Binding Pattern Elucidation of NNK and NNAL Cigarette Smoke Carcinogens with NER Pathway Enzymes: an Onco-Informatics Study

Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/ NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.


Introduction
Lung cancers are powerfully linked with cigarette smoke carcinogens like NNK (4-(Methylnitrosamino)-1-(3pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) (Xue et al., 2014). One of the preliminary critical actions is most likely damage of the hereditary material (DNA) by a cigarette smoke carcinogen. This damage can, beneath the definite status, be repaired by cellular DNA repair mechanisms (Raphael Ceccaldi et al., 2015). Though, if not repaired, cells will try to duplicate their DNA during cell division, but are obstructed by the damage and will do fault duplication progression leading to gene mutations brought onto a trail of uncontrolled cell division leading to a tumor growth. Studies show that in ordinary cells, NER removes numerous types of DNA lesions, defending cell integrity (Rouillon et al., 2011).
However, in cancer cells uncovered to DNA
Computational tools such as molecular docking are important to understand the binding capabilities of NNK and NNAL with enzymes involved in NER pathways (Xia et al., 2012). It has never been explored through in silico approaches. Therefore, we used protein-protein docking to know the functional loss of the enzymes due to their interaction with NNK and NNAL. In order to perform protein-protein interaction it is necessary to find out the co-operated functional enzymes encoded by genes using STRING 9.0.5 database (Szklarczyk et al., 2011) and their 3D structures. At last the comparative analysis (Protein-Protein docking vs Protein-Complex*) was completed by ZDOCK protocol using Discovery Studio Client 2.5 (Accelrys Software Inc, 2013).

Preparation of protein structures
The structures of enzymes involved in the NER pathways were obtained from Protein Data Bank (Berman et al., 2000) (Table 1). Published structures were edited to remove HETATM and water molecule using Discovery Studio Client 2.5. Energy minimization was performed by the implementation of CHARMm force field (D. T Mirijanian et al., 2014) after addition of hydrogen atoms to the selected enzymes using Accelyrs Discovery studio client 2.5.

Docking studies
Molecular Docking studies were performed to analyze the binding affinity of NNK/NNAL with enzymes involved NER pathways. Autodock (Version 4.2) suite (Morris et al., 1998;2009) and Cygwin interface was used in the Microsoft Windows 7 professional, operating System on Intel® Xeon® Processor E3-1220 v3 (Quad Core, 3.10GHz Turbo, 8MB) and 256GB 2.5inch Serial ATA Solid State Drive of Dell Precision T1700 Workstation was used to dock the NNK/NNAL on binding site of the enzymes. Molecular docking methods followed by the searching the best conformation of enzymes and carcinogens complex on the basis of binding energy. Water molecules were removed from the 3D X-ray crystallography structures of enzymes before docking and hydrogen atoms were added to all target enzymes. Kollman united charges and salvation parameters were added to the enzymes. Gasteiger charge was added to the ligands. Grid box was set to cover the maximum part of enzymes and ligands. The values were set to 60×60×60 Å in X, Y and Z axis of a grid point. The default grid points spacing was 0.375 Å. Lamarckian Genetic Algorithm (LGA) (Goodsell et al., 1996;Tsai et al., 2012) was used for enzymes-ligands flexible docking calculations. The LGA parameters like population size (ga_pop_size), energy evaluations (ga_num_generation), mutation rate, crossover rate and step size were set to 150, 2500000, 27000, 0.02, 0.8 and 0.2 Å, respectively. The LGA runs were set at 50 runs. All obtained conformations of enzymes and ligand complex were analyzed the interactions and binding energy of the docked structure using Discovery Studio 2.5 molecular visualization software.

Protein-protein interaction analysis
We found the interacting proteins (used as ligands) of selected enzymes using STRING 9.0.5 database that predict interacting interactions incorporate direct (physical) and indirect (functional) associations derived  DOI:http://dx.doi.org/10.7314/APJCP.2015.16.13.5311 Binding Pattern of Cigarette Smoke Carcinogens NNK and NNAL with NER Pathway Enzymes: an Onco-informatics Study from four sources, i.e. genomic context, high throughput experiments (conserved) co-expression and previous knowledge of proteins against your query ( Figure:5 A,B,C and D). We used discovery studio Client 2.5 for Zdock (Dock Proteins) Protocol. Zdock scores obtained for both Protein-Protein interactions as well as for Protein-Complex (ligand protein+NNK/NNAL) interaction.

