Systematic Evaluation of Protein-Small Molecule Hybrids on the Yeast Surface

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 the 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 by 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 functions 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 multiple 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.

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library

“…Interestingly, this even includes small molecules with metal-binding functionality that could interfere with CuAAC; this observation is consistent with other surveys of chemical reactivity performed on the yeast surface in our lab 29 . However, not all small molecules react efficiently at large scale.…”

“…For example, covalent antibody discovery could be conducted using either the photocrosslinkable ncAA AzF ( 1 ) 30,31 or the spontaneously crosslinkable ncAA OBeY ( 6 ) 31 . Additionally, both the azide side chain of AzF ( 1 ) and alkyne functionality of OPG ( 2 ) enable bioorthogonal click chemistry reactions 29,30 .…”

“…While in previous work we have routinely conducted CuAAC at small scale on the yeast surface (2×10 6 cells in 250 µL reaction) 29,42 , chemistry-driven sorts require modification of 1×10 10 cells to ensure tenfold oversampling of a billion-member library during the initial round of screening. Because CuAAC reaction efficiency can change as a function of cell density 29,[43][44][45] , we evaluated CuAAC conjugations at small and large scales for a range of small molecules (Figure 2B and C). Reactions with 9 small molecules containing a range of chemical moieties (Figure 2B) were conducted on cells from the library in 250 µL total reaction volume at low cell density (2×10 6 modified cells; 8×10 6 cells/mL) and in 50 mL total reaction volume at high cell density (1×10 10 modified cells; 2×10 8 cells/mL).…”

“…The copyright holder for this preprint (which this version posted May 29, 2024. ; https://doi.org/10.1101/2024.05. 29.596443 doi: bioRxiv preprint spontaneous crosslinking in the clones isolated here. Nonetheless, these efforts validate discovery of chemically expanded antibodies in yeast display format, with the library design providing a range of opportunities to pursue chemistry-driven antibody discovery.…”

“…Early work with phage display and E. coli display platforms demonstrated the technical feasibility of engineering ncAAcontaining antibodies with expanded chemical functionalities in high throughput 13,27,28 . In addition, our lab has integrated yeast display with ncAAs and demonstrated semirational strategies for endowing antibodies and other binding proteins with bioorthogonal chemistries 29 , photocrosslinking 30,31 , and spontaneous crosslinking capabilities 31 . The power of expanding chemical functionality into display technologies has been demonstrated in myriad ways using peptide-based screens in mRNA display [32][33][34] and phage display formats 35 .…”