Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases

Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Pedro Miguel Miranda Araújo Mestrado em Bioquímica Departamento de Química e Bioquímica 2014 Advisor Professor Doutor Joaquim C.G. Esteves da Silva, Associated Professor with Aggregation, Faculdade de Ciências da Universidade do Porto Co-Advisor Master Luís Pinto da Silva, Phd Student, Faculdade de Ciências da Universidade do Porto II All the corrections determined by the Jury, and only those, were made. The Jury President, Porto, ______/______/_________ III Abstract Protein phosphorylation is one of the most important mechanisms within living cells, having a central role in the cellular signalling pathways. One of the most important functions of protein kinases is the control of checkpoints during cell cycle. The outcome of checkpoints activation depends on the DNA damage severity. If it is possible to repair the cell cycle is arrested giving the cellular repair mechanism the needed time to act but if the damage is too extended the result is apoptosis. The three most relevant checkpoints are located in late G1 (start checkpoint), before entering mitosis (G2/M checkpoint) and during mitosis (M checkpoint). Protein p53 is an essential factor in the G1 checkpoint. However, it is absent in many tumors leading to the loss of this checkpoint. The inhibition of G2/M checkpoint in p53-deficient cells conjugated with common radio or chemotherapy would create and increase DNA damage leading to a catastrophic mitotic event that would result in apoptosis. Through this mechanism inhibition of G2/M checkpoint was proposed as a sensitization therapy in p53-dificient cells. G2/M checkpoint is regulated by ATM and ATR pathways with the downstream effectors Checkpoint kinase 1 and 2. More recently the role of MK2 in the regulation of G2/M checkpoints was also revealed. Aiming the reduction of time and costs in drug development computational tools started to be employed in this field, being the processes in which they took part commonly called Computer aided drug design (CADD). These methods can be separated in two different types of approaches, structure based (SBDD) and ligand based (LBDD). In this work SBDD methods were used. Starting from x-ray crystallography structures semi-empirical energy calculation were performed to study the binding sites of protein kinases Chk1 and MK2. In the first stage of this work the binding between Checkpoint kinase 1 and two molecules with known inhibitory potential, C39 and C40 was studied. Similarly to the experimental data C39 showed the best binding potential towards Chk1, being this inhibition mode highly dependent on the interactions with the residues Lys38 and Glu91. In C40 binding mode residue Glu91 was also very important. Using this knowledge 10 novel molecules were designed based on C39, Modified molecule 1 to 10 (MD1-10). When compared to C39, MD8 and MD9 showed significant improvements in the binding energy. MD9 achieved the best improvement (21%) and MD8 the second best (19%). Due to difficulties in the study of MK2 a closely related protein from the same family, MK3, was suggested as a model structure. Thus the binding characteristics of two potential inhibitors, P4O and 05B, to MK2 and MK3 was studied. This work revealed the importance of binding site water molecules and the similarity in the binding profile of both enzymes with the same central characteristics. From the work present in this thesis two oral communications were performed, one book chapter and two papers in peer-reviewed journals were published, while other paper is currently submitted: IV  P. Araújo, L. Pinto da Silva and J.C.G. Esteves da Silva, Molecular Design of Potential Chk1targeting Anti-Cancer Drugs, IJUP 14 - Encontro de Jovens Investigadores da Universidade do Porto (2014);  P. Araújo, L. Pinto da Silva and J.C.G. Esteves da Silva, Computational Study of G2 Checkpoint Protein Kinases-Inhibitor Complexes, 4º PYCheM - 4th Portuguese Young Chemists Meeting (2014);  Pedro M. M. Araújo, Luís Pinto da Silva and Joaquim C. G. Esteves da Silva, Computational Chemistry Theories, Methods and Applications, Chapter 1: Protein Kinase-targeting drug Discovery and Design: Computational Chemistry as an Indispensable Tool, Nova Publishers (2014) 1-22;  Pedro M.M. Araújo, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva, Comparative theoretical study of the binding of potential cancer-treatment drugs to Checkpoint kinase 1 Chemical Physics Letters 591 (2014) 273–276;  Pedro M.M. Araújo, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva, Theoretical Modelling of Potential Chk1 Inhibitors, Letters in Drug Design & Discovery, 2015. V Resumo A fosforilação proteica é um dos mecanismos mais relevantes nas células vivas, tendo um papel fulcral nas vias de sinalização celulares. Um das funções mais importantes das proteínas cinases é a de controlo dos checkpoints durante o ciclo celular. O resultado da ativação dos checkpoints depende da severidade do dano do ADN. Se for possível o seu reparo o ciclo celular é parado criando o tempo necessário para os mecanismos de reparo do ADN atuarem no entanto se os danos forem muito extensos o resultado é apoptose. Os três checkpoint mais relevantes estão localizados em G1 (checkpoint de inicio), antes de entrar em mitose (G2/M checkpoint) e durante a mitose (M checkpoint). A proteína p53 é uma fator essencial do checkpoint G1. No entanto, está ausente em muitos tumores levando a perda deste checkpoint. A inibição do checkpoint G2/M em células sem p53 funcional conjugada com a radio ou quimioterapia comum iria criar e aumentar danos no ADN levando a um evento mitótico catastrófico que resultaria na apoptose. Através deste mecanismo a inibição do checkpoint G2/M foi proposta como uma terapia de sensibilização para células deficientes em p53. O checkpoint G2/M é regulado pelas vias de sinalização ATM e ATR tendo como efetores downstream a Checkpoint kinase 1 e 2. Mais recentemente foi descrito o papel da MK2 na regulação do checkpoint G2/M. Tendo como objetivo a redução do tempo e custos no desenvolvimento de novos fármacos as ferramentas computacionais começaram a ser aplicadas nesta área, sendo os processos nos quais estão envolvidas chamados Computer aided drug design (CADD). Estes métodos podem ser divididos em dois tipos diferentes de abordagens, baseadas na estrutura (SBDD) ou baseadas no ligando (LBDD). No presente trabalho métodos de SBDD foram usados, começando a partir de estruturas de cristalografia raio-x cálculos semi-empíricos de energia foram feitos para estudar os locais de ligação das cinases Chk1 e MK2. Na primeira fase deste trabalho a ligação à Chk1 de duas moléculas com potencial inibitório conhecido, C39 e C40, foi estudada. Similarmente aos resultados experimentais C39 mostrou o melhor potencial de inibição da Chk1, sendo o seu modo de inibição é altamente dependente das interações com os resíduos Lys38 e Glu91. De forma semelhante no modo de ligação da C40 o resíduo Glu91 também foi muito importante. Usando o conhecimento obtido foram criadas 10 novas moléculas baseadas na C39, Modified molecule 1 a 10 (MD1-10). Quando comparadas com a C39 as moléculas MD8 e MD9 mostraram melhorias significantes na energia de ligação. MD9 atingiu o melhor resultado (21% de melhoria) enquanto a MD8 o segundo melhor (19% de melhoria). Devido a dificuldades no estudo da MK2 uma proteína semelhante da mesma família, MK3, foi sugerida como modelo. Desta forma foram estudas as características da ligação de dois potenciais inibidores, P4O e 05B, a MK2 e MK3. Este estudo revelou a importância das moléculas de água presentes nos locais ligação e a semelhança entre os perfis de ligação das duas enzimas, que apresentaram as mesmas características centrais. VI A partir do trabalho presente nesta tese duas comunicações orais foram realizadas, um capítulo de livro e dois artigos em revistas com revisão por pares foram publicados, enquanto um outro artigo se encontra submetido:  P. Araújo, L. Pinto da Silva and J.C.G. Esteves da Silva, Molecular Design of Potential Chk1targeting Anti-Cancer Drugs, IJUP 14 - Encontro de Jovens Investigadores da Universidade do Porto (2014);  P. Araújo, L. Pinto da Silva and J.C.G. Esteves da Silva, Computational Study of G2 Checkpoint Protein Kinases-Inhibitor Complexes, 4º PYCheM - 4th Portuguese Young Chemists Meeting (2014);  Pedro M. M. Araújo, Luís Pinto da Silva and Joaquim C. G. Esteves da Silva, Computational Chemistry Theories, Methods and Applications, Chapter 1: Protein Kinase-targeting drug Discovery and Design: Computational Chemistry as an Indispensable Tool, Nova Publishers (2014) 1-22;  Pedro M.M. Araújo, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva, Comparative theoretical study of the binding of potential cancer-treatment drugs to Checkpoint kinase 1 Chemical Physics Letters 591 (2014) 273–276;  Pedro M.M. Araújo, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva, Theoretical Modelling of Potential Chk1 Inhibitors, Letters in Drug Design & Discovery, 2015. VII Acknowledgments To my family for all the love and support. To Sara Vaz for always being there when I needed most. To my colleagues Celso, Daniel and Inês for the support and help during the two years of my master degree. To Bruno, Diana, Dilson, Joel, Margarida, Mariana, Paulo and Teresa for the good environment created in group, the good times, the laughs and the amazing lunch times. To my co-advisor Master Luís Pinto Silva for all the patience, knowledge transferred, good laughs and for the assistance in all the work performed during this year, I could only do this with your help. To my advisor Professor Doctor Joaquim C.G. Esteves da Silva for the opportunity to participate in this work and join this group and for all the advising and help. VIII Contents List of Abbreviations IX Chapter 1Introduction 1 1.1. – Kinases 1 1.2. – Cell Cycle Checkpoints 1 1.3. – G1 Checkpoint and p53-deficient cells 2 1.4. – DNA Damage Response mechanism 2 1.5. – Objectives 3 1.6. – References 4 Chapter 2 - Protein Kinase-Targeting Drug Discovery and Design: Computational Chemistry as an Indispensable Tool 7 Chapter 3 – Checkpoint kinase 1 binding site and inhibition model 30 3.1. – Comparative theoretical study of the binding of potential cancer-treatment drugs to Checkpoint kinase 1 30 3.2. – Theoretical Modelling of Potential Chk1 Inhibitors 35 Chapter 4 - Theoretical Analysis of the Binding of Potential Inhibitors to Protein Kinases MK2 and MK3 42 Chapter 5 – Conclusions and Future perspectives 56 5.1. – Conclusion 56 5.2. – Future perspectives 57 IX List of Abbreviations Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3 related C39 Compound 39 C40 Compound 40 CADD Computer aided drug design Cdc25 Cell division cycle Cdk1 Cyclin-dependent kinase 1 Cdk2 Cyclin-dependent kinase 2 Chk1 Checkpoint kinase 1 Chk2 Checkpoint kinase 2 CK2 Casein kinase 2 CML Chronic myeloid leukemia CYS Cysteine DDR DNA damage response DNA Deoxyribonucleic acid G1 Gap 1 (cell cycle) G2 Gap 2 (cell cycle) Gadd45α Growth arrest and DNA-damage-inducible protein GADD45 alpha GIST Gastrointestinal stromal Tumor Gln Glutamine Gly Glycine Glu Glutamic acid GPU Graphics processing unit GSK Glycogen synthase kinase GTP Guanine triphosphate H-bond Hydrogen Bound His Histidine HIV Human immunodeficiency virus HPV Human Papilloma Virus HTS High throughput screening IC50 Half maximal inhibitory concentration Ile Isoleucine IUPAC International Union of Pure and Applied Chemistry JAK Janus kinase LBDD Ligand based drug design LBVS Ligand based virtual screening Leu Leucine LHS Left Hand Side Lys Lysine M phase Mitosis (cell cycle) MAPK Mitogen-activated protein kinases MD Molecular dynamics MD1 - MD10 Modified molecule 1 to 10 MET Methionine MK2/MAPKAPK2 MAP kinase-activated protein kinase 2 FCUP 6 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases [30] D.A. Case, T.E. Cheatham, T. Darden, H. Gohlke, R. Luo, K.M. Merz, et al., The Amber biomolecular simulation programs., J. Comput. Chem. 26 (2005) 1668–1688. [31] Y. Duan, C. Wu, S. Chowdhury, M.C. Lee, G. Xiong, W. Zhang, et al., A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations., J. Comput. Chem. 24 (2003) 1999–2012. [32] T. Yanai, D.P. Tew, N.C. Handy, A new hybrid exchange?correlation functional using the Coulomb-attenuating method (CAM-B3LYP), Chem. Phys. Lett. 393 (2004) 51–57. [33] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, et al., Gaussian 09, Revision A.02, Gaussian Inc Wallingford CT. 34 (2009) Wallingford CT. [34] J. