蛋白激酶 ( protein kinase ) 是生物體訊息傳導途徑的重要成員之一，藉由磷酸化特定蛋白質，調控細胞酵素及傳遞訊息。
本研究中，我們的目的是利用分子動力學模擬方法，幫助設計PKA的抑製劑。首先我們從蛋白質資料庫(PDB)中選擇4個具有IC50值的PKA配體複合物，當中有其它類似的配體分子，再利用MM/PBSA計算小分子和PKA之間的結合自由能。
四個結晶結構當中，除了2C1A模擬結果，與原有的IC50值相比不具正相關，其它三個結構與現有IC50值比，則得到良好的相關性。經過模擬計算，我們進行討論和分析，取代配體當中的那些官能基能提高整體的親合力。基於這些結果，我們進一步設計五個新配位分子。最後，得到了幾點改變官能基的建議：（1）添加甲基在鹵化苯基的環上，可以增加與疏水性空腔間的凡得瓦作用力。（2）添加羥基於小分子骨架，此羥基上的氧原子容易與ASN 168/ ASP 184產生強而穩定的氫鍵作用力。

Protein kinase A plays significant role in a number of signaling pathways in cell, and is thought to be a potential drug target for treatments of several diseases by inhibiting its kinase activity.

In the present study, we aimed to use molecular dynamics simulation method to aid in design of PKA inhibitors. We selected 4 ligand-PKA complexes, in which their ligands are analogous, from Protein data bank with available IC50 values, MM/PB(GB)SA was employed to investigate the binding between ligand and PKA. The calculated binding energies are in good accord with the available IC50 values of examined complexes except one. The calculated results were analyzed and discussed to aid in understanding which functional group substitutions can enhance the binding affinity. Based on these results,we further designed 5 new ligands to predict their binding affinities.

The calculated results suggest the fillowing points to change the functional groups:
(1) Adding a methyl group at the R1 or R3 position (see Figure in the text) of the single ring in the ligand can enhance the binding affinity. The van der Waals interaction is the main contributor in the total binding energy .
(2) Adding a hydroxyl group at R5 position of the singl ring gave stable hydrogen bonds with the LYS 168 and ASP 184, increasing the binding affinity. These results together with the analysis above should be able to aid in design of PKA inhibitors analogous to the ligands investigated in the present study.

1. Olsen, J.V. et al. Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks. Cell 127, 635-648 (2006).
2. Pickin, K.A., Chaudhury, S., Dancy, B.C.R., Gray, J.J. & Cole, P.A. Analysis of Protein Kinase Autophosphorylation Using Expressed Protein Ligation and Computational Modeling. Journal of the American Chemical Society 130, 5667-5669 (2008).
3. Benson, J.D. et al. Validating cancer drug targets. Nature 441, 451-456 (2006).
4. Sebolt-Leopold, J.S. & English, J.M. Mechanisms of drug inhibition of signalling molecules. Nature 441, 457-462 (2006).
5. Fabian, M.A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotech 23, 329-336 (2005).
6. Manning, G., Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The Protein Kinase Complement of the Human Genome. Science 298, 1912-1934 (2002).
7. Taylor, S.S. et al. Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1784, 16-26 (2008).
8. Iyer, G.H., Moore, M.J. & Taylor, S.S. Consequences of Lysine 72 Mutation on the Phosphorylation and Activation State of cAMP-dependent Kinase. Journal of Biological Chemistry 280, 8800-8807 (2005).
9. Yonemoto, W., McGlone, M., Grant, B. & Taylor, S. Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. Protein Eng. 10, 915-925 (1997).
10. Toner-Webb, J., van Patten, S.M., Walsh, D.A. & Taylor, S.S. Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase. Journal of Biological Chemistry 267, 25174-25180 (1992).
11. Kim, C., Xuong, N.-H. & Taylor, S.S. Crystal Structure of a Complex Between the Catalytic and Regulatory (RI{alpha}) Subunits of PKA. Science 307, 690-696 (2005).
12. Kinderman, F.S. et al. A Dynamic Mechanism for AKAP Binding to RII Isoforms of cAMP-Dependent Protein Kinase. Molecular Cell 24, 397-408 (2006).

