allowing for both increased charge content and the S28N mutations, while restricting changes to positions 30 and 33 that were largely unsuccessful. While the S28N mutation may be specific to EZH2 inhibitor design, the increased charge constraint may be characteristic of general histone-modifying enzyme inhibitor design and is worthy of further exploration. Analysis of the template-based constraints demonstrate how one can use the results from this study to guide future EZH2 and other histone-modifying enzyme design. In order to guide future peptide inhibitor design more generally, however, one must analyze the influence of the fold specificity and approximate YYA-021 Binding affinity metrics on the capability of the method to correctly identify peptidic inhibitors. Hence, it is useful to focus on the 923564-51-6 peptides derived from Run 4 that stood out in the endpoint assay and IC50 results. In analyzing the quantitative results from the Fold Specificity and Approximate Binding Affinity validation stages, these three peptides are the top three ranked peptides in Fold Specificity and three of the top four in Approximate Binding Affinity. Since these values are used simply as a ranking metric, this demonstrates the usefulness that both metrics have in producing designed peptides with a high probability of success. Besides analyzing the influence of input biological constraints and the selection metrics, it is also important to analyze the results of the method from a structural perspective. This allows us to determine how well the validation stages predict consistent contacts that may play an important role in the inhibitory properties of the peptides. Such knowledge is important for future design studies. Taking the four lowest energy structures from the docking runs for the top peptide SQ037, the contacts observed consistently across the four structures were identified. The PDB files used in the analysis are provided in File S1. The analysis of the structures reveal three distinct contacts, found in all or most of the four structures. These include a contact between K24 of the peptide and D41 of the protein, W26 of the peptide and M57 of the protein, and N32 of the peptide and D108/Y109. While all of these contacts are interesting in interpreting the r