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, Development and testing of a general amber force field., J. Comput. Chem. 25 (2004) 1157–1174. [35] J.C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, et al., Scalable molecular dynamics with NAMD., J. Comput. Chem. 26 (2005) 1781–802. [36] U. Essmann, L. Perera, M.L. Berkowitz, T. Darden, H. Lee, L.G. Pedersen, A smooth particle mesh Ewald method, J. Chem. Phys. 103 (1995) 8577–8593. [37] J.J.P. Stewart, Optimization of parameters for semiempirical methods V: Modification of NDDO approximations and application to 70 elements, J. Mol. Model. 13 (2007) 1173–1213. [38] V.M. Anisimov, C.N. Cavasotto, Quantum Mechanical Binding Free Energy Calculation for Phosphopeptide Inhibitors of the Lck SH2 Domain, J. Comput. Chem. 1 (2011) 1–8. [39] K.D. Dubey, R.P. Ojha, Binding free energy calculation with QM/MM hybrid methods for AblKinase inhibitor., J. Biol. Phys. 37 (2011) 69–78. [40] N. Nunthaboot, F. Tanaka, S. Kokpol, H. Chosrowjan, S. Taniguchi, N. Mataga, Quantum mechanical study of photoinduced charge transfer in FMN binding protein., J. Phys. Chem. B. 112 (2008) 15837–15843. [41] Y.-Z. Xiong, P.-Y. Chen, ONIOM DFT/PM3 calculation on the interaction between STI-571 and abelson tyrosine kinase., J. Mol. Model. 14 (2008) 1083–1086. [42] L. Pinto da Silva, J. Vieira, J.C.G. Esteves da Silva, Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase, Chem. Phys. Lett. 543 (2012) 137–141. FCUP 7 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Chapter 2 - Protein Kinase-Targeting Drug Discovery and Design: Computational Chemistry as an Indispensable Tool The following chapter was written in response to the invitation from Nova Publishers to the group of Professor Joaquim C.G. Esteves da Silva. The design of the structure and the selection of the topics covered was conducted by the three authors of the publication according to the publisher suggestion to address the topic of “computational drug design”. The text was written by the author Pedro Araújo. The supervision, revisions and suggestions of improvement were added by Luis Pinto da Silva and Professor Joaquim Esteves da Silva. FCUP 8 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 9 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 10 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 11 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 12 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 13 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 14 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 15 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 22 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 23 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 24 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 25 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 26 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 27 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 28 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 29 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 30 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Chapter 3 – Checkpoint kinase 1 binding site and inhibition model 3.1. - Comparative theoretical study of the binding of potential cancertreatment drugs to Checkpoint kinase 1 The following article published in Chemical Physics Letters was outlined by the authors Luis Silva Pinto and Professor Joaquim Esteves da Silva. The bibliographic research was performed by Luis Pinto Silva and Pedro Araújo. The theoretical calculations were executed by Luís Pinto Silva and Pedro Araújo. The text writing of Theoretical Methods section was performed by Luís Pinto Silva and the remaining document by Pedro Araújo. Revision of the manuscript, suggestions of improvement and correction were added by Luis Pinto Silva and Professor Joaquim Esteves da Silva. The supplementary information of this document can be seen in Appendix 1. The succeeding paper reports our initial studies regarding the Chk1 ATP binding site. FCUP 31 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 38 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 39 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 40 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 41 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases FCUP 42 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Chapter 4 - Theoretical Analysis of the Binding of Potential Inhibitors to Protein Kinases MK2 and MK3 In the work Theoretical Analysis of the Binding of Potential Inhibitors to Protein Kinases MK2 and MK3 the planning, the bibliographic research, all the calculations and the text writing was performed by Pedro Araújo. The supervisor, revision of the manuscript, suggestions of improvement and corrections were added by the authors Luis Pinto Silva and Professor Joaquim Esteves da Silva. The document has not yet published version. The supplementary information of this document can be seen in Appendix 3. In this work we report our observations of the ATP binding site of MK2 and MK3. FCUP 43 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Theoretical Analysis of the Binding of Potential Inhibitors to Protein Kinases MK2 and MK3 Pedro M.M. Araújo, Luís Pinto da Silva e Joaquim C.G. Esteves da Silva* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciência da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. *Corresponding author: Joaquim C.G. Esteves da Silva, E-mail: [email protected]; Tel.: 351 226082869; Fax: 351 226082959. Abstract MK2 (or MAPKAPK2) was already known for its role in the inflammatory response, however recent studies indicate the involvement of this protein kinase in the DNA damage response mechanism. Within its kinase family MK3 shows a high degree of similarity to MK2. In this article we report a theoretical study of the binding of two molecules, 05B and P4O, to MK2 and MK3. The data here obtained clarifies which are the most relevant residues in the binding of potential inhibitors to these kinases and the contribution of the binding site water molecules. Keywords Cell cycle checkpoints; DNA damage response (DDR); Enzyme-Inhibitor Interactions; MK2; MK3; Semi-empirical calculations. FCUP 44 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases 1. Introduction The DNA damage response (DDR) can be divided into two main kinase-based-pathways, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) with the downstream effectors Chk2 (Checkpoint kinase 2) and Chk1 (Checkpoint kinase 1) respectively. Those pathways are capable of critically influence the cell cycle checkpoints [1,2]. Checkpoints are cellular mechanisms able to respond when the conditions to the progression of the cycle are not ideal, for instance if there is DNA damage. Resulting from the activation of the checkpoints the cell cycle can be stalled in late G1 (start checkpoint), before mitosis (G2/M) or before sister-chromatid separation (M phase). This time gap gives the cell the time needed for the DNA repair mechanisms to act, or if the damage is too severe initiating the apoptotic process [1,3,4]. Protein p53 is a key factor in G1 checkpoint, however it is usually mutated or inactivated in human tumours. Without p53 G1 checkpoint is lost, increasing the importance of M and G2/M checkpoints in the protection of DNA stability. Downstream from ATM and ATR was identified the p38/MK2 pathway as the third kinase-based mechanism to take part in DDR. The Yaffe group reported the finding of two G2/M checkpoint events in p53-deficient cells, spatially and temporally distinct. The one mediated by Chk1 occur in the nucleus while the event mediated by MK2 (or MAPKAPK2) is more delayed and took place in the cytoplasm [1]. This enzyme was already known to take part in the inflammatory response due to its presence in the synthesis of inflammatory cytokines and tumour necrosis factor α (TNFα) [3,5]. Morandell et al. created an animal model in which was possible to simultaneously generate MK2-expressing and MK2-deficient tumours, showing that in the absence of p53 MK2 is essential to the survival of NSCLC (non-small cell lung cancer) tumours [4]. Kooper et al. reported that the impairment in the DNA replication caused by gemcitabine (used in chemotherapy) and Chk1 was stopped by MK2. Making this protein in the first cellular effector whose deletion leads to the progression of DNA replication under unfavourable conditions [6]. The inhibition of G2/M checkpoint in p53-deficient tumour cells would increase the DNA instability rapidly, creating catastrophic mitotic events that would lead to the death of those cells [3]. Checkpoint kinase 1 emerged as a target to this purpose since it takes part in G2/M checkpoint. The search for Chk1 inhibitors is in an advanced state with some molecules already in clinical trials [3,7]. However, with the recent findings in this field [1,2,4,8] there is strong evidence that MK2 needs to be inhibited in order to stop G2/M checkpoint. The development of compounds targeting MK2 inhibition has been slowed due to the absence of a high resolution 3D structures of this enzyme. Since the binding domains of MK2 and MK3 have high similarity it was proposed the usage of MK3, that has published 3D structures with higher resolution, as a model to understand the binding of small molecules to MK2 [5]. In the present work we theoretically evaluated the ATP binding site characteristics of MK2 and MK3 when bond to two different ligands: 05B and P4O (Fig. 1), through semi-empirical calculations in an implicit FCUP 45 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases solvent. The purpose of our study is to obtain relevant information about the binding mode of potential inhibitors to these two kinases, which may help create novel and more specific inhibitors. Figure 1: Two dimensional representation of A: Molecule 05B; B: Molecule P4O. 2. Theoretical Methods The PDB ID of the 3D structures used in this study were 3R2B [9] for MK2 bond to 05B, 2JBP [10] for MK2 bond to P4O, 3R1N [9] for MK3 bond to 05B and 3FHR [5] for MK3 bond to P4O. Each complex was study with and without the water molecules from the crystallography structure, deleting the information about those molecules in the PDB file before energy minimizations. The hydrogen atoms, the missing atoms, and TIP3P water molecules up to 15 Å were added by the LEAP module of the AMBER suite of programs [11]. The ff03 force field was used for intramolecular interactions [12]. Due to the high number of atoms present in the molecules P4O and 05B their geometries were obtained with the CAM-B3LYP/6-31G method [13]. The geometry optimizations were made with the GAUSSIAN09 software package [14]. The CAM-B3LYP/6-31G obtained geometries were used in the parameterization of the ligands with the ANTECHAMBER module of AMBER and the general AMBER force field [15]. The parameterization was made by performing a CAM-B3LYP/6-311G (d,p) single point energy calculation, at the CAM-B3LYP/6-31G geometries. Three phases of energy minimizations were performed for each enzyme–ligand complex, using the Not (just) Another Molecular Dynamics program (NAMD) molecule dynamic code with AMBER potential functions, parameters, and the file formats. The two initial minimizations had 30 000 steps each, in the first