The Development of In Vivo Phage-Assisted Directed Evolution Platforms to Modulate Protein-Protein Interactions

Though biomolecular interactions regulate virtually all cell processes, controlling biomolecular interactions, both for the purposes of scientific inquiry and for correcting aberrant interactions, remains a difficult challenge. In the first chapter of my dissertation, I highlight the ways that current directed evolution platforms have been able to generate toolsto control interactions among the biomolecules in the central dogma and suggest current challenges and future opportunities. As others have noted in their deeming of protein-protein interactions (PPIs) as "undruggable," PPIs have proven particularly challenging to modulate, not only due to their complex and varied biophysical properties, but also because hitting off-target interactions routinely poses an issue. In chapter 2, I describe a platform I developed that aims to address such specificity issues by enabling the directed evolution of specific protein binders. We use the technology, termed PPI specificity phage-assisted continuous evolution (PACE), to evolve varied binding profiles of extant and ancestral BCL-2 family proteins, which enabled further insight regarding the roles of chance and contingency in the evolution of this protein family. I went on to adapt the PPI specificity PACE technology to directly select for inhibition of a PPI rather than protein binding alone, a property that does not always confer inhibition. This work is detailed in the creation of PANCS-PPIi in chapter 3, where I establish platform parameters with 3 distinct model PPIs and go on to both improve PPI inhibition of an existing inhibitor and also identify a de novo PPI inhibitor. I conclude in chapter 4 by summarizing and contextualizing my work and looking to future opportunities in the field.