Leaf, C. M.; Qi, P.; Gandhi, Y. P.; Jalali-Yazdi, F.; Ong, J. N.; Takahashi, T. T.; Kalia, R.; Roberts, R. W.

In vitro selection and directed evolution technologies such as mRNA display, explore large libraries (10^14 variants) and generate thousands to millions of functional polypeptide ligands to a variety of targets. Denoising diffusion implicit machine learning models (DDIMs) trained using display-derived deep sequencing data can greatly expand these functional sequences beyond what is accessible experimentally. However, methods are needed to predict peptide properties such as binding free energies ({Delta}G{degrees}). Here, we applied machine learning methods to predict binding free energies of both experimental and DDIM-generated peptide ligands against a target of interest, the oncogenic protein Bcl-xL. To do this, we trained a Closed-form Continuous (CfC) neural network using a dataset of 15,700 peptide ligands where pairs of sequences and their corresponding binding free energies ({Delta}G{degrees}) were used as inputs. This type of model was chosen due to its ability to represent irregular series. The resulting CfC model accurately predicts the rank order, within error, and binding free energies ({Delta}G{degrees}) for both experimental and DDIM-generated peptides, identifying five DDIM-generated peptides with single-digit picomolar affinities. Combining trained DDIM and CfC models offers a unified route to expand the scope of experimental ligand discovery, predict the molecular properties of both experimental and generated ligands, and highlights the utility of large quantitative datasets for making accurate in silico predictions of high-affinity peptide candidates.