STRUCTURAL BIOINFORMATICS
Definition Biological Intro: nucleic acids and protein structures Data bank and modeling tools Computational tool: Molecular Dynamics Research project: a docking analysis
Teresa Rutigliano Definition Structural bioinformatics: prediction and analysis of the three-dimensional structure of biological macromolecules
Generalizations about 3D : molecular folding, evolution, binding interactions, and structure/function relationships
Working both from experimentally solved structures (obtained e.g., by NMR or CD or X-ray) and from computational models DNA structure Deoxyribonucleic acid = DNA
Wikipedia DNA stores biological information RNA structure Ribonucleic acid = RNA
Wikipedia RNA catalyzes biological reactions (controlling gene expression, or sensing and communicating responses to cellular signals) Protein structure
Lysine
Wikipedia
Proteins are essential parts of organisms and participate in every process within cells From DNA to Protein Synthesis
YouTube Just an easy explanation !! Data bank and tools The reference site for protein structures, crystallographic and NMR, is The Protein Data Bank http://www.wwpdb.org/
(NCBI for FASTA! https://www.ncbi.nlm.nih.gov/)
Download Tool: Structures, Sequences, and Ligands RCSB PDB - Download Files
Have a look at the file format! Databases and tools
• Download the PDB: http://www.ebi.ac.uk/pdbe/
• Visualization and modeling: DeepView - Swiss-PdbViewer C Ascalaph Designer Definition Structural bioinformatics: prediction and analysis of the three-dimensional structure of biological macromolecules
Generalizations about 3D : molecular folding, evolution, binding interactions, and structure/function relationships
Working both from experimentally solved structures (obtained e.g., by NMR or CD or X- ray) and from computational models Computational tools
• Quantum Mechanics (QM) electronic structure based on Schrödinger Equation accurate, expensive
• Classical Molecular Mechanics (MM)(MD) Empirical forces based on Newton’s Laws less accurate, fast Molecular Dynamics
• Molecules interact with others (protein or nucleic acid or ligands..), their binding induce conformational changes that influence the activity or shape or accessibility ..
• MD studies the Evolution In Time of a System Molecular Dynamics
INPUT: -Topological properties (connectivity) -Structural properties (conformation) -Energetical properties (force Field) -Thermodynamical properties Aims of Molecular Dynamics
• Extend exploration of interactions • Study of system properties • Analysis of the conformational changes • Study of mutants in silico • Analysis of the structural flexibility • Drug discovery • Refinement of the 3D structure models • Useful guide to validate or explain experimental data Research project: a docking analysis INTRO Nervous system
neurotransmitters Immune system
White Blood Cell, cytokines (IL-2) Endocrine system
Hormones (melatonin)
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 INTRO
A complex network between the nervous system, the endocrine and immune system, a bidirectional interrelationship, may affect their functions e.g. cytokines produced by activated immune cells can affect, positively or negatively, the secretion of hormones and vice versa
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 OUR AIM
To validate if the immune system is simultaneously enhanced by the elements of the neuroendocrine system and other stimulating agents of the immune system itself
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 OUR AIM
We used structural bioinformatics approach to analyse such interactions at a membrane receptor-ligand level
1
Examples of membrane receptors: 1. Ligands 2. Receptors
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The Biological model
On a bilayer membrane
we emulated a malignant POPE membrane with 340 lipids epithelial tissue where cells http://people.ucalgary.ca/~tieleman/download.html contain villin, ezrin and fimbrin proteins forming finger-like structures (microvilli)
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model
We docked:
1) Ligands: • One of the neuroendocrine system: Melatonin • One of the immune system: IL-2 • One from external environment able to activate immune system: Lipopolysaccharide
2) Related receptors
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model Melatonin (MLT)
• It is a hormone secreted by the pineal gland • Its secretion increases in darkness and decreases during exposure to light • It is implicated in the regulation of mood, learning and memory, dreaming, fertility and reproduction • Melatonin is an effective antioxidant
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model
MLT exerts both direct and indirect anti-cancer effects in synergy with other molecules
Studies carried out an immunotherapy by administering high dosages of Melatonin (MLT), and low dosages of Interleukin 2 (IL-2)
20% tumor regression
Melatonin in chemotherapy is useful to reduce toxicity
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model Interleukin-2 (IL-2)
• It is necessary for immune response • It is a signalling molecule • It activates certain White Blood Cells, which fight diseases and infections
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model
IL-2
IL-2 has been used in clinical trials for: • The treatment of chronic viral infections • As a booster (adjuvant) for vaccines • Anti cancer therapies
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model Bacterial lipopolysaccharide (LPS):
• It is the major outer surface membrane components present in almost all Gram-negative bacteria • It is an endotoxin, it is also an exogenous pyrogen
LPS
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model LPS:
• It is cause of infection and inflammation
• In presence of neoplastic cells and bacterial infection the immune system is overstimulated
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Simulation method
• Biological functions are mediated by interactions between molecules • Interactions are determined by geometric, chemical-physical and structural complementarity • Interactions induce conformational changes
We want to analyze interactions between these molecules and known receptors Simulation techniques allow this exploration
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Simulation method
• The docking tools allow to study in-silico conformational changes and the involved attractive forces, considering molecules both individually or in combination.
