Extraction of molecular features for the drug discovery targeting protein‐protein interaction of Helicobacter pylori CagA and tumor suppressor protein ASSP2

Half of the world population is infected by the Gram‐negative bacterium Helicobacter pylori (H. pylori). It colonizes in the stomach and is associated with severe gastric pathologies including gastric cancer and peptic ulceration. The most virulent factor of H. pylori is the cytotoxin‐associated gene A (CagA) that is injected into the host cell. CagA interacts with several host proteins and alters their function, thereby causing several diseases. The most well‐known target of CagA is the tumor suppressor protein ASPP2. The subdomain I at the N‐terminus of CagA interacts with the proline‐rich motif of ASPP2. Here, in this study, we carried out alanine scanning mutagenesis and an extensive molecular dynamics simulation summing up to 3.8 μs to find out hot spot residues and discovered some new protein‐protein interaction (PPI)‐modulating molecules. Our findings are in line with previous biochemical studies and further suggested new residues that are crucial for binding. The alanine scanning showed that mutation of Y207 and T211 residues to alanine decreased the binding affinity. Likewise, dynamics simulation and molecular mechanics with generalized Born surface area (MMGBSA) analysis also showed the importance of these two residues at the interface. A four‐feature pharmacophore model was developed based on these two residues, and top 10 molecules were filtered from ZINC, NCI, and ChEMBL databases. The good binding affinity of the CHEMBL17319 and CHEMBL1183979 molecules shows the reliability of our adopted protocol for binding hot spot residues. We believe that our study provides a new insight for using CagA as the therapeutic target for gastric cancer treatment and provides a platform for a future experimental study.

[1]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[2]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[3]  H. Berendsen,et al.  Essential dynamics of proteins , 1993, Proteins.

[4]  R. Rappuoli,et al.  Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin , 1995, Infection and immunity.

[5]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[6]  Peter A. Kollman,et al.  Computational alanine scanning of the 1:1 human growth hormone–receptor complex , 2002, J. Comput. Chem..

[7]  Takeshi Azuma,et al.  Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Itohara,et al.  Causal Relationship between the Loss of RUNX3 Expression and Gastric Cancer , 2002, Cell.

[9]  M. Selbach,et al.  Src Is the Kinase of the Helicobacter pylori CagA Protein in Vitro and in Vivo * , 2002, The Journal of Biological Chemistry.

[10]  R. Rappuoli,et al.  c‐Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs , 2002, Molecular microbiology.

[11]  M. Blaser,et al.  Helicobacter pylori and gastrointestinal tract adenocarcinomas , 2002, Nature Reviews Cancer.

[12]  Vadim M Govorun,et al.  Protein‐protein interactions as a target for drugs in proteomics , 2003, Proteomics.

[13]  Flavio Seno,et al.  Geometry and symmetry presculpt the free-energy landscape of proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[14]  L. Vassilev,et al.  In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 , 2004, Science.

[15]  Jaw-Town Lin,et al.  Helicobacter pylori Infection and CagA Protein Translocation in Human Primary Gastric Epithelial Cell Culture , 2006, Helicobacter.

[16]  S. Brandt,et al.  Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering. , 2007, Gastroenterology.

[17]  S. Wessler,et al.  Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility , 2007, Oncogene.

[18]  Julie C. Mitchell,et al.  An automated decision‐tree approach to predicting protein interaction hot spots , 2007, Proteins.

[19]  Naomi Ohnishi,et al.  Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse , 2008, Proceedings of the National Academy of Sciences.

[20]  Ozlem Keskin,et al.  Restricted mobility of conserved residues in protein-protein interfaces in molecular simulations. , 2008, Biophysical journal.

[21]  Solène Grosdidier,et al.  Identification of hot-spot residues in protein-protein interactions by computational docking , 2008, BMC Bioinformatics.

[22]  P. Mas,et al.  Expression of Helicobacter pylori CagA domains by library‐based construct screening , 2009, The FEBS journal.

[23]  M. Blaser,et al.  Coadaptation of Helicobacter pylori and humans: ancient history, modern implications. , 2009, The Journal of clinical investigation.

[24]  Shaomeng Wang,et al.  Small-molecule inhibitors of the MDM2-p53 protein-protein interaction to reactivate p53 function: a novel approach for cancer therapy. , 2009, Annual review of pharmacology and toxicology.