Z dock calculations
Discovery studio Client 2.5 was used to complete protein-protein docking using ZDOCK is an initial stage, rigid body molecular docking algorithm that uses a fast Fourier transform (FFT) algorithm to improve performance for searching in translational space (Chen et al., 2003;Pierce et al., 2014). All of the available structures from PDB were used to calculate the docking poses and the structures obtained were subjected to energy minimization using the smart minimize algorithm (Max steps 200, RMS gradient 0.01) in the program Discovery studio 2.5. The resulting Zdock scores with the highest value were used as appropriate conformational pose (Jamal et al., 2012).

Results and Discussion
In the achievement of the current investigation molecular docking techniques were adopted to explore the binding capabilities of NNK and NNAL with enzymes encoded by respective genes of NER pathways. Primarily the 1IRD (Crystal Structure of Human Carbonmonoxy-Haemoglobin at 1.25 Å Resolution) was used as a positive control and 3CI9 (Human heat shock factor-binding protein 1) as a negative control to validate our docking analysis. Molecular interaction results of these enzymes showed that 1IRD docked with NNK, observed binding energy was -6.68 Kcal/Mol, it docked with NNAL and observed binding energy was -6.31 Kcal/Mol. 3CI9 docked with NNK with the experimental binding energy of -3.91 Kcal/Mol, it interacted with NNAL with binding energy of +2.09 Kcal/Mol. We performed docking analysis between 16 enzymes and NNK/NNAL separately. The observed docking energy between NER pathway enzymes and NNK were ranging from -4.28 kcal/mol to -8.05 kcal/ mol (Table 1) similarly between NER pathways enzymes and NNK were ranging from -5.33 kcal/mol to -7.94 kcal/ mol. In the completion of next step of our hypothesis, we selected top four NER enzymes encoded by respective genes from Table 1 and Table 2 on the basis of their highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/ mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 are -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively (Table 1).
Furthermore, the active site characterization analysis of top four enzymes also revealed that NNAL and NER  Phe87, Lys88, Tyr91, Leu92, Leu200, Thr201, Asp202, Leu205, Leu258, and Tyr262. The estimated inhibition constant of NNAL and CCNH docked complex was 29.57 uM ( Table 3 Figure In the further analysis, the protein-protein docking was adopted using ZDOCK protocol in Discovery Studio Client 2.5. Initially, we have found out the cooperated the enzymes encoded by genes for four selected enzymes, i.e. RAD23A, CCNH, CDK7, and CETN2 by STRING 9.0.5 database. The found Best closely related enzymes for RAD23A PDB ID: 1DV0 (RAD23 Homolog A) was PDB ID: 2KDE (PSMD4 MCB1, 26S proteasome non-ATPase regulatory subunit 4), for CCNH PDB ID: 1KXU (CDK-Activating Kinase Complex Subunit) was PDB ID: 1UA2 (CDK7,Cell division protein kinase 7), for CDK7 PDB ID: 1UA2 (Cyclin H) was PDB ID: 1KXU (CDK-Activating Kinase Complex Subunit) and for CETN2 PDB ID:1ZMZ (Centrin, EF-Hand Protein, 2) was PDB ID: 2GGM (XPC, DNA-repair protein complementing XP-C cells) (Figure 5 A (Table 4). The results shown that Zdock score of protein complex (contain enzymes and cigarette smoke carcinogens conformation) interaction were higher than protein-protein interaction. Analysis clearly revealed that when NNK/NNAL interacts with NER enzymes their metabolic activity to form complex with its cooperated enzymes reduces significantly. Thus, NNK and NNAL were capable to damage the DNA repair machinery and its will lead to the functional loss of NER enzymes encoded by genes RAD23A, CCNH, CDK7, and CETN2.

Conclusion
This hypothesis able to provide better understanding to explore the molecular interaction of NNK and NNAL with enzymes involved in NER pathways. It is also helpful to understand the biological insights of NNK and NNAL binding efficacy in the progression of cancer. The study revealed that the enzymatic activity of these enzymes RAD23A, CCNH, CDK7, and CETN2 affected by NNK and NNAL. Therefore, the possibility of DNA damage will be increased because these enzymes have an important role in the DNA damage control. Once the DNA repair machinery altered due to interaction of cigarette smoke carcinogens NNK and NNAL the whole biological process will lead to uncontrolled tumor growth and finally cancer will be developed. For the further confirmation of study the in vivo and in vitro validation needed.