all the non-water atoms were fixated while in the second the remaining atoms were fixated but not the water atoms. The third minimization had 45 000 steps and none atoms fixated [16]. In this process, the Particle Mesh Ewald method was used to include the long-range interactions [17]. FCUP 46 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases After the energy minimizations protocol the Root-mean-square deviations (RMSD) between the atom positions of the PDB structures and the new obtained positions were calculated. The deviations values indicated that MK2 structures were more rearranged than those involving MK3. Nevertheless, the largest rearrangements occurred in regions away from the binding site not affecting its characteristics (Supplementary Information Figure 1 to 4). These results were expected since MK2 structures had a lower resolution. The RMSD values obtained were 2.17 Å for 3R2B, 1.64 Å for 2JBP, 1.18Å for 3R1N and 1.14Å for 3FHR. The deviation of each residue in the four different structures can be observed in the Supplementary Information (Graphic 1 to 4). Note that the residues numeration in the graphics does not correspond to the PDB file, residue number 1 corresponds to the first residue in the file and so on. In the end of the energy minimizations process, one selection of binding site residues were withdrawn from the complexes bond to 05B and another for those bond to P4O. For 05B the selected residues were: Leu52, Lys69, Lys73, Met118, Met121, Glu122, Glu170, Asn171, Thr186, Asp187 and the two water molecules in the binding site for MK3 (Fig. 2A); and: Leu72, Lys89, Lys93, Met138, Leu141, Asp142, Glu190, Asn191, Thr206 and Asp207 for MK2 (Fig. 2B). For P4O the selected residues were: Leu50, Lys73, Cys120, Met121, Glu122, Glu170, Asn171, Lys177, Thr186, Asp187 and the two water molecules in the binding site for MK3 (Fig. 2C); and: Leu70, Lys93, Cys140, Leu141, Asp142, Glu190, Asn191, Lys197, Thr206, Asp207 and the water molecule in the binding site for MK2 (Fig. 2D). The selections were made based in our observation of the binding sites and based on the reports of published 3D structures of MK2 and MK3 [5,9,10]. The energies of association of the ligands with the two enzymes were estimated through single point energy calculations with the semi-empirical method PM6, with the implicit solvent diethyl ether in the SMD solvation model [18]. To assess the binding energy for each complex (ΔEass), three calculations were performed (Eq.(1)): PM6 single point energy calculations on the model composed by the ligand and all the chosen active site residues (Ecomplex); PM6 single point energy calculations on a model including only the chosen active site molecules (Eenzyme); PM6 single point calculations on the ligand (Eligand). After the calculation of ΔEass for each complex, the energetic contribution to the ΔEass of each active site molecule was evaluated. To estimate the contribution of an active site residue X the energy of association was calculated without the residue X, in the same manner as previously described. By comparing the ΔEass obtained for the model in the absence of that molecule, with the results obtained with all the active site molecules present, it is possible to get a quantitative picture of the contribution of each molecule to the associating energy of the ligands (ΔΔEass, Eq. (2)). The removal of the molecules that contribute favourably to the interaction of the ligands with the active site will lead to less stable complexes and consequently to more positive ΔΔEass. The family of PMx methods was used due to the very high number of atoms present in these models, and due to its use in previous studies where interaction/ binding energies were calculated [19–23]. The single point energy calculations were performed with the GAUSSIAN 09 program package [14]. FCUP 47 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases (Equation 1) (Equation 2) Figure 2. A: Schematic representation of 05B and the selected residues from MK3 (3R1N structure); B: Schematic representation of 05B and the selected residues from MK2 (3R2B structure); C: Schematic representation of P4O and the selected residues from MK3 (3FHR structure); D: Schematic representation of P4O and the selected residues from MK2 (2BJP structure). 3. Results and Discussion FCUP 54 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Table 1: Cartesian coordinates of MK3 bound to 05B. Table 2: Cartesian coordinates of MK3 bound to 05B without binding site water molecules. Table 3: Cartesian coordinates of MK2 bound to 05B. Table 4: Cartesian coordinates of MK3 bound to P4O. Table 5: Cartesian coordinates of MK3 bound to P4O without binding site water molecules. Table 6: Cartesian coordinates of MK2 bound to P4O. Table 7: Cartesian coordinates of MK2 bound to P4O without binding site water molecules. References [1] Reinhardt, H. C.; Hasskamp, P.; Schmedding, I.; Morandell, S.; van Vugt, M. A. T. M.; Wang, X.; Linding, R.; Ong, S. E.; Weaver, D.; Carr, S. A.; et al. DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization. Mol. Cell, 2010, 40, 34–49. [2] Höpker, K.; Hagmann, H.; Khurshid, S.; Chen, S.; Schermer, B.; Benzing, T.; Reinhardt, H. C. Putting the Brakes on p53-Driven Apoptosis. Cell Cycle, 2012, 11, 4122–4128. [3] Meng, Z.; Ciavarri, J. P.; McRiner, A.; Zhao, Y.; Zhao, L.; Reddy, P. A.; Zhang, X.; Fischmann, T. O.; Whitehurst, C.; Arshad Siddiqui, M. Potency Switch between CHK1 and MK2: Discovery of imidazo[1,2A]pyrazineand imidazo[1,2-C]pyrimidine-Based Kinase Inhibitors. Bioorg. Med. Chem. Lett., 2013, 23, 2863–2867. [4] Morandell, S.; Reinhardt, H. C.; Cannell, I. G.; Kim, J. S.; Ruf, D. M.; Mitra, T.; Couvillon, A. D.; Jacks, T.; Yaffe, M. B. A Cre-Versible Approach Identifies Synthetic Lethal Interactions between MK2 and p53 in the DNA Damage Response in Vivo. Cell Rep., 2013, 5, 868–877. [5] Cheng, R.; Felicetti, B.; Palan, S.; Toogood-Johnson, I.; Scheich, C.; Barker, J.; Whittaker, M.; Hesterkamp, T. High-Resolution Crystal Structure of Human Mapkap Kinase 3 in Complex with a High Affinity Ligand. Protein Sci. , 2010, 19, 168–173. [6] Köpper, F.; Bierwirth, C.; Schön, M.; Kunze, M.; Elvers, I.; Kranz, D.; Saini, P.; Menon, M. B.; Walter, D.; Sørensen, C. S.; et al. Damage-Induced DNA Replication Stalling Relies on MAPK-Activated Protein Kinase 2 Activity. Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 16856–16861. [7] Ma, C.; Cai, S.; Li, S.; Ryan, C. Targeting Chk1 in p53-Deficient Triple-Negative Breast Cancer Is Therapeutically Beneficial in Human-in-Mouse Tumor Models. J. Clin., 2012, 122, 1541–1552. [8] Reinhardt, H. C.; Aslanian, A. S.; Lees, J. a; Yaffe, M. B. p53-Deficient Cells Rely on ATMand ATR-Mediated Checkpoint Signaling through the p38MAPK/MK2 Pathway for Survival after DNA Damage. Cancer Cell, 2007, 11, 175–189. [9] Barf, T.; Kaptein, A.; De Wilde, S.; Van Der Heijden, R.; Van Someren, R.; Demont, D.; SchultzFademrecht, C.; Versteegh, J.; Van Zeeland, M.; Seegers, N.; et al. Structure-Based Lead Identification of ATP-Competitive MK2 Inhibitors. Bioorganic Med. Chem. Lett., 2011, 21, 3818–3822. FCUP 55 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases [10] Hillig, R. C.; Eberspaecher, U.; Monteclaro, F.; Huber, M.; Nguyen, D.; Mengel, A.; MullerTiemann, B.; Egner, U. Structural Basis for a High Affinity Inhibitor Bound to Protein Kinase MK2. J. Mol. Biol., 2007, 369, 735–745. [11] Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber Biomolecular Simulation Programs. J. Comput. Chem., 2005, 26, 1668–1688. [12] Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; et al. A Point-Charge Force Field for Molecular Mechanics Simulations of Proteins Based on Condensed-Phase Quantum Mechanical Calculations. J. Comput. Chem., 2003, 24, 1999–2012. [13] Yanai, T.; Tew, D. P.; Handy, N. C. A New Hybrid Exchange-correlation functional using the Coulomb-Attenuating Method (CAM-B3LYP). Chem. Phys. Lett., 2004, 393, 51–57. [14] Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, Revision A.02. Gaussian Inc Wallingford CT, 2009, 34, Wallingford CT. [15] Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem., 2004, 25, 1157–1174. [16] Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem., 2005, 26, 1781– 1802. [17] Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys., 1995, 103, 8577–8593. [18] Stewart, J. J. P. Optimization of Parameters for Semiempirical Methods V: Modification of NDDO Approximations and Application to 70 Elements. J. Mol. Model., 2007, 13, 1173–1213. [19] Dubey, K. D.; Ojha, R. P. Binding Free Energy Calculation with QM/MM Hybrid Methods for Abl-Kinase Inhibitor. J. Biol. Phys., 2011, 37, 69–78. [20] Nunthaboot, N.; Tanaka, F.; Kokpol, S.; Chosrowjan, H.; Taniguchi, S.; Mataga, N. Quantum Mechanical Study of Photoinduced Charge Transfer in FMN Binding Protein. J. Phys. Chem. B, 2008, 112, 15837–15843. [21] Xiong, Y.-Z.; Chen, P.-Y. ONIOM DFT/PM3 Calculation on the Interaction between STI-571 and Abelson Tyrosine Kinase. J. Mol. Model. 2008, 14, 1083–1086. [22] Pinto da Silva, L.; Vieira, J.; Esteves da Silva, J. C. G. Comparative Theoretical Study of the Binding of Luciferyl-Adenylate and Dehydroluciferyl-Adenylate to Firefly Luciferase. Chem. Phys. Lett., 2012, 543, 137–141. [23] Araújo, P. M. M.; Pinto da Silva, L.; Esteves da Silva, J. C. G. Comparative Theoretical Study of the Binding of Potential Cancer-Treatment Drugs to Checkpoint Kinase 1. Chem. Phys. Lett., 2014, 591, 273–276. FCUP 56 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Chapter 5 – Conclusions and Future perspectives 5.1. – Conclusion As a final remark a comparison between the proposed objectives at the beginning of this work and the obtained results shall be made. Initially, the focus of this work was Checkpoint kinase 1. The studies targeting this enzyme are in the chapter 4 of this document in two published papers. The first document reports our observations studying the binding mode of two molecules to Chk1, Compound 39 and Compound 40. Within this work it was successfully observed the major characteristics in the formation of those enzyme-inhibitor complex, the most relevant residues for those bindings and it was concluded that Compound 39 has the best inhibitory potential of the two. The second publication consists of our attempts to improve the binding potential of Compound 39-like molecules to Chk1. We created 10 novel molecules in a two-phased study based in the knowledge obtained in the previously referred publication. The obtained result was an improvement around 20% in the binding energy of Modified molecules 8 and 9 (MD8 and MD9) to Checkpoint kinase 1, when compared to Compound 39 in the same conditions. After a general look it is safe to say that the objectives proposed in the study of Chk1 were accomplished. We successfully understood the binding of potential inhibitors to this kinase and it was also possible to create compounds with improved binding energy to the enzyme. The study of MK2 naturally emerged after entering in contact with research that reports second G2/M checkpoint event in the p53-deficient tumors cells, as stated in the introduction of this document. Similarly to the Chk1 work, in the study of MK2 the objective was also to understand the binding mode of potential inhibitors to this enzyme. However this task was expected to be more challenging than Chk1 due to the absence of high resolution crystallography structures. In order to understand the binding mode of potential inhibitors to MK2 another enzyme, MK3, was added to the study. MK3 belongs to the same family of kinases and has a high degree of similarity to MK2. With the inclusion of MK3 in the study, which has crystallography structures with better resolution, the understanding of MK2's ATP binding site could possibly be enrich. Our studies in MK2 and MK3 resulted in the work presented in the Chapter 4, were it is described the most relevant residues for the binding