13. Dodge-Kafka, K.L. et al. The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature 437, 574-578 (2005).
14. Taylor, S.S. et al. Dynamics of signaling by PKA. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1754, 25-37 (2005).
15. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis. Science 270, 1326-1331 (1995).
16. Favata, M.F. et al. Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase. Journal of Biological Chemistry 273, 18623-18632 (1998).
17. Kolch, W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6, 827-837 (2005).
18. Logan, C.Y. & Nusse, R. THE WNT SIGNALING PATHWAY IN DEVELOPMENT AND DISEASE. Annual Review of Cell and Developmental Biology 20, 781-810 (2004).
19. Tan, Y.-H. et al. Cytotoxicity and Proteomics Analyses of OSU03013 in Lung Cancer. Clinical Cancer Research 14, 1823-1830 (2008).
20. Sun, Y.-C. Examination of effects of GSK3beta phosphorylation, beta-catenin phosphorylation, and beta-catenin degradation on kinetics of Wnt signaling pathway using computational method. Theoretical Biology and Medical Modelling 6, 13 (2009).
21. Aye, T.T. et al. Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins. Molecular & Cellular Proteomics 8, 1016-1028 (2009).
22. Seino, S. & Shibasaki, T. PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis. Physiol. Rev. 85, 1303-1342 (2005).
23. Tetsu, O. & McCormick, F. [beta]-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422-426 (1999).
24. Clevers, H. Wnt/[beta]-Catenin Signaling in Development and Disease. Cell 127, 469-480 (2006).
25. De Clercq, E. Antiviral drugs in current clinical use. Journal of Clinical Virology 30, 115-133 (2004).
26. Russell, R.J. et al. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443, 45-49 (2006).
27. Capdeville, R., Buchdunger, E., Zimmermann, J. & Matter, A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 1, 493-502 (2002).
28. Congreve, M., Murray, C.W. & Blundell, T.L. Keynote review: Structural biology and drug discovery. Drug Discovery Today 10, 895-907 (2005).
29. Kuhn, B. & Kollman, P.A. Binding of a Diverse Set of Ligands to Avidin and Streptavidin: An Accurate Quantitative Prediction of Their Relative Affinities by a Combination of Molecular Mechanics and Continuum Solvent Models. Journal of Medicinal Chemistry 43, 3786-3791 (2000).
30. Kollman, P.A. et al. Calculating Structures and Free Energies of Complex Molecules: Combining Molecular Mechanics and Continuum Models. Accounts of Chemical Research 33, 889-897 (2000).
31. Swanson, J.M.J., Henchman, R.H. & McCammon, J.A. Revisiting Free Energy Calculations: A Theoretical Connection to MM/PBSA and Direct Calculation of the Association Free Energy. Biophysical Journal 86, 67-74 (2004).
32. Ryckaert, J.-P., Ciccotti, G. & Berendsen, H.J.C. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics 23, 327-341 (1977).
33. Nicholls, A. & Honig, B. A rapid finite difference algorithm, utilizing successive over-relaxation to solve the Poisson-Boltzmann equation. Journal of Computational Chemistry 12, 435-445 (1991).
34. Tomasi, J. & Persico, M. Molecular Interactions in Solution: An Overview of Methods Based on Continuous Distributions of the Solvent. Chemical Reviews 94, 2027-2094 (1994).
35. Sitkoff, D., Sharp, K.A. & Honig, B. Accurate Calculation of Hydration Free Energies Using Macroscopic Solvent Models. The Journal of Physical Chemistry 98, 1978-1988 (1994).
36. Still, W.C., Tempczyk, A., Hawley, R.C. & Hendrickson, T. Semianalytical treatment of solvation for molecular mechanics and dynamics. Journal of the American Chemical Society 112, 6127-6129 (1990).
37. Page, C.S. & Bates, P.A. Can MM-PBSA calculations predict the specificities of protein kinase inhibitors? Journal of Computational Chemistry 27, 1990-2007 (2006).
38. Tripathi, A. et al. Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity. Bioorganic & Medicinal Chemistry 16, 2235-2242 (2008).