Yeast Biosensors for Cyclic Peptide Drug Discovery By Directed Evolution

Small molecule drug discovery typically concentrates on diversity screening of constantly growing chemical libraries prior to lead optimisation using medicinal chemistry. However, failure to find hits against highly validated targets is forcing drug discovery efforts to explore new modalities such as proteins, peptides, and oligonucleotides. Discovery of these biology-based modalities can be enhanced by the power of natural selection via directed evolution, where genetic selections enable higher-throughput screening than possible with classical approaches. To harness this power of directed evolution, we developed a synthetic biology platform in the yeast Saccharomyces cerevisiae where genetic circuits can detect cyclic peptides generated in vivo that disrupt disease state protein-protein interactions important in human health. By using genetically encoded biosensors, each individual cell can act as a laboratory, thus creating a system where the analyses of billions of experiments can occur concurrently within a single culture. To do this, we developed a variation of the repressed transactivator yeast two-hybrid system to screen protein-protein interaction inhibitors. This selection incorporates a life or death pre-screen to purge negative hits. Building on this, we engineered a fluorescent readout dependent on the strength of protein interaction inhibition, enabling fine-tuned selection of potent inhibitors. As proof of concept, we show our system can detect the in vivo generated cyclic peptide cyclo-CLLFVY. This peptide is a known inhibitor of hypoxia inducible factor-1 (HIF-1) dimerisation, an important protein heterodimer regulator of hypoxia response in cancers. Future work will screen in vitro generated peptide libraries against other protein-protein interactions that are medically relevant, with the goal to move to the in vivo mutagenesis of peptide libraries for diversity creation. We argue this system will be generally applicable to any protein-protein interaction, ultimately aiming to deliver a versatile plug and play system for the continuous directed evolution and discovery of cyclic peptide drugs.