De novo design of a protein fold for small-molecule binding through aromatic π stacking

Authors

Ouchida, S. T., Horst, M. T., Gou, X., Bakanas, I., Hatstat, A. K., Schnaider, L., Diolaiti, M. E., Ashworth, A., DeGrado, W. F.

Abstract

The de novo design of proteins that bind chemically complex small molecules has broad chemical and biological implications, but strategies typically rely on a small set of protein scaffolds and require extensive experimental screening. Here, we computationally designed proteins around a minimal aromatic {pi}-stacking motif to bind the anthracycline anticancer drug doxorubicin. Experimental characterization of twelve proteins revealed a M doxorubicin binder; two additional design cycles improved scaffold stability and binding affinity to yield an 85-residue protein that binds doxorubicin with a dissociation constant of 85 nM. An X-ray crystal structure of the protein-drug complex confirmed the accuracy of the designed {pi}-{pi} stacking interactions. The designed protein could act to protect cultured cells from doxorubicin-induced cytotoxicity. Unlike previous ligand-binding protein designs based on repeat proteins or naturally occurring folds, the designed protein adopts a previously unobserved 5-helix globular fold, indicating that a broader space of folded, functional proteins exists even for compact tertiary structures smaller than 100 residues. These results demonstrate that motif-guided generative protein design can discover compact de novo protein folds capable of high-affinity recognition of chemically complex small molecules.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 09 Aug 2026.

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