[25]  Doheon Lee,et al.  A feature-based approach to modeling protein–protein interaction hot spots , 2009, Nucleic acids research.

[26]  Nir London,et al.  Can self‐inhibitory peptides be derived from the interfaces of globular protein–protein interactions? , 2010, Proteins.

[27]  A. Barabasi,et al.  Network medicine : a network-based approach to human disease , 2010 .

[28]  R. Peek,et al.  Helicobacter pylori CagA targets gastric tumor suppressor RUNX3 for proteasome-mediated degradation , 2010, Oncogene.

[29]  R. Rappuoli,et al.  Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host , 2011, Proceedings of the National Academy of Sciences.

[30]  A. Louche,et al.  Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin , 2012, Proceedings of the National Academy of Sciences.

[31]  Holger Gohlke,et al.  MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. , 2012, Journal of chemical theory and computation.

[32]  Ross C. Walker,et al.  An overview of the Amber biomolecular simulation package , 2013 .

[33]  Irina S Moreira,et al.  Computational Alanine Scanning Mutagenesis-An Improved Methodological Approach for Protein-DNA Complexes. , 2013, Journal of chemical theory and computation.

[34]  Peter M. Kasson,et al.  GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit , 2013, Bioinform..

[35]  Ozlem Keskin,et al.  Hot spots in protein-protein interfaces: towards drug discovery. , 2014, Progress in biophysics and molecular biology.

[36]  A. Panchenko,et al.  Predicting the Impact of Missense Mutations on Protein–Protein Binding Affinity , 2014, Journal of chemical theory and computation.

[37]  Xin Lu,et al.  Structure of the Helicobacter pylori CagA oncoprotein bound to the human tumor suppressor ASPP2 , 2014, Proceedings of the National Academy of Sciences.

[38]  Sangdun Choi,et al.  In silico mechanistic analysis of IRF3 inactivation and high-risk HPV E6 species-dependent drug response , 2015, Scientific Reports.

[39]  M. Sternberg,et al.  The Contribution of Missense Mutations in Core and Rim Residues of Protein–Protein Interfaces to Human Disease , 2015, Journal of molecular biology.

[40]  Tom L. Blundell,et al.  Flexibility and small pockets at protein–protein interfaces: New insights into druggability , 2015, Progress in biophysics and molecular biology.

[41]  U. Ghoshal,et al.  Association of heterogenicity of Helicobacter pylori cag pathogenicity island with peptic ulcer diseases and gastric cancer , 2017, British journal of biomedical science.

[42]  S. Backert,et al.  Type IV Secretion and Signal Transduction of Helicobacter pylori CagA through Interactions with Host Cell Receptors , 2017, Toxins.

[43]  F. Mégraud,et al.  Helicobacter pylori Strains and Gastric MALT Lymphoma , 2017, Toxins.

[44]  Kang-Yell Choi,et al.  Screening-based approaches to identify small molecules that inhibit protein–protein interactions , 2017, Expert opinion on drug discovery.

[45]  E. Merino,et al.  Functional interaction and structural characteristics of unique components of Helicobacter pylori T4SS , 2017, The FEBS journal.

[46]  Weiliang Zhu,et al.  Inhibiting mechanism of small molecule toward the p53‐MDM2 interaction: A molecular dynamic exploration , 2018, Chemical biology & drug design.

[47]  Lei Deng,et al.  Machine Learning Approaches for Protein–Protein Interaction Hot Spot Prediction: Progress and Comparative Assessment , 2018, Molecules.

[48]  P. Marimuthu,et al.  Prediction of Hot Spots at Myeloid Cell Leukemia-1–Inhibitor Interface Using Energy Estimation and Alanine Scanning Mutagenesis , 2018, Biochemistry.

[49]  Asma Sindhoo Nangraj,et al.  Structural-dynamic insights into the H. pylori cytotoxin-associated gene A (CagA) and its abrogation to interact with the tumor suppressor protein ASPP2 using decoy peptides , 2018, Journal of biomolecular structure & dynamics.

[50]  Chi‐Huey Wong,et al.  2‐anilino‐4‐amino‐5‐aroylthiazole‐type compound AS7128 inhibits lung cancer growth through decreased iASPP and p53 interaction , 2018, Cancer science.

[51]  J. Fernández-Recio,et al.  Hot-spot analysis for drug discovery targeting protein-protein interactions , 2018, Expert opinion on drug discovery.