In this step, we mutated residues in EETI-II-sub at the protein�Cprotein interface of the complex via implicit solvent models based on the Generalized Born approximation. The last step consisted in the experimental testing of the inhibitor by an enzymatic inhibitory assay specific for the PvSUB1 recombinant enzyme. A preliminary free energy calculation was performed with snapshots from multiple MD simulations of EETI-II-sub docked onto PvSUB1 to obtain more Ruxolitinib consistent MM/GBSA results. A free-energy decomposition shows the contribution of each single residue to the total free energy of binding. The biggest contribution to the free energy of binding came from the main-chain contacts of residues P4, P3, P2 and P1. This is in agreement with previous observations of important interactions between a protein-inhibitor and a serine protease active site, where important contacts are made by main-chain atoms. For the case of EETI-II-sub the highest contribution originates from the cysteine in P3 and its main-chain, accounting for 24.34 kcal/ mol. We then tried to identify the most favorable SCH772984 ERK inhibitor mutations that could improve the binding affinity of EETI- II-sub to PvSUB1. The cysteine in P3 cannot be mutated because its side-chain is involved in a disulphide bridge that has an important function in stabilizing the EETI-II scaffold and maintaining the loop rigid, whereas the alanine in P2 already contributes with 24.17 kcal/ mol to the total binding energy. We also looked at the parasite sequences that are natural substrates of PvSUB1 or PfSUB1 to suggest positions to introduce mutations in the EETI-II scaffold. Table 4 lists these sequences for PfSUB1 and PvSUB1. While the sequences of several PfSUB1 substrates were experimentally determined, few were identified for PvSUB1. Considering the evolutionary proximity of P. vivax and P. falciparum, with active sites displaying.60% sequence identity, these predicted sequences can be considered reliable. Comparing the cleavage sites, we observed that only alanine and glycine appeared in P2, suggesting that only small residues are tolerated in this position. Position P19 has a negative contribution to the energy and therefore is an interesting position to mutate. However, considering the lack of a specific pocket for this residue we can consider this position almost a secondary contact residue and we decided to keep the wild�Ctype residue. Finally, SUB1 cleavage site sequences have a fairly high similarity at the P1 and P4 positions and we therefore focused on these positions to mutate the EETI-II inhibitory loop. It is worth mentioning that the contribution of these P1 and P4 positions within the substrate-PfSUB1 interaction has recently been experimentally established. We performed 106100 ps MD simulations and MM/GBSA free energy calculations for all possible residues in position P4 and P1 independently, assuming that the effect of the two mutations would be additive. The free energy calculations for the mutants in P4 showed that hydrophobic and bulky residues were preferred for this position. This result fits with the fact that pocket S4 is composed of six hydrophobic residues and seems to have enough space to accommodate larger hydrophobic side-chains than valine. Position P1 instead presents as favorable mutations aromatic residues with polar groups, glutamate and positively charged residues. Surprisingly we found as favorable mutations some positively charged residues, whereas most of sequences recognized by the homologous PfSUB1 present negatively charged or neutral polar sidechains at P1. This might be explained by either the low substrate specificity common to some subtilisins or imprecisions in the structure of the complex.