Although enzymes play many critical roles in normal human physiology and pathological conditions ranging from cancer to infectious diseases, specific inhibition of a single enzyme remains a crucial challenge in drug discovery. We are establishing yeast-based approaches to overcome the limitations of current enzyme inhibitor discovery platforms in the following ways: 1) integrating small molecule chemical functionality within binding proteins and 2) streamlining the yeast display screening process. Expanding the range of chemical functionality present in biomolecules may lead to inhibitors with properties that are difficult to access using conventional small molecules or biologics. In order to utilize both chemical and biological functionality during inhibitor discovery, we have combined noncanonical amino acid incorporation technology with yeast display. This approach has enabled us to present reactive chemical handles within single-chain variable fragment (scFv) and antibody constant region (Fc) domains on the yeast surface. Flow cytometric analyses indicate that encoded azide and alkyne functionalities can be selectively addressed, and that ncAA-containing scFvs are capable of undergoing reactions with small molecules containing functional groups capable of interfering with broad classes of enzymes such as metalloproteinases and serine hydrolases. To streamline yeast display, we have used a previously established synthetic antibody library to evaluate magnetic bead-based screening approaches. Both singleplex and multiplexed screens with this library appear to be very efficient and in many cases can be performed without the use of flow cytometry. Ongoing multiplexed screening efforts with a naïve antibody library against a panel of antigens exhibit enrichment efficiencies comparable to singleplex enrichment efficiencies for numerous antigens. We anticipate that combining multiplexed screening with yeast display libraries containing an expanded range of chemical functionality will support the rapid discovery of novel enzyme inhibitors against a broad range of enzyme targets.

Enhancing the chemical versatility and throughput of yeast display

253rd National Meeting (2017)

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