importance to the further development of the peptide design framework. Retrospective analysis of the structural template and biological constraints used as input into the sequence selection stage can demonstrate what types of constraints are useful for future methyltransferase design, as well as peptidic inhibitor design as a whole. The computational, three-stage de novo peptide design Bax inhibitor peptide V5 method used in this study was focused on the development of novel peptidic inhibitors of enhancer of zeste homolog 2. The first stage of the method is a sequence selection stage that uses biologically relevant constraints in an integer linear optimization model to produce a rank ordered list of sequences with the lowest potential energy in a given template structure. The second stage takes the top sequences from the sequence selection stage and determines the specificity that the candidate sequences have for the target peptide template structure. The sequences with the top fold specificity values are then run through a computationally rigorous third stage to calculate the order Degarelix approximate binding affinity of the sequences to the target protein. Those peptides with the highest predicted binding affinity to the target protein are then validated experimentally. Through the stages of this general methodology, the sequence complexity of the problem is reduced in tandem with increased computational complexity. This results in a small number of candidate peptides for experimental validation. The full framework of the method is shown in Figure 1. The computational details of each stage are described in subsequent sections. EZH2 is a SET domain-containing methyltransferase that catalyzes the di- and trimethylation of the lysine in position 27 of histone H3. The methyltransferase is a catalytic subunit of a larger complex called the polycomb repressive complex 2. Besides EZH2, several non-catalytic subunits of the complex are necessary for correct catalytic function. The SET domain has an unusual ����thread-theneedle���� structure, called a pseudoknot. While the substrate and cofactor bind on opposite ends of the domain, their binding pockets are connected by an inner chamber where the methyl transfer occurs. There are currently no crystal or NMR structures available for the human EZH2 protein. For this reason, a template structure had t
