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A perfect match for the complete thermodynamic and kinetic characterization of molecular interactions.

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  1. 1. AFFINImeter & ITC: A perfect match for the complete thermodynamic and kinetic characterization of molecular interactions 28th November 2015
  2. 2. Overwiew 1. Our Company 2. Our purpose 3. Our product: AFFINImeter o A brief description o Main functionalities: • KinITC • Model builder • Global fitting o Online Demo: APPLICATIONS TO THE PHARMACEUTICAL INDUSTRY
  3. 3. 1. Our company • Established in November 2013 as a spin off of the University of Santiago de Compostela (Spain). • Supported by a highly qualified staff with vast experience in R+D of areas devoted to software development and with expertise experimental biophysical techniques and methods. • We are particularly specialized in providing analytical tools for the study of complex molecular interactions. Santiago de Compostela
  4. 4. 1. Our company Our team • 7 people working in the company with interdisciplinary background: expertise in physics, chemistry, engineering and computer programming. • Broad experience in the application of various biophysical techniques Isothermal Titration Calorimetry (ITC), Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance (NMR) and Molecular Dynamics Simulations. Partners • Academia & Industry
  5. 5. Drug Discovery & Development Ligand – receptor interactions: characterization • Thermodynamics • Kinetics • Structure & mechanism • How strong? • How fast? • Where/how? 2. Our purpose
  6. 6. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) How to get the information? Monitor the interaction (Biophysical technique) Ligand – receptor interaction 2. Our purpose
  7. 7. 3. Our product: AFFINImeter A software for analysis of Isothermal Titration Calorimetry (ITC) data New tools for data analysis User friendly Exploit the full potential of ITC Expand the range of applications
  8. 8. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) ITC Isothermal Titration Calorimetry ITC Gold-standard technique for the thermodynamic characterization of interactions 3. Our product: AFFINImeter
  9. 9. ligand receptor The ITC titration: Power vs. Time plot (Thermogram) Isotherm KA , DH, stoichiometry DG, DS Thermodynamics Kinetics peak integration kon , koff 3. Our product: AFFINImeter
  10. 10. Obtaining kinetic information from ITC Main functionalities: KinITC
  11. 11. Obtaining kinetic information from ITC • IS THERE KINETIC INFORMATION IN MY THERMOGRAM?: A VISUAL INSPECTION Mid titration: Peak broadening Main functionalities: KinITC
  12. 12. AFFINImeter & KinITC heat vs time (kinetic information!) KA = kon/koff kon = KA·koff Main functionalities: KinITC • P. Dumas et al. kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J. Am. Chem. Soc. 201211; 134(1):559-65. • Implementation of KinITC into AFFINImeter. Microcal application Note, 2015
  13. 13. Structure & Mechanism Simple 1:1 interaction • Competitive experiments. • Species dimerization. • Multi-site binding • Cooperative effects • etc Complex data, usually explained by the presence of several equilibria Main functionalities: Model Builder and Global fitting
  14. 14. Tailored binding models Global fitting ROBUST ANALYSIS • Structural information. • Binding mechanism. • Cooperativiy. • Competitive experiments. Model builder AFFINImeter advanced tools Main functionalities: Model Builder and Global fitting
  15. 15. Automatic raw data processing KinITC (1:1 interactions) Generation of dataseries Advanced analysis of isotherms 3. Our product: AFFINImeter Raw data Processed data Isotherm & ETC curve Dataseries (isotherms) Model builder & Global fitting AUTOMATED, FAST, EASY
  16. 16. AFFINImeter demo
  17. 17. EDTA +M Ca+2 Ba+2 M = , Practical case 1
  18. 18. ITC isotherms of Ba2+/Ca2+ mixtures binding to EDTA GLOBAL FITTING Practical case 1
  19. 19. Bioactive pentasaccharide (anticoagulant activity) + Antithrombin (AT) HIGH AFFINITY (Bioactive sequence) Other sequences + Antithrombin (AT) LOW AFFINITY Heterogeneous mixtures of ligands Heparin – protein interactions Heparin Practical case 2
  20. 20. Laboratorios farmacéuticos ROVI • Determination of percentage of pentasaccharide in Low Molecular Weight Heparins. LMW-2 LMW-3 LMW-4 LMW-5 LMW-6 LMW-7 LMW-8LMW-1 Application in the pharmaceutical industry GLOBAL FITTING Practical case 2
  21. 21. Software as a Service annual plans adapted to different needs Costumer support service Data Analysis Consulting Local instalation Update service AND + + +
  22. 22. Analytical tools to study complex ITC data
  23. 23. kon (M-1s-1) 106 M-1s-1 8.4·105 M-1s-15·103 M-1s-1 KinITC SPR 10-5 s-1 koff (s-1) 6.5·10 -4 s-1 1.1·10-2 s-1 0.1s-1 KinITC SPR 103 M-1s-1 Small-Fragment Screening Kon= 3.7 e4 Koff= 1.1 E-2 Kd= 287 nM Enzyme-Subtrate Kon=1.7E4 Koff=1.3E-2 Kd= 764 nM DNA-Ligand Kon= 8.4 E5 Koff= 1.6 E-3 Kd= 1.8 nM Protein-Ligand Kon= 3.04 E4 Koff= 6.5E-4 Kd= 21.6 nM Detection limits of kinetic rate constants: KinITC vs SPR
  24. 24. “Essentially, KinITC is based upon linking the kinetics of the reaction to heat power production in the measurement cell” = = = = concentration of titrand in the cell 1:1 binding Heat power KinITC: obtaining kinetic information from ITC AFFINImeter & KinITC
  25. 25. Wieseman parameter Stoichiometric ratio for current injection KinITC: obtaining kinetic information from ITC Equilibration time Dissociation rate constant AFFINImeter & KinITC