of two different molecules, P4O and 05B, to these two kinases. In this report special attention was given to the role of water molecules in the ATP binding sites. Within this chapter the binding mode of potential inhibitors to MK2 and MK3 was defined as previously proposed in the objectives, the description of the central role of some water molecules, the differences between MK2 and MK3 and also the core similarities between the most relevant residues for both protein kinases was observed. FCUP 57 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases 5.2. - Future perspectives The work here reported can be seen as a starting point, a gathering of data which can be used for future studies targeting the inhibition of Chk1 or MK2. The information about the key features of Chk1 binding site here obtained can be used in any project targeting this enzyme. In the second published work (topic 3.2.) presented in Chapter 3 we tried to perform the suggestions of improvement that resulted from our first work (topic 3.1.). Although we achieved some improvement the possibility of best solutions for those questions still exist. The study of more molecules with known Chk1 inhibitor potential would be a good source of information for further improvement. The inhibitory potential of the molecules proposed by us (topic 3.2.) needs to be verified experimentally, if those results show to be promising those molecules may enter in more advanced testing. The data obtained for MK2 and MK3 showed impressive similarities, which indicates that compound with high specificity for only one of this kinases will be hard to obtain. The usage of MK3 as MK2 model already happens and in the absence of structures with better resolution for MK2 this appears to be a reasonable solution. Further studies should consider the possibility to use induced mutagenesis of MK3 to create an ATP binding site without differences from MK2, either computational or experimental. Since Chk1 and MK2 have been reported with a central function in p53-deficient tumor cells would be interesting to study compound with inhibitory potential for both of them. One molecule with the ability to successful inhibit Chk1 and MK2 would be extremely competitive in the field of cancer treatment for chemosensitization and radiosensitization. FCUP 58 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Appendix 1 Comparative Theoretical Study of the Binding of Potential CancerTreatment Drugs to Checkpoint Kinase 1 Pedro M.M. Araújo, Luís Pinto da Silva and Joaquim C.G. Esteves da Silva* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. *Corresponding Author: Joaquim C.G. Esteves da Silva; Email: [email protected]; Tel.: +351 226082869; Fax: +351 22608259; Table 1: Cartesian coordinates of Checkpoint kinase 1 complexed with compound 39. Table2: Cartesian coordinates of Checkpoint kinase 1 complexed with compound 40. FCUP 59 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Table1: Cartesian coordinates of Checkpoint kinase 1 complexed with compound 39 N 2.10900 -8.08600 -11.99600 H 1.09900 -8.12300 -11.95500 C 2.80200 -7.32400 -10.94600 H 3.86900 -7.26100 -11.17100 C 2.20700 -5.89600 -10.93900 H 1.17800 -5.96000 -10.58600 H 2.76500 -5.29300 -10.22100 C 2.20700 -5.16300 -12.30500 H 1.63200 -5.74300 -13.02700 C 1.52100 -3.79700 -12.17700 H 1.48400 -3.30600 -13.14900 H 0.50100 -3.92100 -11.81800 H 2.07400 -3.16700 -11.48600 C 3.62600 -4.96700 -12.85900 H 3.58300 -4.42400 -13.80400 H 4.23200 -4.40200 -12.14900 H 4.09600 -5.93200 -13.04400 C 2.70400 -7.99800 -9.55700 O 3.36700 -7.56700 -8.61300 H 2.38900 -7.72300 -12.90700 N 1.89400 -9.06100 -9.44100 H 1.42000 -9.35600 -10.28500 C 1.63100 -9.83800 -8.21700 H 2.44500 -10.54700 -8.07400 H 1.61900 -9.17900 -7.34800 C 0.31000 -10.61800 -8.26000 O -0.65900 -10.16600 -8.87400 H 0.23200 -11.52500 -7.77300 N -5.91800 -12.50200 -3.51100 H -4.91000 -12.54000 -3.49500 C -6.53800 -11.26000 -4.01600 H -7.51900 -11.14800 -3.55000 C -5.68000 -10.07000 -3.54900 H -6.13800 -9.14600 -3.90000 H -5.72900 -10.04000 -2.45900 C -4.21500 -10.06200 -3.96500 C -3.84200 -9.83300 -5.30600 H -4.59600 -9.68000 -6.06400 C -2.48200 -9.77600 -5.66600 H -2.19400 -9.59800 -6.69000 C -1.48100 -9.92700 -4.68300 O -0.16700 -9.83200 -5.02400 H 0.40400 -9.91700 -4.25800 C -1.85000 -10.15700 -3.34000 H -1.08900 -10.25900 -2.58000 C -3.21200 -10.22800 -2.98700 FCUP 60 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -3.48700 -10.37800 -1.95200 C -6.80300 -11.22200 -5.54300 O -7.17900 -10.17500 -6.07200 H -6.27000 -13.29100 -4.05300 H -6.66500 -12.07000 -6.11400 N -2.79800 -8.72800 -10.05500 H -2.01200 -9.27100 -9.69500 C -2.54600 -7.31300 -10.40600 H -3.50400 -6.83400 -10.59600 C -1.86500 -6.55000 -9.24400 H -0.90700 -7.02800 -9.02900 C -1.60600 -5.06700 -9.56200 H -1.10900 -4.59200 -8.71600 H -0.96000 -4.97000 -10.43100 H -2.54700 -4.55200 -9.75800 C -2.73600 -6.60600 -7.97700 H -2.21800 -6.10700 -7.16400 H -3.69100 -6.10800 -8.15300 H -2.92200 -7.63500 -7.67700 C -1.69200 -7.23500 -11.68300 O -0.73600 -7.99100 -11.83300 H -3.18400 -9.20600 -10.86900 H -1.92300 -6.54600 -12.41500 N -6.53700 -4.72600 -11.49000 H -7.36400 -4.17600 -11.71000 C -6.71700 -5.90300 -10.61000 H -5.78800 -6.47400 -10.58000 C -7.01600 -5.40700 -9.17900 H -6.23800 -4.69300 -8.90100 H -7.97100 -4.87900 -9.17200 C -7.04600 -6.52200 -8.11500 H -7.86200 -7.21400 -8.33000 H -6.10300 -7.07000 -8.14500 C -7.24200 -5.92900 -6.70700 H -6.46000 -5.19000 -6.52500 H -8.20800 -5.42200 -6.65800 C -7.17800 -7.00100 -5.60700 H -8.00700 -7.70400 -5.73900 H -6.24300 -7.55700 -5.70900 N -7.24600 -6.38300 -4.25800 H -6.49800 -5.70900 -4.13000 H -8.12100 -5.86700 -4.12700 H -7.18000 -7.05300 -3.49900 C -7.81300 -6.84200 -11.17300 O -8.88600 -6.38800 -11.57100 H -5.83200 -4.11300 -11.08100 H -7.64200 -7.85900 -11.21500 N -14.52400 -4.20800 -2.22700 FCUP 61 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -14.48800 -5.20500 -2.38600 C -13.37200 -3.40400 -2.66400 H -13.03900 -2.78500 -1.82900 C -12.21700 -4.34500 -3.04500 H -12.02800 -5.01200 -2.20300 H -12.51700 -4.94100 -3.90500 C -10.92300 -3.59600 -3.38800 H -11.10000 -2.96800 -4.26100 H -10.64600 -2.95600 -2.55000 C -9.75400 -4.52900 -3.70700 O -9.82500 -5.74100 -3.42100 O -8.74200 -4.04500 -4.25800 C -13.74400 -2.46500 -3.82400 O -13.39000 -1.28900 -3.79400 H -15.36900 -3.83600 -2.66100 H -14.28900 -2.82200 -4.62500 N -4.04600 5.45800 -4.52800 H -3.75300 4.78800 -3.80700 C -4.33200 4.93400 -5.88900 H -3.68500 5.47200 -6.58400 C -3.99300 3.42300 -6.01200 H -4.42700 2.90700 -5.15400 C -4.51800 2.73600 -7.28700 H -4.29300 1.67100 -7.24100 H -5.59900 2.83600 -7.37100 H -4.04900 3.15900 -8.17200 C -2.46800 3.22100 -5.97500 H -2.23700 2.16000 -6.06100 H -2.00000 3.75100 -6.80600 H -2.05200 3.59500 -5.04100 C -5.79200 5.18400 -6.30700 O -6.72500 4.73400 -5.63900 H -4.86500 5.96500 -4.19200 H -5.99200 5.73500 -7.15600 N -7.02600 0.89600 -12.65400 H -6.40800 0.39100 -13.28300 C -6.37700 1.62500 -11.55100 H -7.11200 2.23900 -11.03200 C -5.79400 0.58100 -10.56900 H -5.10700 -0.05500 -11.12800 H -5.21000 1.09800 -9.80600 C -6.81400 -0.33200 -9.85300 H -7.46100 -0.80800 -10.58800 C -6.06800 -1.44400 -9.10500 H -6.78300 -2.10100 -8.60800 H -5.48000 -2.03600 -9.80700 H -5.40500 -1.00600 -8.35900 C -7.68800 0.45100 -8.86400 FCUP 62 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -8.37600 -0.22700 -8.35900 H -7.06200 0.94300 -8.11900 H -8.27000 1.20100 -9.39200 C -5.26200 2.58300 -12.02300 O -4.71600 2.44200 -13.11700 H -7.71100 0.24700 -12.26700 H -4.96800 3.35800 -11.40800 N -1.67000 3.82300 -12.47600 H -2.02700 4.38200 -13.23300 C -0.32700 3.24400 -12.62500 H -0.33500 2.24600 -12.18500 C -0.01000 3.10200 -14.12500 H -0.67700 2.35300 -14.55200 H -0.23000 4.05200 -14.61500 C 1.42200 2.70700 -14.45500 C 1.97700 1.52700 -13.92200 H 1.37600 0.88400 -13.29600 C 3.31400 1.18000 -14.20100 H 3.74000 0.27700 -13.78900 C 4.10100 2.00300 -15.03500 O 5.39500 1.66600 -15.29300 H 5.83300 2.27200 -15.90900 C 3.54100 3.17700 -15.58600 H 4.13600 3.81300 -16.22500 C 2.20900 3.52900 -15.28700 H 1.79000 4.43800 -15.69500 C 0.76300 4.05500 -11.89200 O 0.84700 5.27500 -12.03300 H -2.33200 3.07200 -12.28100 N 1.63500 3.35200 -11.16200 H 1.48100 2.35400 -11.08900 C 2.72400 3.90900 -10.35100 H 2.65700 5.00000 -10.31400 C 2.53100 3.36000 -8.92900 H 2.52100 2.26900 -8.96300 H 3.36100 3.68100 -8.30000 S 0.96900 3.97400 -8.23000 H 0.83600 3.07800 -7.24600 C 4.10600 3.55500 -10.94500 O 4.70600 2.53300 -10.61100 N 4.62800 4.37900 -11.85400 H 4.10700 5.22500 -12.08500 C 5.85900 4.06100 -12.61300 H 5.74500 3.05800 -13.02300 C 6.04000 5.01100 -13.81000 H 6.80300 4.60200 -14.47400 H 5.10100 5.05900 -14.36100 O 6.41500 6.32800 -13.43300 FCUP 63 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 7.38600 6.34800 -13.25400 C 7.16100 4.00400 -11.79400 O 8.13600 3.40600 -12.25600 H 7.21800 4.46000 -10.87000 N 7.18300 2.32100 -8.92100 H 6.41600 2.66500 -9.48900 C 7.06000 0.96000 -8.37300 H 6.22500 0.46100 -8.86000 H 7.97600 0.40500 -8.58000 C 6.79500 0.88700 -6.86800 O 6.31100 1.83800 -6.26200 H 7.34400 2.97300 -8.15300 N 7.08700 -0.27300 -6.27800 H 7.48900 -0.99600 -6.85000 C 6.91000 -0.56200 -4.84400 H 5.94100 -0.18100 -4.52500 C 6.92400 -2.08600 -4.61300 H 7.83200 -2.49900 -5.05500 H 6.94300 -2.28400 -3.54000 C 5.69400 -2.78800 -5.21600 H 4.81100 -2.50600 -4.64300 H 5.55400 -2.45800 -6.24500 C 5.81100 -4.31400 -5.21600 O 6.56100 -4.89900 -4.40700 O 5.14300 -4.97500 -6.04400 C 7.97500 0.11800 -3.96000 O 9.13900 0.24600 -4.34800 H 7.70900 0.47200 -3.02800 N 5.20100 -3.05400 1.15300 H 5.29800 -3.53300 2.05100 C 4.70300 -3.85800 0.02300 H 5.43700 -3.81300 -0.78300 C 4.54500 -5.33100 0.44400 H 3.72700 -5.42700 1.16100 H 4.26900 -5.89400 -0.44700 C 5.80800 -5.99200 1.01300 H 5.81600 -7.02800 0.67400 H 6.69600 -5.50600 0.60900 C 5.85500 -5.98900 2.54300 O 5.66100 -4.91600 3.15200 O 6.07900 -7.08000 3.12700 C 3.35800 -3.37800 -0.56200 O 3.03200 -3.71400 -1.69800 H 4.59700 -2.24100 1.27400 N 2.56100 -2.61400 0.19400 H 2.89100 -2.36700 1.11800 C 1.27200 -2.04600 -0.24600 H 0.95000 -2.54700 -1.16100 FCUP 70 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases N 2.42300 2.53300 -8.83600 H 2.31700 1.53700 -8.94700 C 3.47300 2.97800 -7.91000 H 3.50600 4.06700 -7.88700 C 3.09200 2.47600 -6.50600 H 2.93500 1.39700 -6.53100 H 3.91100 2.68300 -5.81500 S 1.59000 3.31600 -5.92100 H 1.33400 2.52400 -4.87400 C 4.87900 2.49900 -8.34800 O 5.44400 1.55400 -7.80300 N 5.46500 3.14100 -9.35500 H 4.92200 3.86600 -9.82000 C 6.77400 2.77800 -9.94800 H 6.74000 1.72000 -10.20900 C 7.00200 3.54900 -11.26200 H 7.86700 3.12900 -11.77900 H 6.13200 3.41400 -11.90000 O 7.20100 4.94200 -11.07200 H 8.09400 5.08800 -10.68800 C 8.01100 2.92400 -9.03400 O 9.12800 2.64200 -9.47300 H 7.90300 3.25500 -8.06300 N 7.75700 1.19700 -6.00400 H 7.02800 1.48600 -6.65000 C 7.60800 -0.12200 -5.36300 H 6.86500 -0.70200 -5.90900 H 8.56100 -0.64900 -5.40400 C 7.15500 -0.08400 -3.89900 O 6.59300 0.90000 -3.43300 H 7.85600 1.90700 -5.27900 N 7.40000 -1.17100 -3.16800 H 7.92300 -1.91800 -3.61300 C 7.09300 -1.30400 -1.73100 H 6.10800 -0.88700 -1.52600 C 7.06900 -2.79500 -1.33800 H 8.04400 -3.21400 -1.58400 H 6.92700 -2.87900 -0.26000 C 5.95900 -3.60700 -2.04300 H 4.99100 -3.30300 -1.64200 H 5.96700 -3.38900 -3.11100 C 6.12700 -5.12200 -1.88000 O 7.27800 -5.60100 -1.77100 O 5.12800 -5.87400 -1.91300 C 8.11300 -0.53800 -0.86200 O 9.31700 -0.56600 -1.13700 H 7.78700 0.00200 -0.04500 N 5.21000 -3.19600 3.83500 FCUP 71 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 5.24600 -3.65200 4.74700 C 4.72000 -4.01300 2.71500 H 5.43200 -3.95000 1.89100 C 4.61700 -5.48300 3.14800 H 3.87700 -5.59300 3.94000 H 4.26300 -6.05100 2.28700 C 5.95000 -6.10100 3.58400 H 5.91700 -7.15900 3.31800 H 6.76700 -5.64300 3.02400 C 6.24400 -6.00700 5.08300 O 5.83900 -5.03100 5.75700 O 6.92300 -6.93100 5.58900 C 3.34800 -3.58800 2.16300 O 3.05300 -3.86400 1.00600 H 4.64000 -2.35300 3.90600 N 2.49900 -2.92600 2.95500 H 2.80100 -2.70900 3.89600 C 1.20300 -2.37900 2.50200 H 0.88000 -2.90900 1.60400 C 0.15400 -2.63800 3.59900 H 0.50000 -2.21400 4.54200 H -0.78200 -2.14900 3.33100 C -0.15000 -4.12300 3.79400 O 0.07700 -4.96400 2.93800 N -0.65400 -4.50900 4.94600 H -0.78700 -3.85000 5.70300 H -0.87900 -5.48500 5.08800 C 1.29500 -0.88500 2.10200 O 0.29600 -0.25200 1.73900 H 2.20900 -0.40800 2.13900 N 3.28200 1.82000 -0.44800 H 2.40500 2.31500 -0.33500 C 3.93000 1.91800 -1.75700 H 4.76300 1.21800 -1.81500 C 2.88400 1.55000 -2.82300 H 2.00800 2.18200 -2.67000 H 3.28500 1.77500 -3.80900 C 2.45000 0.06900 -2.80200 H 2.27400 -0.25800 -1.77600 C 1.13900 -0.07100 -3.57100 H 0.80300 -1.10700 -3.52900 H 0.38000 0.55700 -3.11100 H 1.27900 0.23700 -4.60500 C 3.50900 -0.83800 -3.44300 H 3.15700 -1.86800 -3.45700 H 3.71600 -0.51700 -4.46500 H 4.42400 -0.79900 -2.86300 C 4.51000 3.31900 -1.99900 FCUP 72 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases O 4.12800 4.28500 -1.33900 H 3.93500 2.13400 0.27000 H 5.23100 3.45300 -2.72600 N -2.17800 1.18600 0.61600 H -1.64500 0.89300 1.42500 C -2.76700 0.08400 -0.17600 H -3.69200 0.41700 -0.64800 C -1.78700 -0.31600 -1.29100 H -2.28000 -0.99700 -1.98400 H -1.50500 0.58100 -1.84100 O -0.61600 -0.94700 -0.79200 H -0.41200 -0.60400 0.09400 C -3.12500 -1.15100 0.68300 O -2.86200 -1.16400 1.88600 H -2.91900 1.82100 0.91200 N -3.74200 -2.17200 0.07500 H -3.94500 -2.07000 -0.91600 C -4.19000 -3.43100 0.69800 H -4.72000 -3.97800 -0.08300 C -3.00100 -4.32700 1.11100 H -2.28800 -4.36900 0.28700 H -2.49200 -3.88200 1.96700 C -3.42000 -5.76500 1.46900 O -4.63500 -6.09000 1.42900 O -2.53900 -6.57600 1.83900 C -5.22100 -3.21400 1.82700 O -4.95300 -3.42900 3.01200 H -6.16600 -2.87800 1.58200 C 5.33500 -4.29300 -5.80300 C 4.03100 -4.57100 -5.37900 C 2.95600 -3.77600 -5.79200 C 3.22600 -2.67700 -6.62000 C 4.50000 -2.44400 -7.04300 C 5.58400 -3.22100 -6.66200 C 4.43200 -1.33500 -7.91500 N 2.44300 -1.70500 -7.19100 N 3.17000 -0.87400 -8.01700 C 1.10500 -1.50300 -6.87200 S 0.35300 -0.05200 -7.41100 C -1.00500 -0.51200 -6.44900 C -0.83000 -1.70400 -5.77300 N 0.40600 -2.28400 -6.04400 C -1.89300 -2.19700 -4.86400 N -1.61100 -3.43100 -4.40000 C -2.39600 -4.28100 -3.55900 C -3.79200 -4.26100 -3.62300 C -4.55200 -5.10100 -2.81300 C -3.92000 -6.02800 -1.99200 FCUP 73 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C -2.53400 -6.09700 -1.95800 C -1.76200 -5.21400 -2.72600 N -0.39100 -5.25800 -2.65100 C 0.27900 -6.55000 -2.64300 C 0.28500 -4.27100 -1.82100 C 1.75800 -4.16100 -2.17700 N 2.32100 -5.50000 -2.04200 C 1.74300 -6.42000 -3.00900 O -2.90500 -1.59500 -4.54600 H 6.15300 -4.93200 -5.47700 H 3.86000 -5.42300 -4.72600 H 1.95900 -4.03900 -5.46000 H 6.58600 -3.01500 -7.03300 H 5.23300 -0.85100 -8.46200 H -1.85100 0.16400 -6.46300 H -0.72000 -3.81700 -4.66700 H -4.29400 -3.56800 -4.29000 H -5.63700 -5.02400 -2.81900 H -4.49500 -6.68900 -1.35400 H -2.05600 -6.81800 -1.29400 H -0.20500 -7.20900 -3.36000 H 0.21300 -6.99100 -1.64900 H 0.20100 -4.56500 -0.77500 H -0.17900 -3.29200 -1.94000 H 2.25700 -3.47500 -1.49200 H 1.87500 -3.80100 -3.19900 H 3.34200 -5.46000 -2.17700 H 2.23500 -7.39000 -2.92700 H 1.84900 -6.03700 -4.02500 FCUP 74 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Appendix 2 Modelling of Potential Chk1 Inhibitors Aiming for Cancer Treatment Pedro M.M. Araújo, Luís Pinto da Silva and Joaquim C.G. Esteves da Silva* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. *Corresponding Author: Joaquim C.G. Esteves da Silva; Email: [email protected]; Tel.: +351 226082869; Fax: +351 22608259; Table 1: Cartesian coordinates of Checkpoint kinase 1 complexed with MD8. Table2: Cartesian coordinates of Checkpoint kinase 1 complexed with MD9. FCUP 75 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Table 1: Cartesian coordinates of Checkpoint kinase 1 complexed with MD9 N 2.54900 -8.13900 -11.70000 H 1.56400 -8.36100 -11.62000 C 3.11600 -7.24900 -10.67900 H 4.18300 -7.10300 -10.85900 C 2.38300 -5.89000 -10.77500 H 1.34600 -6.04600 -10.47200 H 2.83200 -5.20300 -10.05700 C 2.38400 -5.21400 -12.16600 H 1.98200 -5.90400 -12.90800 C 1.46500 -3.98700 -12.14400 H 1.36800 -3.58800 -13.15100 H 0.47300 -4.27200 -11.80000 H 1.86900 -3.22100 -11.48400 C 3.79300 -4.79400 -12.60600 H 3.74300 -4.29900 -13.57700 H 4.22500 -4.10700 -11.87800 H 4.43400 -5.67000 -12.69800 C 2.98700 -7.82600 -9.25000 O 3.55700 -7.26600 -8.31600 H 2.72500 -7.74000 -12.62200 N 2.24300 -8.92800 -9.07300 H 1.82900 -9.33700 -9.90100 C 1.95400 -9.57400 -7.78000 H 2.77500 -10.2490 -7.53900 H 1.90500 -8.82800 -6.98500 C 0.65100 -10.3800 -7.77400 O -0.3150 -10.009 -8.44500 N 0.61700 -11.4720 -7.00500 H 1.41700 -11.6840 -6.43000 C -0.4890 -12.443 -6.97900 H -1.4170 -11.874 -6.95400 C -0.4990 -13.280 -8.27400 H -1.4220 -13.860 -8.31800 H -0.4980 -12.605 -9.12700 C 0.69300 -14.2380 -8.41500 H 1.62200 -13.7060 -8.20800 H 0.59400 -15.0590 -7.70400 C 0.74700 -14.7980 -9.83200 O -0.2060 -15.487 -10.26600 O 1.69500 -14.4940 -10.58300 C -0.5060 -13.355 -5.73600 O 0.48400 -13.4730 -5.00500 H -1.3650 -13.880 -5.50900 N -5.1370 -12.531 -2.73300 H -4.1840 -12.586 -3.05900 C -5.9310 -11.357 -3.16500 FCUP 76 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -6.8710 -11.342 -2.61200 C -5.1610 -10.076 -2.78000 H -5.7230 -9.2100 -3.12900 H -5.1510 -10.016 -1.69100 C -3.7290 -9.9410 -3.28400 C -3.4470 -9.8210 -4.66300 H -4.2480 -9.8300 -5.38600 C -2.1200 -9.6590 -5.10800 H -1.9050 -9.5590 -6.16100 C -1.0640 -9.5950 -4.17400 O 0.21700 -9.40800 -4.59500 H 0.81800 -9.29800 -3.85400 C -1.3430 -9.6970 -2.79400 H -0.5420 -9.6210 -2.07200 C -2.6690 -9.8740 -2.35600 H -2.8760 -9.9300 -1.29600 C -6.3400 -11.345 -4.66300 O -6.8740 -10.345 -5.14600 H -5.6290 -13.382 -3.00500 H -6.1650 -12.173 -5.25300 N -2.4970 -8.7700 -9.73700 H -1.6550 -9.2420 -9.40300 C -2.3460 -7.4240 -10.33500 H -3.3210 -7.0990 -10.68700 C -1.8480 -6.3670 -9.31700 H -0.7900 -6.5400 -9.11600 C -2.0160 -4.9440 -9.88300 H -1.6260 -4.2150 -9.17300 H -1.4710 -4.8370 -10.81700 H -3.0710 -4.7340 -10.06600 C -2.6050 -6.4320 -7.97900 H -2.2490 -5.6400 -7.32200 H -3.6760 -6.3050 -8.14400 H -2.4260 -7.3860 -7.48300 C -1.3870 -7.4770 -11.53500 O -0.3060 -8.0490 -11.43500 H -2.9690 -9.3760 -10.40800 H -1.6530 -7.0310 -12.42700 N -2.6570 0.16300 -14.42700 H -3.1200 0.94300 -13.96200 C -2.6320 -1.1140 -13.70000 H -1.9120 -1.7910 -14.16200 C -2.1680 -0.8290 -12.26400 H -2.1640 -1.7480 -11.67600 H -1.1620 -0.4110 -12.26600 H -2.8350 -0.1060 -11.79200 C -4.0090 -1.8170 -13.74200 O -5.0280 -1.1640 -13.95300 FCUP 77 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -1.7010 0.43300 -14.65700 H -4.0720 -2.8360 -13.59300 N -6.1560 -5.0020 -11.45100 H -6.8050 -4.2180 -11.44500 C -6.4300 -6.1310 -10.53800 H -5.5050 -6.6880 -10.39100 C -6.8540 -5.5800 -9.15800 H -6.1000 -4.8590 -8.83800 H -7.8010 -5.0510 -9.25400 C -6.9850 -6.6540 -8.05900 H -7.7510 -7.3790 -8.33800 H -6.0320 -7.1740 -7.95500 C -7.3630 -6.0170 -6.70800 H -6.6660 -5.2050 -6.49700 H -8.3680 -5.5940 -6.77400 C -7.3160 -7.0240 -5.54600 H -8.0650 -7.8050 -5.70900 H -6.3310 -7.4990 -5.52900 N -7.5590 -6.3470 -4.24500 H -6.8980 -5.5910 -4.09800 H -8.4830 -5.9120 -4.21500 H -7.4770 -6.9660 -3.44500 C -7.4560 -7.1070 -11.16100 O -8.5060 -6.6960 -11.65600 H -5.2160 -4.6520 -11.26800 H -7.2570 -8.1200 -11.16200 N -14.660 -3.848 -2.36300 H -14.627 -4.847 -2.51100 C -13.499 -3.052 -2.79400 H -13.159 -2.452 -1.94800 C -12.347 -3.994 -3.18700 H -12.124 -4.638 -2.33400 H -12.662 -4.620 -4.02200 C -11.070 -3.239 -3.58100 H -11.246 -2.699 -4.51100 H -10.827 -2.521 -2.79800 C -9.8800 -4.1750 -3.79200 O -10.097 -5.334 -4.20100 O -8.7220 -3.7500 -3.57000 C -13.858 -2.077 -3.93200 O -13.495 -0.907 -3.85900 H -15.498 -3.477 -2.81100 H -14.399 -2.405 -4.74800 N -4.0070 5.56100 -4.60800 H -3.7670 4.90100 -3.85700 C -4.2840 5.00100 -5.95600 H -3.6460 5.53100 -6.66600 C -3.9320 3.48900 -6.03500 FCUP 78 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -4.3520 2.99500 -5.15800 C -4.4630 2.75300 -7.28100 H -4.1990 1.69700 -7.22200 H -5.5470 2.82000 -7.34000 H -4.0270 3.17000 -8.18500 C -2.4040 3.31600 -6.00600 H -2.1510 2.26000 -6.09000 H -1.9490 3.85100 -6.84200 H -1.9940 3.70100 -5.07400 C -5.7470 5.23500 -6.37500 O -6.6770 4.90100 -5.64200 H -4.8080 6.11900 -4.31200 H -5.9510 5.67900 -7.28400 N -6.7130 0.64000 -12.59500 H -6.0880 0.12100 -13.20400 C -6.0720 1.34800 -11.46700 H -6.8030 1.99200 -10.97800 C -5.5870 0.27400 -10.46200 H -4.9680 -0.4360 -11.01200 H -4.9490 0.74300 -9.71000 C -6.6920 -0.5200 -9.72800 H -7.4420 -0.8600 -10.43800 C -6.0820 -1.7700 -9.08300 H -6.8500 -2.3210 -8.54000 H -5.6650 -2.4180 -9.85500 H -5.2900 -1.4810 -8.39500 C -7.3860 0.33100 -8.65600 H -8.1680 -0.2520 -8.16900 H -6.6630 0.65100 -7.90500 H -7.8410 1.20900 -9.10900 C -4.8930 2.26400 -11.88400 O -4.2840 2.08400 -12.94000 H -7.4270 0.00800 -12.23100 N -4.5550 3.23700 -11.02900 H -5.1380 3.34200 -10.20700 C -3.4630 4.21300 -11.22500 H -3.5670 4.62400 -12.22800 C -3.6300 5.39000 -10.23800 H -4.6470 5.76300 -10.34700 H -3.5150 5.04300 -9.21300 C -2.6500 6.55500 -10.46400 H -1.6870 6.31200 -10.01300 H -2.4960 6.70000 -11.53300 C -3.1900 7.86100 -9.87300 O -4.1580 8.41500 -10.44300 O -2.6750 8.34700 -8.83400 C -2.0560 3.58700 -11.14500 O -1.5330 3.30800 -10.06700 FCUP 79 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases N -1.4150 3.39300 -12.30400 H -1.8970 3.62100 -13.15900 C -0.0580 2.83600 -12.39000 H -0.0560 1.91000 -11.81300 C 0.27600 2.47800 -13.84600 H -0.5830 0 1.98200 -14.29200 H 0.45800 3.39100 -14.41500 C 1.47600 1.55500 -13.97200 C 1.28100 0.16200 -14.00500 H 0.28100 -0.24200 -13.99400 C 2.38500 -0.70800 -14.04100 H 2.24400 -1.77400 -14.05800 C 3.69400 -0.18900 -14.06700 O 4.75300 -1.04100 -14.09400 H 5.59000 -0.58200 -13.97300 C 3.89500 1.20800 -14.04200 H 4.89800 1.60400 -14.05000 C 2.78500 2.07700 -13.99800 H 2.93700 3.14700 -13.96600 C 1.02600 3.75700 -11.79200 O 1.06500 4.95800 -12.05600 N 1.94900 3.16000 -11.03300 H 1.84000 2.16400 -10.89100 C 3.00900 3.83000 -10.27200 H 2.89200 4.91600 -10.32300 C 2.80600 3.38700 -8.81300 H 2.80300 2.29600 -8.75600 H 3.62800 3.75500 -8.20100 S 1.24300 4.04800 -8.15400 H 0.38900 3.57700 -9.08300 C 4.41800 3.49000 -10.81900 O 4.95900 2.41300 -10.57000 N 5.02500 4.39100 -11.59400 H 4.53300 5.26900 -11.76000 C 6.28400 4.13900 -12.34100 H 6.17200 3.20000 -12.88000 C 6.52900 5.23800 -13.38800 H 7.40600 4.97900 -13.98400 H 5.66700 5.27800 -14.05700 O 6.71900 6.52500 -12.81600 H 7.56900 6.56200 -12.31600 C 7.57200 3.95700 -11.51900 O 8.56400 3.46200 -12.06100 N 7.56600 4.31500 -10.23100 H 6.71500 4.73200 -9.86200 C 8.65000 4.04700 -9.27300 H 9.60900 4.02600 -9.79200 H 8.65800 4.83500 -8.52200 FCUP 86 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -2.81400 0.85700 -14.06800 C -2.27000 -1.1930 -13.83600 H -1.54800 -1.8530 -14.31800 C -1.79500 -0.9310 -12.40100 H -1.77700 -1.8600 -11.83000 H -0.79200 -0.5050 -12.40700 H -2.46200 -0.2220 -11.90900 C -3.63800 -1.9100 -13.87800 O -4.65900 -1.2740 -14.12700 H -1.36200 0.40500 -14.73300 H -3.69400 -2.9240 -13.69700 N -5.79400 -5.1250 -11.51600 H -6.47200 -4.3660 -11.53100 C -6.03200 -6.2520 -10.58400 H -5.10400 -6.8140 -10.46800 C -6.40600 -5.6850 -9.19500 H -5.63600 -4.9690 -8.90500 H -7.35200 -5.1490 -9.26700 C -6.51400 -6.7490 -8.08200 H -7.31100 -7.4530 -8.32300 H -5.57100 -7.2940 -8.02000 C -6.81100 -6.1000 -6.71600 H -6.05300 -5.3430 -6.51400 H -7.78700 -5.6100 -6.74900 C -6.80400 -7.1310 -5.57400 H -7.61200 -7.8520 -5.73200 H -5.85800 -7.6790 -5.59900 N -6.96300 -6.4770 -4.24800 H -6.25600 -5.7680 -4.09300 H -7.86800 -6.0090 -4.16200 H -6.90200 -7.1260 -3.46900 C -7.08800 -7.2350 -11.14700 O -8.15600 -6.8280 -11.60200 H -4.87000 -4.7360 -11.33100 H -6.89200 -8.2480 -11.14000 N -14.2910 -4.285 -2.47700 H -14.2360 -5.290 -2.57300 C -13.1270 -3.486 -2.89600 H -12.8560 -2.812 -2.08100 C -11.9270 -4.412 -3.15100 H -11.7220 -4.972 -2.23800 H -12.1910 -5.115 -3.93900 C -10.6590 -3.647 -3.56000 H -10.8160 -3.195 -4.53900 H -10.4730 -2.856 -2.83500 C -9.42600 -4.5460 -3.65000 O -9.58000 -5.7480 -3.94300 O -8.29000 -4.0480 -3.47500 FCUP 87 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C -13.4500 -2.617 -4.12300 O -13.1020 -1.440 -4.13800 H -15.1150 -3.952 -2.97700 H -13.9550 -3.020 -4.92700 N -3.72700 5.20400 -4.82400 H -3.49900 4.54000 -4.07200 C -3.98800 4.65800 -6.18000 H -3.34800 5.20000 -6.88000 C -3.62100 3.15100 -6.26800 H -4.07000 2.64000 -5.41600 C -4.10500 2.44600 -7.54800 H -3.82800 1.39200 -7.51300 H -5.18800 2.50000 -7.63400 H -3.64900 2.89900 -8.42500 C -2.09500 2.97900 -6.18500 H -1.84000 1.92300 -6.26500 H -1.61000 3.52000 -6.99900 H -1.71800 3.35600 -5.23500 C -5.45200 4.88400 -6.60200 O -6.37900 4.46200 -5.91100 H -4.52900 5.76400 -4.53600 H -5.65800 5.39500 -7.47400 N -6.38200 0.49000 -12.80100 H -5.74300 -0.0200 -13.40400 C -5.76200 1.19500 -11.66100 H -6.50700 1.82400 -11.17900 C -5.27100 0.12300 -10.65700 H -4.62800 -0.5690 -11.20100 H -4.65800 0.59900 -9.89100 C -6.37000 -0.7000 -9.94900 H -7.08200 -1.0730 -10.68000 C -5.73800 -1.9170 -9.26200 H -6.50900 -2.4990 -8.75600 H -5.25100 -2.5490 -10.00500 H -4.99900 -1.5870 -8.53400 C -7.13500 0.13200 -8.91200 H -7.90900 -0.4780 -8.44500 H -6.45200 0.49000 -8.14200 H -7.60900 0.98400 -9.39300 C -4.59100 2.13100 -12.05800 O -3.98400 1.98000 -13.11900 H -7.09500 -0.1500 -12.45100 N -4.25900 3.09000 -11.18700 H -4.83100 3.16600 -10.35400 C -3.18500 4.08700 -11.38000 H -3.30500 4.50500 -12.37800 C -3.36600 5.24900 -10.38000 H -4.39300 5.59800 -10.47000 FCUP 88 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -3.22600 4.89500 -9.36000 C -2.42100 6.43900 -10.61900 H -1.44500 6.22100 -10.18300 H -2.28700 6.58800 -11.69100 C -2.98600 7.73100 -10.02000 O -3.93300 8.29600 -10.61400 O -2.50600 8.19500 -8.95500 C -1.76600 3.49000 -11.31000 O -1.26900 3.12600 -10.24600 N -1.08600 3.41900 -12.46000 H -1.54100 3.72700 -13.30400 C 0.27400 2.87100 -12.56000 H 0.26500 1.91000 -12.04400 C 0.62000 2.60500 -14.03200 H -0.24500 2.16100 -14.51800 H 0.83000 3.55000 -14.53500 C 1.78900 1.65600 -14.21100 C 1.55200 0.26900 -14.24800 H 0.54300 -0.10500 -14.19200 C 2.62700 -0.63200 -14.34300 H 2.44900 -1.69400 -14.36100 C 3.94700 -0.14800 -14.42900 O 4.98100 -1.02600 -14.51600 H 5.83300 -0.58500 -14.44600 C 4.19000 1.24400 -14.40000 H 5.20300 1.61200 -14.45500 C 3.11000 2.14400 -14.29000 H 3.29200 3.20900 -14.25100 C 1.35700 3.74200 -11.88700 O 1.34300 4.96900 -11.97900 N 2.33100 3.07800 -11.25600 H 2.24200 2.07000 -11.23500 C 3.39400 3.67100 -10.43500 H 3.33200 4.76200 -10.45300 C 3.12800 3.19000 -8.99800 H 3.09600 2.09900 -8.97500 H 3.94100 3.51800 -8.35200 S 1.56400 3.86400 -8.35400 H 0.71500 3.40900 -9.29500 C 4.80800 3.28100 -10.93700 O 5.33300 2.22000 -10.60500 N 5.43900 4.12100 -11.76000 H 4.95900 4.99000 -11.99000 C 6.71400 3.81800 -12.46400 H 6.59400 2.87000 -12.98600 C 7.00800 4.89200 -13.52600 H 7.93200 4.64100 -14.04900 H 6.19600 4.88500 -14.25400 FCUP 89 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases O 7.11600 6.20500 -12.98900 H 7.92700 6.28800 -12.43400 C 7.97700 3.61700 -11.60500 O 8.97200 3.08500 -12.11000 N 7.94500 3.99600 -10.32200 H 7.09600 4.44000 -9.98300 C 9.00300 3.72500 -9.33800 H 9.97200 3.69100 -9.83500 H 9.00200 4.52100 -8.59400 C 8.82700 2.40100 -8.58600 O 9.69100 2.03900 -7.78800 N 7.72600 1.67400 -8.81900 H 7.04400 2.05600 -9.46800 C 7.43500 0.36900 -8.20500 H 6.58500 -0.08500 -8.71200 H 8.30600 -0.27700 -8.31000 C 7.07900 0.45500 -6.72000 O 6.41700 1.39400 -6.29000 N 7.48800 -0.55800 -5.95400 H 8.06600 -1.26100 -6.38300 C 7.25000 -0.68200 -4.50500 H 6.29300 -0.22500 -4.25800 C 7.18400 -2.17100 -4.11000 H 8.05900 -2.68200 -4.51500 H 7.21200 -2.25700 -3.02200 C 5.90500 -2.85700 -4.61500 H 5.05700 -2.50600 -4.02700 H 5.73400 -2.58700 -5.65900 C 5.97900 -4.38100 -4.52100 O 6.54800 -4.93900 -3.55900 O 5.46800 -5.07300 -5.43400 C 8.32000 0.03400 -3.66200 O 9.50000 0.07600 -4.02000 H 8.04400 0.48900 -2.77800 N 5.52500 -3.09600 0.97700 H 5.60300 -3.57300 1.87800 C 5.01900 -3.89600 -0.15200 H 5.72300 -3.80700 -0.98200 C 4.93400 -5.38300 0.23900 H 4.10500 -5.53500 0.93200 H 4.71100 -5.94500 -0.66800 C 6.21500 -5.98100 0.84000 H 6.30200 -7.00600 0.47900 H 7.08800 -5.43100 0.48600 C 6.19500 -6.00700 2.37100 O 5.97100 -4.94600 2.98900 O 6.39100 -7.10700 2.94700 C 3.63600 -3.45700 -0.68100 FCUP 90 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases O 3.25300 -3.84700 -1.78000 H 4.93800 -2.27000 1.08800 N 2.86500 -2.67300 0.08300 H 3.23500 -2.39200 0.98200 C 1.56900 -2.09700 -0.33000 H 1.22700 -2.58400 -1.24500 C 0.53000 -2.38100 0.77400 H 0.91000 -2.01900 1.73000 H -0.38800 -1.8360 0 0.55500 C 0.14600 -3.85400 0.91400 O 0.32400 -4.68300 0.03000 N -0.40400 -4.2350 0 2.04700 H -0.54800 -3.5750 0 2.80200 H -0.74000 -5.1830 0 2.13800 C 1.66000 -0.58100 -0.66100 O 0.65000 0.06200 -0.97100 N 2.86600 0.00200 -0.59500 H 3.65400 -0.59700 -0.38300 C 3.16100 1.41600 -0.87100 H 2.23300 1.96400 -1.04200 C 3.88300 2.01800 0.35300 H 4.77500 1.41900 0.54900 H 4.21200 3.02900 0.10800 C 3.02700 2.07300 1.63900 H 2.61800 1.08700 1.84800 C 3.90100 2.48100 2.83400 H 3.29700 2.51100 3.74200 H 4.69900 1.75200 2.97400 H 4.33900 3.46400 2.66300 C 1.84700 3.04300 1.50200 H 1.31300 3.11200 2.45000 H 2.20500 4.03000 1.21500 H 1.15200 2.67600 0.74900 C 3.99400 1.55600 -2.15800 O 5.03500 0.91900 -2.30900 N 3.51400 2.38600 -3.08600 H 2.66300 2.89000 -2.86800 C 4.02400 2.51800 -4.45800 H 4.87600 1.85500 -4.58600 C 2.90000 2.09100 -5.42100 H 2.06400 2.77500 -5.29300 H 3.26000 2.19200 -6.44300 C 2.37100 0.65600 -5.24000 H 2.07800 0.49000 -4.20300 C 1.12700 0.48000 -6.10900 H 0.71900 -0.51900 -5.95900 H 0.37100 1.20900 -5.82100 H 1.38300 0.63000 -7.15700 FCUP 91 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 3.42600 -0.38300 -5.63400 H 3.00200 -1.38200 -5.56600 H 3.77200 -0.20600 -6.65300 H 4.26600 -0.32200 -4.95000 C 4.52800 3.94000 -4.77400 O 4.30800 4.85500 -3.98300 H 5.04800 4.12300 -5.64600 N -2.01300 1.58300 -1.78600 H -1.43100 1.25400 -1.02800 C -2.68500 0.52600 -2.57000 H -3.59800 0.92500 -3.01600 C -1.77100 0.06700 -3.71800 H -2.35500 -0.5270 -4.41900 H -1.40200 0.94800 -4.24200 O -0.66400 -0.7080 -3.28000 H -0.32800 -0.3430 -2.44400 C -3.11500 -0.6860 -1.71300 O -2.66200 -0.8280 -0.57600 H -2.71400 2.22500 -1.41700 N -4.01400 -1.5120 -2.26400 H -4.32500 -1.2640 -3.19900 C -4.77100 -2.6170 -1.64400 H -5.22400 -3.1770 -2.46400 C -3.88500 -3.6200 -0.87000 H -2.99500 -3.8290 -1.46700 H -3.56300 -3.1830 0.07600 C -4.59600 -4.9550 -0.57600 O -5.85000 -5.0000 -0.51300 O -3.89700 -5.9870 -0.45400 C -5.92600 -2.0850 -0.77500 O -5.70700 -1.4700 0.27000 N -7.16600 -2.3770 -1.17700 H -7.27900 -2.9120 -2.03300 C -8.38200 -2.0660 -0.41500 H -8.08700 -1.7840 0.59300 C -9.06400 -0.8370 -1.04200 H -9.19600 -0.9940 -2.11200 H -10.0510 -0.703 -0.60000 C -8.26300 0.43500 -0.81200 C -7.32100 0.87200 -1.76300 H -7.19300 0.32800 -2.68800 C -6.52300 2.00100 -1.50300 H -5.79400 2.32300 -2.23400 C -6.66600 2.69900 -0.29100 H -6.04500 3.55900 -0.08700 C -7.61100 2.27200 0.65800 H -7.71400 2.79700 1.59500 C -8.40800 1.14300 0.39700 FCUP 92 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -9.11800 0.80700 1.13700 C -9.28300 -3.3030 -0.21700 O -10.4770 -3.179 0.07200 N -8.69800 -4.5050 -0.31600 H -7.71100 -4.5120 -0.57200 C -9.32900 -5.8020 -0.02300 H -10.3790 -5.780 -0.31200 H -8.81200 -6.5760 -0.59100 C -9.25900 -6.2020 1.45600 O -10.1520 -6.902 1.93200 H -8.47200 -5.8880 2.04500 N 2.63900 -5.19800 -5.08000 C 1.81600 -6.09600 -5.90600 C 0.52500 -6.38900 -5.12800 N -0.19900 -5.1510 -4.79800 C 0.63300 -4.15800 -4.09900 C 1.96700 -3.91000 -4.82000 C -1.56400 -5.1650 -4.57900 C -2.12700 -6.1400 -3.76600 C -3.49100 -6.1590 -3.56300 N -4.31500 -5.2710 -4.14700 C -3.76800 -4.3140 -4.92500 C -2.40100 -4.2270 -5.18800 N -1.84100 -3.2810 -6.12400 C -2.36900 -2.1850 -6.72100 C -1.41500 -1.6080 -7.70300 N -1.78700 -0.5000 -8.37700 C -0.10300 -2.0880 -8.03400 C 0.46600 -1.26400 -8.95200 S -0.64700 0.01000 -9.33100 N 1.78000 -1.43700 -9.38600 C 2.42000 -0.67400 -10.31300 C 2.66900 -2.48400 -8.81500 C 3.96900 -2.22300 -9.52900 C 3.80600 -1.17700 -10.39800 C 4.84600 -0.72700 -11.19900 C 6.08600 -1.36800 -11.11400 C 6.25500 -2.44000 -10.21500 C 5.19100 -2.87500 -9.41100 C 7.21800 -0.90600 -11.98200 O 7.13100 -0.01500 -12.80900 O -3.48200 -1.7670 0 -6.45400 O 1.95300 0.24500 -10.95700 H 3.57000 -5.03300 -5.49800 H 2.35500 -7.03000 -6.08700 H 1.58300 -5.62800 -6.86800 H 0.79200 -6.89600 -4.19700 H -0.11100 -7.0540 -5.71600 FCUP 93 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 0.85100 -4.52400 -3.09300 H 0.09700 -3.20900 -4.00000 H 1.78000 -3.39500 -5.76600 H 2.60500 -3.27700 -4.19900 H -1.50700 -6.8750 -3.26600 H -3.95900 -6.9130 -2.93400 H -4.48700 -3.6350 -5.37700 H -0.89100 -3.4960 -6.38100 H 0.34000 -2.96300 -7.57200 H 2.27800 -3.48000 -9.04200 H 2.77200 -2.35000 -7.73500 H 4.69500 0.11700 -11.86600 H 7.21600 -2.94700 -10.14700 H 5.31300 -3.70600 -8.71700 H 8.17700 -1.45100 -11.83800 FCUP 94 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Appendix 3 Theoretical Analysis of the Binding of Potential Inhibitors to Protein Kinases MK2 and MK3 Pedro M.M. Araújo, Luís Pinto da Silva and Joaquim C.G. Esteves da Silva* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal. *Corresponding Author: Joaquim C.G. Esteves da Silva; Email: [email protected]; Tel.: +351 226082869; Fax: +351 22608259; Figure 1: Three dimensional representation of ligand 05B bond to MK2. Yellow for 05B; blue for MK2 residues with RMSD values inferior to 3Å; red for MK2 residues with RMSD values superior to 3Å. FCUP 95 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Figure 2: Three dimensional representation of ligand P4O bond to MK2. Yellow for P4O; blue for MK2 residues with RMSD values inferior to 3Å; red for MK2 residues with RMSD values superior to 3Å. Figure 3: Three dimensional representation of ligand 05B bond to MK3. Yellow for 05B; blue for MK3 residues with RMSD values inferior to 3Å; red for MK3 residues with RMSD values superior to 3Å. FCUP 102 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases N -0.4440 1.7770 -5.97100 H -0.9770 2.5720 -6.29900 C 0.77500 1.46400 -6.74200 H 0.84900 2.25400 -7.49000 C 2.05900 1.55800 -5.87700 H 1.94900 2.38700 -5.17700 H 2.17800 0.64700 -5.28900 C 3.33700 1.81800 -6.69700 O 4.44700 1.41900 -6.27300 O 3.27000 2.52100 -7.73500 C 0.64900 0.15200 -7.54300 O 1.27700 -0.8610 -7.23400 H 0.02100 0.12200 -8.36100 O -0.9980 3.1230 -9.72300 H -0.3270 3.2360 -10.44400 H -1.8170 3.2660 -10.23100 O -1.3800 4.1450 -7.24300 H -0.4920 4.5690 -7.25400 H -1.3880 3.7780 -8.16200 C 2.20500 6.61400 -4.09800 C 1.82700 5.35500 -6.26800 N 3.08500 4.90900 -6.20100 C 4.06400 5.31500 -5.17300 C 3.49500 5.88700 -3.80700 C 3.17400 4.76500 -2.80000 C 4.43600 4.05400 -2.32400 N 5.37100 5.04100 -1.76600 O 1.15200 5.00000 -7.20700 C 1.43400 6.29000 -5.19200 N 1.52400 7.53300 -3.32100 C 0.31500 7.75400 -3.94100 C 0.20800 6.99600 -5.08000 C -0.6690 8.6820 -3.43600 N -0.3180 9.4460 -2.40900 C -1.2120 10.336 -1.98900 N -2.4340 10.515 -2.50100 C -2.7920 9.7290 -3.51500 C -1.9110 8.8060 -4.01200 C -0.8150 11.192 -0.80500 C -1.5160 12.400 -0.54200 C 0.25100 10.8110 0.06500 C 0.54700 11.6330 1.14000 C -1.1690 13.215 0.53900 C -0.1360 12.799 1.36400 FCUP 103 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases O 1.52300 11.4550 2.07800 O 0.36400 13.4510 2.45400 C 1.45800 12.6200 2.95200 C 5.76500 6.09100 -2.71200 C 4.51200 6.84600 -3.15600 H 3.31600 4.13600 -6.81700 H 4.65700 6.10200 -5.64700 H 4.73700 4.47300 -5.00500 H 2.50100 4.03600 -3.25200 H 2.66300 5.18300 -1.93000 H 4.91200 3.52300 -3.14900 H 4.16900 3.32700 -1.55700 H 6.21200 4.53100 -1.48500 H 1.82700 7.94800 -2.44500 H -0.6220 6.9330 -5.78100 H -3.7850 9.8600 -3.93900 H -2.2110 8.1650 -4.82900 H -2.3240 12.730 -1.19400 H 0.83300 9.90000 -0.06900 H -1.6810 14.150 0.73700 H 2.40900 13.1750 2.90400 H 1.24900 12.3030 3.98600 H 6.27000 5.64600 -3.57300 H 6.45500 6.78200 -2.22100 H 4.77200 7.63500 -3.86000 H 4.06400 7.32200 -2.28300 H 4.06400 7.32200 -2.28300 Table 2: Cartesian coordinates of MK3 bound to 05B