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bioRxiv · 10.1101/2023.05.12.540568

Systemic evaluation of protein-small molecule hybrids on the yeast surface

Huang, M.·Rueda-Garcia, M.·Harthorn, A.·Hackel, B. J.·Van Deventer, J. A.
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Abstract

Protein-small molecule hybrids are structures that have the potential to combine the inhibitory properties of small molecules and the specificities of binding proteins. However, achieving such synergies is a substantial engineering challenge, with fundamental principles yet to be elucidated. Recent work has demonstrated the power of yeast display-based discovery of hybrids using a combination of fibronectin binding domains and thiol-mediated conjugations to introduce small molecule warheads. Here, we systematically study the effects of expanding the chemical diversity of these hybrids on the yeast surface, investigating a combinatorial set of fibronectins, noncanonical amino acid (ncAA) substitutions, and small molecule pharmacophores. Our results show that previously discovered thiol-fibronectin hybrids are generally tolerant of a range of ncAA substitutions and retain binding to carbonic anhydrases following click chemistry-mediated assembly of hybrids with diverse linker structures. Most surprisingly, we identified several cases where replacement of a potent acetazolamide warhead with a substantially weaker benzenesulfonamide warhead still resulted in the assembly of functional hybrids. In addition to these unexpected findings, we expanded the throughput of our system by validating a 96-well plate-based format to produce yeast-displayed hybrid conjugates in parallel. These efficient explorations of hybrid chemical diversity demonstrate that there are abundant opportunities to expand the functions of protein-small molecule hybrids and elucidate principles that dictate their efficient discovery and design.

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Huang, M., Rueda-Garcia, M., Harthorn, A., Hackel, B. J., Van Deventer, J. A.. 2023-05-14. Systemic evaluation of protein-small molecule hybrids on the yeast surface. https://doi.org/10.1101/2023.05.12.540568

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