• This method is extremely useful when structural information (obtained e.g., by NMR or CD or X-ray) are not available, and for analysis of intermolecular complexes
• Docked conformations and interaction energies were achieved using the docking package HEX
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Docking tool HEX:
• It is an interactive program for calculating and displaying feasible docking of proteins and DNA molecules • It can also calculate protein-ligand docking, assuming the ligand is rigid, and it can superpose pairs of molecules using knowledge of their 3D shapes • It uses spherical polar Fourier (SPF) correlations (reached by means of rotations and translations)
Source: Dave Ritchie-Team Orpailleur, INRIA ,France http://hex.loria.fr/
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Docking tool HEX Rotational Search:
• A Cartesian grid is used to sample the molecular surfaces numerically • Molecules are oriented on the basis of their spherical polar coordinates
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Docking tool HEX Docking Search:
1) Rotating the receptor and ligand around their centroids on the basis of their intermolecular distances 2) The receptor and ligand are each assigned Euler rotational angles 3) The final rotation is defined as a twist of the ligand about the intermolecular axis
4) Perform a full six-dimensional Illustration of spherical polar docking search with respect to the intermolecular axis
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Docking tool
HEX Molecular Mechanics computation:
• Molecular mechanics energies are evaluated using a Newton- like model and energy minimization is applied
• These energies are calculated using hydrogen bond potentials along with an explicit charge-charge electrostatic contribution
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods Docking tool HEX Clustering Docking Results:
• Docking calculations never give a unique solution. The best orientations (lowest energy) are retained for viewing (1-500). The first solution is chosen for our analysis.
• It uses a clustering algorithm to group spatially similar docking orientations: – Each docking solution is first ordered by energy, and the lowest energy solution is taken for the first cluster. – The process is then repeated on the basis of the main- chain alpha-carbon RMS , until all solutions have been assigned to a cluster .
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Material and methods The biological model
IL-2, MLT and LPS, and their known receptors were anchored to a lipid membrane with typical features of adenocarcinoma
Ligands MLT IL-2 LPS
Receptors MT-1 IL-2R TLR4
Proteins forming Villin Ezrin Fimbrin microvilli
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Results IL-2
IL-2 alone. In absence of other ligand IL- Two ligands: IL-2 plus MLT. In presence of 2(water green) is far from its receptor Melatonin (fuchsia) IL-2 ligand (water (yellow) green) moves toward IL-2 receptor (yellow)
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Results MLT
Two ligands: IL-2 plus MLT. In presence of One ligand: Melatonin alone Melatonin (fuchsia) IL-2 ligand (water is far from its receptor (blue) green) moves toward IL-2 receptor (yellow). MLT is again far from its receptor
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Results MLT
Two ligands: MLT plus LPS. MLT (fuchsia) Three ligands: IL-2 plus MLT plus LPS seems to have an inner position in presence of LPS (purple)
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Results LPS
One ligand: LPS alone
Three ligands: IL-2 plus MLT plus LPS
Two ligands: IL-2 plus LPS
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Conclusions
We were able to verify and confirm that IL-2 and LPS bind their known receptors (internal control)
Some ligands, and their receptors, behave in a different way when other structures are present, in fact: LPS, such as IL-2, is affected by the presence of other molecules and is attracted by them where there are at least two.
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Conclusions
• The presence of LPS or IL-2 appears to promote immersion of Melatonin in the membrane
• Melatonin is never located in its receptor
• IL-2 receptor appears to be of fundamental importance for all ligands present in the study. Probably the surface of this receptor presents numerous “hot-spots”
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 Conclusions
• In summary, this study allows to observe that the simultaneous presence of Melatonin, IL-2 and LPS resulted in in docking position changes, and this may explain results obtained in oncology field.
• It can therefore be concluded that, as hypothesized, the model shows the existence of a synergetic interaction between neuroendocrine agents, immune substances and mediators of the inflammatory
• Further studies and in-vitro experiments may shed more light on the effective action of a LPS- MLT- based therapy.
8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014 • M. Szczepanik, Melatonin and its influence on immune system, J Physiol Pharmacol, vol. 58 Suppl 6, 2007, pp. 115-124. • Y.Yamaguchi, A. Ohshita A., Y. Kawabuchi , J. Hihar, E. Miyahara, K. Noma, T. Toge, Locoregional immunotherapy of malignant ascites by intraperitoneal administration of OK-432 plus IL-2 in gastric cancer patients, Japan Anticancer Research, vol. 15,1995, pp. 2201-2206. • L. Nespoli, F. Uggeri, A. Nespoli, F. Brivo, L. Fumagalli, M. Sargenti, L. Gianotti, Modulation of systemic and intestinal immune response by Interleukin-2 therapy in gastrointestinal surgical oncology. Personal experience in the context of current knowledge and future perspectives, Anticancer Res., vol. 32, 2012,pp. 989-96 • P. Bongrand, Ligand-Receptor Interactions, Reports on Progress in Physics , vol. 62, 1999, pp. 921-968. • D. V. Ritchie, V. Venkatraman, Ultra-fast FFT protein docking on graphics processors, Bioinformatics, vol. 26, 2010, pp. 2398–405 • David W Ritchie, Vishwesh Venkatraman, Lazaros Mavridis, Using graphics processors to accelerate protein docking calculations, Stud Health Technol Inform 2010 ;159:146-55
THANKS for the attention 8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014