without binding site water molecules. N 6.86500 8.62100 -5.24900 H 7.03200 8.07200 -4.41800 C 7.19600 7.98200 -6.53600 H 6.80100 8.56300 -7.36700 C 8.73200 7.88300 -6.67100 H 9.10400 7.25700 -5.85700 H 8.96300 7.36500 -7.60400 C 9.51400 9.21400 -6.64700 H 9.28900 9.75100 -5.72500 C 11.02000 8.92300 -6.66600 H 11.58100 9.85700 -6.63500 H 11.29300 8.32400 -5.79600 H 11.28400 8.37600 -7.57200 FCUP 104 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 9.15400 10.11400 -7.83700 H 9.76800 11.01600 -7.81700 H 9.32700 9.58400 -8.77500 H 8.11100 10.41100 -7.77500 C 6.56800 6.58000 -6.62800 O 6.75100 5.77700 -5.71700 H 5.88800 8.91300 -5.26400 H 5.99800 6.31700 -7.44700 N -9.49200 19.1380 -3.39600 H -10.0180 18.811 -2.59900 C -8.06200 18.7820 -3.42700 H -7.54700 19.4030 -4.16400 C -7.46700 19.0820 -2.03700 H -7.68800 20.1170 -1.77300 H -7.94700 18.4340 -1.30100 C -5.94400 18.8700 -1.97700 H -5.69900 17.8550 -2.29200 H -5.45100 19.5710 -2.65100 C -5.42000 19.0640 -0.54800 H -5.64600 20.0720 -0.19700 H -5.91300 18.3410 0.10600 C -3.90600 18.8320 -0.51300 H -3.68000 17.9240 -1.08000 H -3.39200 19.6650 -1.00300 N -3.40700 18.6670 0.87300 H -2.41900 18.4580 0.86200 H -3.53500 19.5060 1.43100 H -3.91400 17.9130 1.34300 C -7.83400 17.3050 -3.81600 O -8.42800 16.4010 -3.21600 H -9.93800 18.8020 -4.25000 H -7.18200 17.0710 -4.58100 N -0.61300 11.3490 -10.30900 H -1.55700 11.0700 -10.56600 C 0.51000 10.55000 -10.82500 H 1.39000 10.77200 -10.22500 C 0.18700 9.05600 -10.64200 H -0.34200 8.92800 -9.69800 H -0.47300 8.72600 -11.44400 C 1.43300 8.15600 -10.60100 H 1.97300 8.21800 -11.54500 H 2.09300 8.48600 -9.79900 C 1.00500 6.70400 -10.34400 H 0.38500 6.66600 -9.44500 FCUP 105 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 0.40500 6.35800 -11.18900 C 2.19700 5.75900 -10.15600 H 2.81600 5.78900 -11.05800 H 2.78800 6.09200 -9.30100 N 1.70400 4.37700 -9.94700 H 2.43700 3.69200 -9.79900 H 1.07600 4.32700 -9.15300 H 1.22300 4.07700 -10.79700 C 0.82700 10.92000 -12.29200 O -0.05200 10.8600 -13.15500 H -0.47400 12.3230 -10.57900 H 1.78000 11.21900 -12.55100 N -6.19600 11.3100 -8.46900 H -5.49500 12.0140 -8.26800 C -6.60700 10.4430 -7.34900 H -7.42100 9.78900 -7.65900 C -5.40500 9.58300 -6.91800 H -4.61700 10.2520 -6.57500 H -5.69300 8.94900 -6.07700 C -4.85000 8.67900 -8.02600 H -5.60000 7.92000 -8.25700 H -4.68000 9.26300 -8.93200 S -3.29300 7.85300 -7.60200 C -2.19000 9.29000 -7.61400 H -1.15900 8.95800 -7.49400 H -2.29700 9.81800 -8.56300 H -2.43900 9.96500 -6.79700 C -7.07900 11.2480 -6.12700 O -6.76400 12.4310 -5.98600 H -5.86800 10.7260 -9.23800 H -7.66900 10.7910 -5.41400 N -4.48200 11.3580 -0.25300 H -4.03000 11.1010 -1.12300 C -4.21600 10.4960 0.92600 H -5.17200 10.2750 1.39500 C -3.60700 9.15300 0.48000 H -2.65400 9.33400 -0.01000 H -3.40100 8.54600 1.36000 C -4.51900 8.35800 -0.46900 H -4.77200 8.98100 -1.32800 H -3.96400 7.50000 -0.84900 S -6.06200 7.75700 0.26100 C -5.39000 6.46200 1.32200 H -6.19600 5.97000 1.86600 FCUP 106 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -4.86600 5.72800 0.71200 H -4.68800 6.89100 2.03200 C -3.37100 11.1720 2.02900 O -2.29900 10.7070 2.41800 H -5.48900 11.4090 -0.40600 N -3.86500 12.2940 2.54800 H -4.78600 12.5690 2.21500 C -3.18000 13.1800 3.50800 H -2.12200 13.1670 3.24900 C -3.65000 14.6310 3.28400 H -3.08600 15.3030 3.93100 H -3.39800 14.8820 2.25400 C -5.15600 14.8650 3.50200 H -5.72600 14.0860 3.00300 H -5.37200 14.8140 4.57000 C -5.65400 16.2020 2.95000 O -6.72600 16.6530 3.42300 O -5.02800 16.7540 2.01400 C -3.22400 12.7070 4.98200 O -3.24000 13.5130 5.91300 H -3.24000 11.6960 5.18800 N 4.47400 -0.11000 2.70800 H 5.15100 -0.86200 2.55600 C 4.68200 1.14400 1.96900 H 4.91700 1.92700 2.68800 C 5.86500 1.01300 0.98700 H 5.58300 0.36200 0.16000 H 6.04900 2.00400 0.56900 C 7.18200 0.51200 1.60000 H 8.01300 0.99500 1.08300 H 7.22400 0.80300 2.64700 C 7.34600 -1.00300 1.47700 O 8.11300 -1.43800 0.59100 O 6.66300 -1.72200 2.23800 C 3.44500 1.59800 1.17600 O 3.12600 2.78400 1.15400 H 3.53800 -0.46200 2.50500 N 2.73600 0.65900 0.53900 H 3.01900 -0.30100 0.65900 C 1.60000 0.94000 -0.35100 H 1.79400 1.87700 -0.87500 C 1.51100 -0.18600 -1.40300 H 1.36700 -1.13900 -0.89400 H 0.64400 -0.01200 -2.04000 FCUP 107 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 2.71900 -0.30200 -2.33200 O 3.58200 0.56000 -2.42100 N 2.82800 -1.40900 -3.03600 H 3.59200 -1.51000 -3.69500 H 2.20000 -2.18100 -2.89000 C 0.25100 1.11900 0.39400 O -0.78500 1.30600 -0.25000 H 0.22000 1.08200 1.42500 N -2.56400 1.16900 -2.90500 H -1.79800 0.78900 -2.36400 C -2.20600 1.94200 -4.11800 H -3.08400 2.02300 -4.75800 C -1.82700 3.37100 -3.71300 H -2.52800 3.71900 -2.95300 C -0.41300 3.49900 -3.16200 H -0.25800 4.51000 -2.78700 H -0.26800 2.79700 -2.34400 H 0.31100 3.28600 -3.94800 O -1.93000 4.20600 -4.83400 H -1.39900 4.98900 -4.65800 C -1.13700 1.26700 -4.98900 O -0.56300 0.25200 -4.60300 H -3.19300 0.41000 -3.16900 N -0.92400 1.86200 -6.16300 H -1.41600 2.73900 -6.25800 C -0.11800 1.43100 -7.32500 H -0.24600 2.23100 -8.05600 C 1.39800 1.37300 -7.01400 H 1.61000 2.00100 -6.14800 H 1.67800 0.35000 -6.75400 C 2.26400 1.86000 -8.19500 O 3.23400 2.63000 -7.99100 O 1.93900 1.56700 -9.36500 C -0.63000 0.15000 -8.03000 O -1.22400 -0.7380 -7.42200 H -0.46700 0.03900 -9.04300 C 1.88200 6.28200 -3.92400 C 1.50100 5.09900 -6.15000 N 2.68500 4.50600 -5.98100 C 3.68800 4.89000 -4.96900 C 3.17400 5.54900 -3.62800 C 2.91200 4.48700 -2.53700 C 4.20600 3.86300 -2.01500 N 5.08600 4.93600 -1.54100 FCUP 108 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases O 0.87700 4.85400 -7.16100 C 1.13700 6.04200 -5.06200 N 1.25700 7.25400 -3.16900 C 0.10400 7.59500 -3.83400 C -0.01800 6.86800 -4.99400 C -0.78500 8.63500 -3.36900 N -0.38000 9.39300 -2.35300 C -1.19400 10.3820 -1.98300 N -2.39400 10.6350 -2.51800 C -2.80600 9.85400 -3.51200 C -2.00000 8.85000 -3.97300 C -0.75200 11.2690 -0.83200 C -1.45700 12.4790 -0.58200 C 0.33800 10.92500 0.02800 C 0.63500 11.77300 1.08900 C -1.11400 13.3150 0.48100 C -0.07700 12.9250 1.30600 O 1.62900 11.64000 2.02500 O 0.40100 13.59700 2.39300 C 1.43900 12.74900 2.96200 C 5.47300 5.86600 -2.60000 C 4.20600 6.56700 -3.08500 H 2.92700 3.78900 -6.65800 H 4.29300 5.63900 -5.47600 H 4.32600 4.02600 -4.77500 H 2.28900 3.69100 -2.93500 H 2.38600 4.94000 -1.69700 H 4.70600 3.29000 -2.80000 H 3.97800 3.18600 -1.19100 H 5.91500 4.51100 -1.13500 H 1.57100 7.64900 -2.28900 H -0.79200 6.92600 -5.75900 H -3.77800 10.0590 -3.95700 H -2.33900 8.22400 -4.78600 H -2.27600 12.7960 -1.22700 H 0.92400 10.01700 -0.09600 H -1.64500 14.2420 0.67200 H 2.37300 13.32000 3.08600 H 1.09800 12.35600 3.93400 H 5.95900 5.32100 -3.41000 H 6.17000 6.60700 -2.19900 H 4.45100 7.29400 -3.85900 H 3.78000 7.11000 -2.24000 FCUP 109 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases Table 3: Cartesian coordinates of MK2 bound to 05B. N 2.53000 6.02200 -22.38800 H 1.67100 5.56700 -22.65900 C 2.40000 7.30900 -21.70000 H 3.19800 7.96300 -22.05700 C 1.06800 7.98400 -22.14100 H 1.07400 9.00100 -21.74400 H 1.10800 8.09100 -23.22700 C -0.31200 7.35600 -21.79100 H -0.45800 7.36300 -20.71300 C -1.42200 8.23100 -22.39200 H -2.39900 7.85600 -22.08800 H -1.31900 9.25800 -22.03900 H -1.36100 8.22100 -23.48100 C -0.51100 5.91100 -22.27600 H -1.54600 5.60700 -22.11900 H -0.27700 5.83400 -23.33800 H 0.12100 5.23300 -21.70300 C 2.67400 7.19300 -20.18200 O 3.76400 6.77700 -19.79400 H 3.11700 6.14400 -23.21400 H 1.94800 7.46300 -19.50000 N 15.80000 -7.0290 -18.43500 H 15.20200 -7.7360 -18.03800 C 15.14800 -5.9590 -19.21100 H 15.74200 -5.7540 -20.10200 C 13.81000 -6.5600 -19.69000 H 14.04400 -7.4600 -20.26200 H 13.22900 -6.8750 -18.82300 C 12.93700 -5.6620 -20.58000 H 12.57300 -4.8160 -20.00000 H 13.52500 -5.2810 -21.41400 C 11.74700 -6.4640 -21.13900 H 12.00300 -6.8090 -22.14300 H 11.55200 -7.3390 -20.51500 C 10.47100 -5.6200 -21.18000 H 10.13800 -5.4520 -20.15300 H 10.69500 -4.6510 -21.63000 N 9.40700 -6.29600 -21.96000 H 9.27300 -7.25300 -21.62600 H 8.52500 -5.81600 -21.86200 H 9.65600 -6.29200 -22.94400 C 15.09600 -4.6030 -18.45200 O 16.08500 -3.8730 -18.46400 FCUP 110 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 16.35400 -6.6070 -17.69000 H 14.24300 -4.3200 -17.94500 N 10.96700 5.67300 -16.96100 H 11.36500 5.40200 -16.06500 C 10.08800 6.86400 -17.01700 H 9.45600 6.76900 -17.89900 C 9.14200 6.92500 -15.79000 H 8.67000 5.95200 -15.66200 H 9.72100 7.14100 -14.89400 C 8.02400 7.98200 -15.94200 H 8.47400 8.94400 -16.17800 H 7.38700 7.70000 -16.78100 C 7.14400 8.16000 -14.68600 H 6.65700 7.21300 -14.43900 H 7.76700 8.46900 -13.84500 C 6.07400 9.23300 -14.96400 H 6.56000 10.12800 -15.36000 H 5.39800 8.85000 -15.73500 N 5.26500 9.61300 -13.77500 H 4.50500 10.22500 -14.06100 H 4.79800 8.82000 -13.33800 H 5.76800 10.15000 -13.06700 C 10.91400 8.15900 -17.18200 O 11.85600 8.40700 -16.42300 H 11.72600 5.79600 -17.63100 H 10.67600 8.82400 -17.93500 N 12.86600 1.59100 -13.61500 H 12.99500 1.57700 -14.62600 C 11.71200 0.85200 -13.07200 H 11.62800 1.03600 -12.00100 C 10.41900 1.35300 -13.73900 H 10.37900 0.99500 -14.76900 H 9.56600 0.93900 -13.19900 C 10.30800 2.88400 -13.76500 H 10.54200 3.28900 -12.78200 H 11.05200 3.27000 -14.46500 S 8.69400 3.49700 -14.28900 C 7.84500 3.47100 -12.69300 H 6.80000 3.75300 -12.83200 H 7.89500 2.47400 -12.26100 H 8.31100 4.18800 -12.01800 C 11.84200 -0.6640 -13.28200 O 12.11800 -1.1150 -14.39600 H 12.81500 2.56000 -13.30200 FCUP 111 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 11.69800 -1.3070 -12.48800 N 8.05200 -5.82000 -15.70400 H 8.19800 -5.16000 -16.45100 C 6.67700 -6.32900 -15.53200 H 6.66300 -7.02700 -14.69600 C 5.75100 -5.13600 -15.18300 H 5.66100 -4.51200 -16.07000 H 4.75300 -5.51400 -14.95700 C 6.21800 -4.24800 -14.00700 H 7.23100 -3.90400 -14.20000 C 5.34700 -2.99400 -13.89700 H 5.86000 -2.26200 -13.27800 H 5.19000 -2.55700 -14.88200 H 4.39000 -3.23900 -13.44300 C 6.19500 -4.98800 -12.66000 H 6.50900 -4.30800 -11.86800 H 5.18900 -5.35200 -12.44800 H 6.88400 -5.82900 -12.67900 C 6.17800 -7.15800 -16.74200 O 5.10000 -6.92700 -17.29000 H 8.36500 -5.40400 -14.82700 N 6.96100 -8.15900 -17.15700 H 7.82700 -8.29300 -16.65400 C 6.65300 -9.11500 -18.24600 H 6.27700 -8.53800 -19.09200 C 7.95500 -9.79600 -18.70700 H 8.44300 -10.2690 -17.85200 H 7.72400 -10.5760 -19.43500 C 8.90900 -8.80200 -19.37300 O 8.80900 -8.60400 -20.60700 O 9.73700 -8.20200 -18.65700 C 5.52600 -10.1350 -17.92000 O 5.47500 -11.2360 -18.47500 H 4.80500 -9.88600 -17.22500 N -4.46000 1.17800 -16.02800 H -5.30400 1.73100 -16.18400 C -3.18300 1.85300 -16.30400 H -2.63900 1.27200 -17.05000 C -3.44700 3.26500 -16.87100 H -3.81300 3.91800 -16.07700 H -2.48800 3.66800 -17.19700 C -4.41200 3.33400 -18.07100 H -4.10700 4.17600 -18.69400 H -4.31800 2.42800 -18.67200 FCUP 118 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases N 2.04100 0.77600 6.12300 H 2.77700 1.38100 6.46100 C 0.86900 0.61900 7.00800 H 0.93800 1.42700 7.74000 C -0.46000 0.82800 6.25500 H -0.31800 1.60900 5.50500 H -0.74600 -0.08800 5.73600 C -1.60400 1.25900 7.17500 O -2.38800 2.14000 6.75500 O -1.73000 0.74100 8.31000 C 0.87300 -0.70600 7.80200 O 0.77500 -1.79500 7.23800 H 0.96000 -0.67700 8.83000 N -1.79500 3.97600 4.68600 C -2.18300 3.59500 3.32900 C -0.93000 5.60600 2.64200 C -0.32200 5.64100 3.85900 C -0.82800 4.85100 4.99200 C -2.15400 4.80200 2.39200 O -0.47200 4.92100 6.15000 C 0.74300 6.59300 3.79100 C 0.67200 7.14400 2.53200 N -0.35700 6.54000 1.82700 C 1.47600 8.25300 2.02600 C 2.55900 8.76100 2.71800 C 3.28100 9.81600 2.20500 N 2.96400 10.37300 1.02900 C 1.91500 9.89500 0.34900 C 1.15300 8.85100 0.82500 C 1.60500 10.54600 -0.94700 C 2.13000 11.79900 -1.27900 C 1.80500 12.36300 -2.50300 C 0.79300 9.90900 -1.88300 N 0.46800 10.43600 -3.07400 C 0.98200 11.64200 -3.37300 C 0.65300 12.20100 -4.60200 C 1.12200 13.46200 -4.96700 C 1.94100 14.17200 -4.09100 C 2.28300 13.63100 -2.85300 H -2.11900 3.39600 5.45000 H -3.18400 3.15200 3.33700 H -1.46800 2.84800 2.97000 H -2.18300 4.46300 1.35500 H -3.03200 5.41400 2.57400 FCUP 119 