(504e) Enhancing the Chemical Versatility of Yeast Display to Target the Tumor Microenvironment

Although cancer-promoting activities of extracellular proteases and peptidases in the tumor microenvironment are well known, the disruption of individual enzymes in this environment remains a fundamental challenge. The development of highly specific enzyme inhibitors would enable characterization of enzyme functions in the microenvironment and could lead to identification of potential leads for therapeutic applications. We are exploring the hypothesis that integrating additional chemical functionality into binding proteins will provide opportunities for discovering potent inhibitors that simultaneously exploit the best features of proteins and small molecules. In order to construct, evaluate, and screen protein-small molecule “hybrids,” we have established a platform enabling yeast display of proteins containing noncanonical amino acids (ncAAs). This platform will enable us to leverage the quantitative and high throughput capabilities of yeast display to determine how best to assemble these structures and, once assembled, identify potent, selective inhibitors. Here, we present advances in the technological capabilities of our ncAA-compatible yeast display platform.

In order to broaden the range of chemical functionality that we can use with our platform, we are pursuing quantitative approaches to evaluate the site-specific addition of a 21st amino acid to proteins in yeast. The use of display-based reporter constructs with tags before and after the intended site for ncAA insertion (via stop codon suppression) enables us to evaluate the efficiency and fidelity of ncAA incorporation. Using a series of orthogonal translation system components that facilitate insertion of aromatic ncAAs into proteins, we observe stop codon readthrough efficiencies of approximately 10 percent and misincorporation of canonical amino acids at levels of 20 percent or lower using flow cytometry-based readouts. This level of performance is sufficient for some applications but will need to be improved in order to realize its full potential. Toward this end, we have begun to investigate several potential strategies for enhancing both stop codon readthrough and ncAA incorporation fidelity. Our findings suggest that changing orthogonal translation system component expression levels or manipulating the yeast genome both appear to be promising strategies for enhancing genetic code manipulation. However, flow cytometry-based readouts also reveal that high cell-to-cell variability and promiscuity of “orthogonal” translation system components can confound uniformly efficient, high fidelity ncAA incorporation. We anticipate that these findings, further quantitative measurements, and screening approaches will reveal important principles of genetic code manipulation and provide opportunities for adding a range of functionalities to proteins displayed on the yeast surface. Expanding the range of genetically encodable chemical groups will facilitate a range of chemical transformation on the yeast surface and the direct encoding of functional groups that can disrupt protease and peptidase activities in ways that the canonical amino acids cannot.

To begin to exploit the existing capabilities of our platform, we are investigating strategies for incorporating ncAAs into antibody fragments at positions located close to antibody complementarity determining regions. We have identified several conserved antibody positions that appear to tolerate aromatic ncAA incorporation and facilitate chemical modification. The insertion of ncAAs at one of several conserved positions results in antibody variants with similar binding properties to those of the parent clones, confirming that the sites we have identified tolerate ncAA substitutions. The introduction of azide or alkyne functionalities at these positions facilitates chemical modification on the yeast surface. Experiments with five antibody variants have uncovered general reactivity trends: 1) conserved sites appear to react similarly in different antibodies; 2) subtle changes in amino acid side chain properties affect chemical reactivity; 3) changes in the structures of small molecules used to derivatize antibody fragments affect apparent reactivity. These findings suggest that chemical modifications of antibodies on the yeast surface are generalizable, provided that empirical tests are used to identify suitable reaction conditions for the individual ncAA-small molecule pair to be employed. We have exploited our understanding of these trends to robustly and consistently construct “hybrid” structures containing functional groups suitable for disrupting major families and superfamilies of enzymes including matrix metalloproteinases and serine hydrolases. Efforts to identify protein-small molecule hybrids that disrupt the activities of members of these groups of enzymes are ongoing. Our approach to incorporating more chemical functionality into proteins displayed on the yeast surface will enable us to explore structures not accessible with current inhibitor discovery technologies in high throughput. These efforts may lead to inhibitors with enhanced specificity and potency with potential applications for evaluating the biology of the tumor microenvironment and could further lead to the identification of new therapeutic candidates.