High-Throughput Platforms to Study Synthetic Receptors, Natural Molecules, and New Pathway Inhibitors

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Project Summary Advances in computational and experimental protein engineering have ushered in a new era of biomolecule development, providing new functional binding proteins, improved enzymes, and rationally designed synthetic receptors. Despite such progress, established techniques lack the ability to develop and study synthetic receptors and inhibitory molecules in high throughput, instead relying on rational or function-agnostic engineering approaches followed by low-throughput characterization for the desired activity. This deficiency in approach results in 1) the development of suboptimal candidate proteins and 2) a costly development process. My long-term goals are to 1) establish new platforms that incorporate protein function as a selective pressure to engineer new molecules and 2) utilize these new platforms to understand the roles of natural and synthetic proteins in cell signaling responses in both normal and disease pathologies. I hypothesize that the incorporation of protein function as a selective pressure in protein engineering campaigns will result in the efficient development of new classes of functional proteins capable of answering key biological questions. The goals during this proposal period are to establish high-throughput screening platforms for inhibitor engineering and for synthetic receptor engineering, as well as develop combined computational and experimental protein engineering pipelines to aid the study of important proteins. Continued work in the described areas has immense potential to aid the study of basic and synthetic biology through 1) greatly expanding the availability of molecules to empower studies of the importance of individual molecules in cell signaling responses, 2) providing a new toolkit for understanding synthetic receptor function, and 3) providing modular platforms to aid discovery of functional engineered molecules. We will pursue three primary directions: Direction 1: Establish a tethered inhibitor engineering platform with yeast surface display. Current directed evolution approaches often lack protein function as a selective pressure, resulting in majority development of passive binding proteins. We will incorporate the concept of tethering from the small molecule screening community to drive yeast-displayed protein selection toward active inhibition. Direction 2. Establish combined computational and experimental protein engineering pipelines. Current protein engineering approaches typically rely on widespread mutagenesis or in-depth computational/rational design to develop functional proteins. We will incorporate accessible computational approaches for protein mutant library design and investigate these approaches for basic mutational studies. Direction 3. Establish a high-throughput platform for studying synthetic receptors. Synthetic receptors have made measurable impacts in both basic and clinical science, but no effective platform exists to study their development in high throughput. We will establish a high-throughput screening platform with proliferation from receptor functions as a selective pressure for engineering new synthetic receptors.