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 1.41100 6.86200 4.60900 H -0.67700 6.76300 0.88300 H 2.85800 8.32800 3.66300 H 4.13900 10.23100 2.73200 H 0.28500 8.51100 0.29300 H 2.76800 12.34900 -0.59600 H -0.00100 11.64500 -5.26500 H 0.83500 13.90100 -5.92300 H 2.30500 15.15800 -4.37400 H 2.91900 14.19300 -2.16900 H 0.36900 8.92500 -1.70300 O 1.56500 3.98700 9.23600 H 2.41100 3.72700 9.64600 H 0.93600 3.69900 9.95000 O 2.08200 4.36300 6.64800 H 1.94800 4.24000 7.61900 H 1.17700 4.64400 6.39600 Table 5: Cartesian coordinates of MK3 bound to P4O without binding site water molecules. N -2.52300 14.22500 2.68800 H -2.35000 14.30000 3.68200 C -1.77700 13.20500 1.93800 H -1.67800 13.51000 0.89400 C -0.37100 13.10400 2.55700 H -0.46200 12.73200 3.57700 H 0.19400 12.36800 1.98900 C 0.43400 14.42200 2.58800 H -0.04900 15.11300 3.27600 C 1.84000 14.12700 3.12400 H 2.41900 15.04900 3.17600 H 1.76900 13.70400 4.12600 H 2.35000 13.41900 2.47200 C 0.51200 15.09800 1.21100 H 1.25600 15.89300 1.21600 H 0.78400 14.36900 0.45600 H -0.45400 15.53100 0.95300 C -2.46100 11.82700 1.90000 O -2.27700 11.08400 0.93400 H -3.52100 14.07900 2.53800 H -3.06900 11.52700 2.67800 N 1.73500 11.64500 10.34000 FCUP 120 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 2.65800 11.36900 10.66700 C 0.58800 10.79200 10.68700 H -0.23300 11.04100 10.01600 C 0.94400 9.32100 10.41800 H 1.39900 9.25000 9.42900 H 1.66800 8.98000 11.16000 C -0.29600 8.41200 10.44400 H -0.76800 8.47300 11.42100 H -1.01400 8.75000 9.69800 C 0.09500 6.95700 10.15200 H 0.58800 6.90800 9.17900 H 0.79900 6.61900 10.91700 C -1.12100 6.02100 10.14600 H -1.60300 6.06700 11.12700 H -1.82700 6.35400 9.38100 N -0.68000 4.62900 9.88800 H -1.42300 3.93900 9.91200 H -0.19900 4.55300 8.99900 H -0.06200 4.35100 10.65200 C 0.10000 11.06000 12.12800 O 0.83200 10.86400 13.10200 H 1.76600 11.75500 9.32600 H -0.85900 11.41000 12.27900 N 7.79100 12.20900 2.51900 H 8.60700 12.79900 2.46600 C 6.59900 12.60900 1.76500 H 5.71600 12.24200 2.29000 C 6.54600 14.14200 1.75700 H 6.75000 14.53300 2.75600 H 7.29600 14.53600 1.06500 S 4.88800 14.65200 1.23400 H 4.20600 14.17300 2.28300 C 6.57300 11.98900 0.35100 O 7.44200 12.26400 -0.47600 H 8.05200 11.26300 2.24200 N 5.56700 11.15900 0.06000 H 4.88500 10.97800 0.78400 C 5.46700 10.37100 -1.18500 H 6.47300 10.07700 -1.47800 C 4.68000 9.07300 -0.92600 H 3.66200 9.31600 -0.63300 H 4.62400 8.50100 -1.85200 C 5.32800 8.20100 0.16400 H 5.37300 8.76600 1.09600 FCUP 121 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 4.69100 7.33600 0.34800 S 7.00100 7.61400 -0.20300 C 6.57700 6.36500 -1.42900 H 7.48000 5.88200 -1.80100 H 5.92500 5.61600 -0.98000 H 6.04700 6.82500 -2.25600 C 4.91200 11.18300 -2.36900 O 3.81600 10.92500 -2.86300 N 5.69400 12.16900 -2.82300 H 6.58100 12.28300 -2.33800 C 5.33800 13.20300 -3.82200 H 4.51200 13.78800 -3.42800 C 6.53200 14.16300 -4.01300 H 7.39300 13.58900 -4.34300 H 6.30700 14.86700 -4.81500 C 6.93700 14.96000 -2.76200 H 6.55300 14.48800 -1.85700 H 8.02600 14.97300 -2.69700 C 6.43300 16.39700 -2.82600 O 7.26200 17.33400 -2.78900 O 5.20100 16.59700 -2.91000 C 4.88000 12.67700 -5.20300 O 4.48300 13.46300 -6.06500 H 4.90700 11.66500 -5.40300 N -3.94000 1.29200 -2.24800 H -4.72600 0.63700 -2.22400 C -3.98100 2.42400 -1.30300 H -4.11400 3.34300 -1.87400 C -5.17100 2.27500 -0.33000 H -4.96900 1.45700 0.36000 H -5.21600 3.18900 0.26400 C -6.55900 2.07000 -0.96400 H -7.30300 2.53000 -0.31300 H -6.60300 2.58000 -1.92600 C -6.91800 0.59100 -1.13400 O -6.36100 -0.03300 -2.06300 O -7.71400 0.07900 -0.31200 C -2.69600 2.58600 -0.46200 O -2.36300 3.69200 -0.04100 H -3.84100 1.65500 -3.19600 N -1.97000 1.48700 -0.22800 H -2.27900 0.63000 -0.66600 C -0.76900 1.42000 0.61200 H -0.72100 2.31800 1.23000 FCUP 122 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C -0.91100 0.20500 1.55600 H -0.99900 -0.70300 0.95900 H -0.01200 0.11700 2.16500 C -2.09800 0.27700 2.52100 O -2.75800 1.28700 2.70500 N -2.42400 -0.82400 3.16200 H -2.00900 -1.70800 2.92300 H -3.18000 -0.79400 3.83900 C 0.55300 1.37200 -0.20400 O 1.61800 1.12600 0.36800 H 0.54000 1.54500 -1.22100 N 7.44700 12.26000 -8.87200 H 7.48500 13.21500 -8.52700 C 7.21600 12.06300 -10.31700 H 6.47500 11.27600 -10.45200 C 6.66100 13.37700 -10.91600 H 6.82000 14.21100 -10.23200 H 7.20600 13.61600 -11.83100 C 5.17200 13.29800 -11.28300 H 4.90600 14.20700 -11.82200 H 5.01300 12.45000 -11.95000 C 4.25300 13.16900 -10.06200 H 4.49800 12.26300 -9.51400 H 4.39700 14.03400 -9.41200 C 2.79400 13.09700 -10.52600 H 2.57800 13.96800 -11.15500 H 2.65500 12.19800 -11.13200 N 1.85700 13.07200 -9.38000 H 0.91300 12.88700 -9.72100 H 2.08600 12.32300 -8.73400 H 1.87300 13.97000 -8.90100 C 8.44700 11.57400 -11.11700 O 8.28300 11.09300 -12.23500 H 8.30700 11.78000 -8.60700 H 9.39500 11.65100 -10.71600 N 3.51500 1.15200 3.08800 H 2.74800 0.73200 2.57800 C 3.14900 1.97000 4.26700 H 4.00200 2.02200 4.94000 C 2.84900 3.40400 3.81900 H 3.64900 3.73500 3.15600 C 1.52400 3.55300 3.07900 H 1.42600 4.57200 2.71100 H 1.50300 2.88000 2.22700 FCUP 123 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 0.69300 3.31700 3.74100 O 2.82500 4.24500 4.94200 H 2.31800 5.02700 4.70300 C 2.00800 1.36100 5.09200 O 1.34800 0.42400 4.65400 H 4.07000 1.72000 2.44800 N 1.83700 1.91000 6.29300 H 2.39600 2.73900 6.43200 C 0.98700 1.48700 7.42500 H 1.08900 2.29100 8.15400 C -0.51600 1.40200 7.06500 H -0.69800 1.95800 6.14800 H -0.78700 0.36300 6.87100 C -1.41600 1.96300 8.18500 O -2.45100 2.60900 7.89500 O -1.04900 1.84700 9.37400 C 1.46300 0.20800 8.15300 O 2.06800 -0.68700 7.56600 H 1.26700 0.10300 9.16100 N -1.63100 4.32700 5.72200 C -2.16800 3.97700 4.40500 C -0.89800 5.97400 3.63700 C -0.21100 5.99100 4.81400 C -0.62800 5.18200 5.97000 C -2.12600 5.16100 3.43400 O -0.16900 5.22400 7.09400 C 0.86000 6.93000 4.68400 C 0.73100 7.47600 3.42700 N -0.34800 6.89000 2.78100 C 1.54900 8.55200 2.87200 C 2.73900 8.93400 3.46100 C 3.48800 9.95200 2.91300 N 3.08000 10.60700 1.82200 C 1.92500 10.26200 1.24300 C 1.14200 9.23400 1.74000 C 1.53900 11.04400 0.03800 C 2.24400 12.19600 -0.34100 C 1.83700 12.89700 -1.46600 C 0.46200 10.65600 -0.75800 N 0.05800 11.31600 -1.85300 C 0.75400 12.41400 -2.20200 C 0.33600 13.11200 -3.32800 C 0.98200 14.28400 -3.72400 C 2.05300 14.76400 -2.97600 FCUP 124 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 2.48900 14.07600 -1.84600 H -1.91700 3.74200 6.49700 H -3.19700 3.61800 4.51100 H -1.56500 3.15900 4.00200 H -2.18300 4.78200 2.41000 H -3.00300 5.78300 3.60300 H 1.56900 7.19600 5.46800 H -0.67500 7.06500 1.84300 H 3.11000 8.42600 4.34200 H 4.43800 10.25800 3.34600 H 0.19700 8.98600 1.29600 H 3.08900 12.57200 0.22900 H -0.50700 12.72900 -3.88900 H 0.65100 14.83000 -4.60800 H 2.56000 15.67900 -3.28000 H 3.33500 14.46200 -1.27700 H -0.13400 9.77600 -0.53400 Table 6: Cartesian coordinates of MK2 bound to P40. N 7.73200 -17.35900 9.29300 H 8.27800 -16.96000 8.54000 C 7.07600 -16.42200 10.22700 H 6.69500 -16.97100 11.09100 C 8.12900 -15.40500 10.72000 H 8.41300 -14.77300 9.88200 H 7.65900 -14.76400 11.46700 C 9.41400 -15.99900 11.33500 H 9.92900 -16.58700 10.57800 C 10.35200 -14.85700 11.75100 H 11.27700 -15.26500 12.15400 H 10.59100 -14.24300 10.88300 H 9.87800 -14.23400 12.50800 C 9.12200 -16.90300 12.54100 H 10.05600 -17.29900 12.93500 H 8.61000 -16.33800 13.32000 H 8.50300 -17.74700 12.24200 C 5.84700 -15.68400 9.64300 O 5.18500 -14.94000 10.36300 H 8.32200 -17.99700 9.82600 H 5.58500 -15.81600 8.65400 N 12.03800 -11.01500 5.34400 H 12.42600 -10.12700 5.65200 FCUP 125 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 11.04400 -11.02400 4.26100 H 10.42800 -11.91500 4.37600 C 10.10600 -9.81300 4.39400 H 9.77800 -9.73000 5.43100 H 10.63900 -8.90300 4.11800 C 8.86900 -9.98900 3.50000 H 9.18400 -10.12200 2.46800 H 8.32500 -10.88300 3.80700 C 7.93800 -8.77800 3.58900 H 7.70400 -8.58300 4.63700 H 8.43500 -7.89900 3.17200 C 6.63800 -9.04600 2.82100 H 6.86300 -9.23600 1.76900 H 6.15700 -9.93500 3.23900 N 5.73000 -7.88800 2.93800 H 4.79200 -8.07400 2.59100 H 5.62000 -7.61600 3.91100 H 6.08400 -7.06500 2.44300 C 11.71700 -11.11600 2.87800 O 12.44300 -10.21100 2.45700 H 12.80700 -11.63300 5.08500 H 11.55800 -11.95100 2.29200 N 12.04500 -9.44000 15.31300 H 12.89100 -9.32800 15.84800 C 11.21300 -10.62100 15.60100 H 10.82800 -11.01400 14.65700 C 12.14100 -11.67300 16.22700 H 13.05300 -11.77500 15.63200 H 12.41500 -11.36000 17.23800 S 11.29000 -13.27300 16.29500 H 11.85000 -13.79400 15.18600 C 9.98900 -10.30400 16.50200 O 10.15100 -9.79600 17.61600 H 11.47500 -8.60100 15.41800 N 8.77400 -10.61900 16.02900 H 8.72600 -11.00200 15.09600 C 7.49000 -10.29700 16.68300 H 7.66800 -9.54600 17.44800 C 6.53800 -9.68900 15.62300 H 6.26700 -10.47800 14.92100 H 5.61900 -9.37900 16.12300 C 7.07600 -8.49300 14.80400 H 7.97800 -8.79300 14.27200 C 6.02600 -8.07800 13.76600 FCUP 126 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H 6.41900 -7.28400 13.13400 H 5.76700 -8.93000 13.13600 H 5.13200 -7.71500 14.27200 C 7.41000 -7.27800 15.67700 H 7.79300 -6.47100 15.05400 H 6.51100 -6.92900 16.18500 H 8.17600 -7.53900 16.40800 C 6.85800 -11.50400 17.42300 O 5.86000 -12.06600 16.97900 N 7.42600 -11.91400 18.56600 H 8.19300 -11.35900 18.92500 C 7.11800 -13.20900 19.21900 H 7.16600 -13.95600 18.42600 C 8.23400 -13.59400 20.22200 H 8.06600 -14.62300 20.54400 H 9.18700 -13.58600 19.69000 C 8.36300 -12.71000 21.47400 O 9.21200 -11.79100 21.47100 O 7.64100 -12.93200 22.47700 C 5.69200 -13.37400 19.81100 O 5.35800 -14.45200 20.31100 H 5.02600 -12.58600 19.78700 N -3.33000 -10.78500 9.89600 H -4.02100 -11.03200 9.18400 C -1.96500 -11.27500 9.66800 H -1.68800 -11.91900 10.50300 C -1.92800 -12.11400 8.37500 H -2.07900 -11.46900 7.51100 H -0.92200 -12.52800 8.29000 C -2.91900 -13.29600 8.31800 H -2.45700 -14.07700 7.71300 H -3.06000 -13.69900 9.32100 C -4.29100 -12.98000 7.69700 O -4.76200 -11.82700 7.81100 O -4.88800 -13.91200 7.09700 C -0.90900 -10.15300 9.58300 O 0.26500 -10.39300 9.85900 H -3.65500 -11.11400 10.80500 N -1.32000 -8.92500 9.24200 H -2.30800 -8.80000 9.07200 C -0.47200 -7.72400 9.21400 H 0.56100 -8.02700 9.04700 C -0.91600 -6.81900 8.04600 H -1.97300 -6.57800 8.15500 FCUP 127 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases H -0.36300 -5.88300 8.10300 C -0.68400 -7.36400 6.64000 O -0.20100 -8.45800 6.39200 N -1.06400 -6.59600 5.64400 H -1.60400 -5.76100 5.81800 H -0.88600 -6.85300 4.68300 C -0.49500 -6.91600 10.53900 O 0.21500 -5.91600 10.65700 H -1.10200 -7.21500 11.31800 N 5.38400 -11.62300 23.78700 H 6.22600 -12.15300 23.56400 C 4.67900 -11.95700 25.03900 H 3.60600 -11.92100 24.86000 C 5.03200 -13.39300 25.48900 H 6.10000 -13.58100 25.37400 H 4.81100 -13.47300 26.55500 C 4.22000 -14.50400 24.80400 H 4.16500 -15.35100 25.49000 H 3.20200 -14.15500 24.62300 C 4.83900 -15.00500 23.49600 H 4.94200 -14.17500 22.79800 H 5.82800 -15.42100 23.70100 C 3.93000 -16.08700 22.89800 H 3.69900 -16.82800 23.67100 H 2.99200 -15.62500 22.57900 N 4.56800 -16.76800 21.74900 H 3.89100 -17.37100 21.28700 H 4.91600 -16.08300 21.08300 H 5.34400 -17.34500 22.07100 C 4.93000 -10.96000 26.20200 O 4.14000 -10.94500 27.14900 H 4.72900 -11.70200 23.00900 H 5.74100 -10.32300 26.18300 N 2.48500 -4.01700 8.85900 H 1.50800 -4.27200 8.80200 C 3.35600 -4.61500 7.81600 H 4.30500 -4.08500 7.77000 C 3.65200 -6.08800 8.14900 H 4.46000 -6.43800 7.50600 C 4.05000 -6.35100 9.60100 H 4.41300 -7.37400 9.69400 H 4.83300 -5.66200 9.90100 H 3.18800 -6.23000 10.26000 O 2.49700 -6.84900 7.91300 FCUP 134 Rational Drug-design of Compounds Targeting Protein Kinase Related Diseases C 5.54100 -14.44700 3.28500 H 3.31300 -7.86600 -6.66000 H 1.87000 -9.60200 -5.87400 H 2.00500 -8.47900 -4.51400 H 2.43700 -10.58900 -3.53400 H 3.39300 -11.14400 -4.90600 H 7.13000 -7.93300 -3.46100 H 4.71100 -11.10000 -1.95400 H 8.14900 -7.86900 -1.49700 H 9.59500 -8.08400 0.49000 H 6.24100 -11.52500 -0.40000 H 5.65200 -12.35000 1.66600 H 8.99700 -15.45100 4.70300 H 6.97000 -16.78200 5.31100 H 4.76100 -16.11000 4.41000 H 4.56600 -14.16100 2.88700 H 9.90900 -11.80300 2.23200