(618d) The in silico Design and Analysis of Darpin – Alpha-Cobratoxin Complexes

Designed Ankyrin Repeats (DARPins) are small, single domain (15 kDa) proteins that can bind to target antigen of interest with high affinity and specificity. They are highly thermostable and soluble molecules. Their small size enables them to have high tumor penetrability and the lack of cysteine residues makes them easy to be engineered as drug conjugates. Such characteristics enable these molecules to be favorably considered for therapeutic applications.

A common strategy for computationally designing binding proteins such as DARPins is to use docking programs to match them to target antigens and subsequently mutate them to improve their affinities. We have previously described MutDock, a computational tool to simultaneously dock and mutate binding proteins. This enables MutDock to identify alternative epitopes that would not bind well initially but can bind well upon mutation. This presentation will describe MutDock’s application to design DARPins to bind alpha-cobratoxin. MutDock was used to design DARPins to bind two epitopes of alpha-cobratoxin: the region that binds the nicotinic acetylcholine receptor and an alternative epitope with many prestabilized residues. After mutation, a total of 3900 designs were identified, computationally analyzed with the Rosetta Suite, and downs elected to 15 designs for experimental testing.