Genetic code expansion for the construction of beyond rule-of-5 compliant macrocyclic peptide libraries

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Project Summary Many intracellular cancer targets involve intracellular protein-protein interactions that are “undruggable” because the binding surfaces are too large and featureless to be blocked by a standard rule-of-5 compliant small molecule. Recently, there have been attempts to catalog molecules that are orally bioavailable but lie beyond the rule of five (bRo5). Macrocyclic peptides can inhabit this bRo5 space, and a key advantage to using peptides as bRo5 molecules is that there are many mature techniques for finding peptide binders from vast libraries. Arguably, the most powerful of these techniques is mRNA display, which allows creation of peptide libraries containing over 10 trillion variants, 6-7 orders of magnitude larger than a standard peptide library prepared on beads. The extreme diversity of these libraries has enabled many successes in inhibitor development. Yet these successes are disconnected from real drug discovery, because the peptides uncovered are much too large to be bRo5 compliant. Libraries that are short in sequence and bRo5 compliant can be created by mRNA display, but these libraries lack the diversity needed to uncover potent inhibitors because standard mRNA display is limited by the genetic code to ~20 variants at each position. In this proposal two strategies to enhance this positional diversity will be pursued. The first involves breaking the degeneracy of the standard genetic code, through isolation of fully modified tRNA isoacceptors. Based on codon reading rules it is predicted that this will allow the addition of 10 non-canonical amino acids (ncAAs) to the code. The second involves insertion of an unnatural base pair (UBP) to the code. The addition of a single UBP into the genetic code at a single codon position opens 32 new empty codons, that can be exploited for the introduction of novel ncAAs to the code. tRNAs that read each of these codons will be prepared and the codon reading preferences will be validated. Putting the two strategies together should allow expansion of the genetic code to the use of 40 monomers at each position. With carefully chosen building blocks, this will allow for the creation of bRo5 compliant libraries containing billions of variants for use in drug discovery.