatò F., Nasi S.. Omomyc, a Potential Myc Dominant Negative, Enhances Myc-Induced Apoptosis. Cancer Res. 2002;62(12):3507–3510. PubMed

Kubota Y., Kim S. H., Iguchi-Ariga S. M. M., Ariga H.. Transrepression of the N-Myc Expression by c-Myc Protein. Biochem. Biophys. Res. Commun. 1989;162(3):991–997. doi: 10.1016/0006-291X(89)90771-7. PubMed DOI

Savino M., Annibali D., Carucci N., Favuzzi E., Cole M. D., Evan G. I., Soucek L., Nasi S.. The Action Mechanism of the Myc Inhibitor Termed Omomyc May Give Clues on How to Target Myc for Cancer Therapy. PLoS One. 2011;6(7):e22284. doi: 10.1371/journal.pone.0022284. PubMed DOI PMC

Groelly F. J., Fawkes M., Dagg R. A., Blackford A. N., Tarsounas M.. Targeting DNA Damage Response Pathways in Cancer. Nat. Rev. Cancer. 2023;23(2):78–94. doi: 10.1038/s41568-022-00535-5. PubMed DOI

Abuetabh Y., Wu H. H., Chai C., Al Yousef H., Persad S., Sergi C. M., Leng R.. DNA Damage Response Revisited: The P53 Family and Its Regulators Provide Endless Cancer Therapy Opportunities. Exp. Mol. Med. 2022;54(10):1658–1669. doi: 10.1038/s12276-022-00863-4. PubMed DOI PMC

Fulda S.. Apoptosis Pathways and Neuroblastoma Therapy. Curr. Pharm. Des. 2009;15(4):430–435. doi: 10.2174/138161209787315846. PubMed DOI

Valter K., Zhivotovsky B., Gogvadze V.. Cell Death-Based Treatment of Neuroblastoma. Cell Death Dis. 2018;9(2):113. doi: 10.1038/s41419-017-0060-1. PubMed DOI PMC

Vernooij L., Kamili A., Ober K., Van Arkel J., Lankhorst L., Vermeulen E., Al-Khakany H., Tax G., Van Den Boogaard M. L., Fletcher J. I., Eising S., Molenaar J. J., Dolman M. E. M.. Preclinical Assessment of Combined BCL-2 and MCL-1 Inhibition in High-Risk Neuroblastoma. EJC Paediatr. Oncol. 2024;3:100168. doi: 10.1016/j.ejcped.2024.100168. DOI

Bierbrauer A., Jacob M., Vogler M., Fulda S.. A Direct Comparison of Selective BH3-Mimetics Reveals BCL-XL, BCL-2 and MCL-1 as Promising Therapeutic Targets in Neuroblastoma. Br. J. Cancer. 2020;122(10):1544–1551. doi: 10.1038/s41416-020-0795-9. PubMed DOI PMC

Fitzgerald M.-C., O’Halloran P. J., Connolly N. M. C., Murphy B. M.. Targeting the Apoptosis Pathway to Treat Tumours of the Paediatric Nervous System. Cell Death Dis. 2022;13(5):460. doi: 10.1038/s41419-022-04900-y. PubMed DOI PMC

Walz S., Lorenzin F., Morton J., Wiese K. E., Von Eyss B., Herold S., Rycak L., Dumay-Odelot H., Karim S., Bartkuhn M., Roels F., Wüstefeld T., Fischer M., Teichmann M., Zender L., Wei C.-L., Sansom O., Wolf E., Eilers M.. Activation and Repression by Oncogenic MYC Shape Tumour-Specific Gene Expression Profiles. Nature. 2014;511(7510):483–487. doi: 10.1038/nature13473. PubMed DOI PMC