**4. Conclusions**

We applied our newly developed Movable Type free energy protocol to the caspase-inhibitor complexing study. Using a Monte Carlo sampling approach, the MT method generated the significant binding modes and calculated the binding free energies using the ratio of the partition functions referencing the bound state and free state protein-ligand systems. Both the large-scaling and carefully set-up small test sets were introduced to provide a comprehensive study regarding the robustness and sensitivity of the MT protocol against such complexing systems. Results revealed good agreements of the calculation predictions with the experimental binding affinities and the global minimum binding modes. Through detailed case studies, we further illustrated the MT protocol mechanism for the free energy extrapolation using a Monte Carlo based sampling method. Moreover, we also took a close look at the global minimum binding mode structures to study how minor changes in the interaction interfaces affecting the binding affinities and how with different interaction interfaces achieved similar binding affinities. Generally, this work provided us useful computational information for the binding affinity prediction using the MT protocol. Future studies including computation-experiment combinatorial research can be expected for the structural based caspase inhibitor design. We also plan to apply the MT protocol to the caspase inhibitor-related inverse docking study.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/19/4850/s1.

**Author Contributions:** Conceptualization, Z.Z.; methodology, Z.Z.; software, S.X. and Z.Z.; validation, S.X. and H.L.; formal analysis, S.X.; investigation, S.X.; resources, Z.Z.; data curation, S.X. and H.L.; writing—original draft preparation, S.X.; writing—review and editing, S.X. H.L. and Z.Z.; visualization, S.X.; supervision, Z.Z.; project administration, Z.Z.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank Taolei Sun from Wuhan University of Technology for useful discussions of the caspase inhibition mechanism and the computational methodology application.

**Conflicts of Interest:** The authors declare no conflict of interest.
