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

1. AFFINImeter & ITC:
A perfect match for the complete thermodynamic
and kinetic characterization of molecular
interactions
28th November 2015

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. 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.
www.software4science.com www.affinimeter.com
Santiago de Compostela

4. 1. Our company
www.software4science.com www.affinimeter.com
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
www.mestrelab.com

5. Drug Discovery &
Development
Ligand – receptor interactions: characterization
• Thermodynamics
• Kinetics
• Structure & mechanism
• How strong?
• How fast?
• Where/how?
2. Our purpose

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. 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. 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. 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. Obtaining kinetic information from ITC
Main functionalities: KinITC

11. Obtaining kinetic information from ITC
• IS THERE KINETIC INFORMATION IN MY THERMOGRAM?: A VISUAL INSPECTION
Mid titration: Peak broadening
Main functionalities: KinITC

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. 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. 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. 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. AFFINImeter demo

17. EDTA
+M
Ca+2 Ba+2
M = ,
Practical case 1

18. ITC isotherms of Ba2+/Ca2+ mixtures binding to EDTA
GLOBAL FITTING
Practical case 1

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. 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. Software as a Service
annual plans adapted
to different needs
Costumer support
service
Data Analysis
Consulting
Local
instalation
Update service
AND
+
+ +

22. Analytical tools to study complex ITC data
https://www.affinimeter.com/

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. “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. Wieseman parameter
Stoichiometric ratio for current injection
KinITC: obtaining kinetic information from ITC
Equilibration time
Dissociation rate constant
AFFINImeter & KinITC