Zero-shot design of drug-binding proteins via neural iterative selection-expansion

The design of proteins that bind to small molecules has been challenging because it requires simultaneous optimization of the protein sequence, protein structure and ligand conformation^1-7. Current deep-learning algorithms have struggled to navigate this landscape, precluding the zero-shot design of binders. Here we show that by combining two neural networks in an iterative design algorithm, small-molecule binding proteins can be created from scratch with high accuracy. We trained a graph neural network-ligand-aware sequence engineering message-passing neural network (LASErMPNN)-to design compatible protein sequences for an input protein backbone and docked ligand. We paired  LASErMPNN with a structure predictor that models a three-dimensional protein-ligand complex for an input protein sequence and ligand identity. The closed-loop iteration of these reciprocal networks optimized sequence-structure-ligand compatibility, and outperformed a comparable design loop using a physics-based energy function. We used our strategy, termed neural iterative selection-expansion (NISE), to design proteins that, using different folds, specifically bind to two chemically distinct small-molecule drugs, exatecan and apixaban, with success rates of 100% and 83%, respectively. The tightest NISE binders had nanomolar-to-picomolar affinities, surpassing those of the next-leading method by 70-fold for exatecan and nearly 10,000-fold for apixaban. LASErMPNN then suggested two amino-acid substitutions that improved the affinity of the tightest exatecan binder by 100-fold without any experimental input. The optimized binder protected the labile lactone ring of exatecan from hydrolysis for days. Our work describes a general recipe for using neural networks to automate the design of small-molecule binding proteins for applications in drug delivery, sensing and catalysis.