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binding stability kinetics

Ultrasensitive Calorimetry for the Life SciencesTM

Microcalorimetry for the Life Sciences

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Why Microcalorimetry?Microcalorimetry is universal detectorHeat is generated or absorbed in every chemical processIn-solutionNo molecular weight limitationsLabel-free Non-optical

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Calorimetry in the Life SciencesBinding Studies

Quick and accurate affinitiesMechanism of action (MOA) screening and conformational changes Structure-function relationshipsSpecific vs. non-specific binding

KineticsKM, Vmax, kcatEnzyme Inhibition

Stability StudiesIntramolecular – e.g. protein unfoldingIntermolecular – e.g. solution optimization, CMCs

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Isothermal Titration Calorimetry

VP-ITC

AutoITC

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67 75.002

4

6

8

10

12

14

16

Time (min)

µcal

/sec

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Differential Scanning Calorimetry

VP-DSC

VP-Capillary DSC Platform

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

How Do They Work?Measuring Temperature Changes in Calorimetry

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Isothermal Titration Calorimetry: A Method for Characterizing

Binding Interactions

Mixture of two components at a set temperatureHeat of interaction is measuredParameters measured from a single ITC experiment

AffinitiesBinding mechanismNumber of binding sitesKinetics

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Isothermal Titration Calorimetry

-14

-12

-10

-8

-6

-4

-2

0

kcal

/mol

e of

inje

ctan

t

0 2 4Molar Ratio

Affinity

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67 75.002

4

6

8

10

12

14

16

Time (min)

µcal

/sec

Typical ITC Data

Stoichiometry

ΔHMechanism

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

ITC – Protein-Small Molecule Interaction

4-carboxybenzene-sulfonomide titrated into carbonic anhydrase IIN = 0.97KD = 730 nMΔH = -11.9 kcal/mol

Day, et al, Protein Sci. 11, 1017-1025 (2002)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

ITC – Protein-Protein Interaction

Pielak and Wang, Biochemistry 40, 422-428 (2001)

A: Wild-type cytochrome c titrated into wild-type cytochrome c peroxidase

B: Mutant cytochrome c titrated into mutant cytochrome c peroxidase

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Multiple Binding Sites

ITC shows differential binding of Mn(II) ions to WT T5 5’ nuclease

-2

0

2

4-10 0 10 20 30 40 50 60 70 80 90 100Time (min)

µcal

/sec

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

2

Molar Ratio

kcal

/mol

e

n = 0.85Ka = 3.0 x 105 M-1

ΔH = -0.59 kcal mol-1

n = 1.3Ka = 1.0 x 104 M-1

ΔH = +1.6 kcal mol-1

Feng, et al, Nat. Struct. Mol. Biol. 11, 450-456 (2004)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Binding EnergeticsMechanism of Action (MoA)

Conformational changesH-bondingHydrophobic interactionsCharge-charge interactions

Multiprobe structure-activity relationship (SAR)Validate in-silico modellingSelectivity and adaptability of drugs

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Binding Mechanism Same affinity but different binding mechanisms and specificity

-1 4

-1 2

-1 0

-8

-6

-4

-2

0

kcal

/mol

e of

inje

ctan

t

0 1 2 3 4

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Enthalpic and Entropic Contributions to Binding Affinity

Enthalpy and Entropy make up the affinity (ΔG=-RTlnK)

-8

-6

-4

-2

0

2

4

6

kcal

mol

-1

ΔH

ΔG

TΔS

ΔG=ΔH-TΔS

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Enthalpy and Entropy

EntropyHydrophobic interactionsWater releaseIon release Conformational changes

EnthalpyHydrogen bonding Protonation eventsMore specific

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Energetic Signatures

A is enthalpy driven. Good H-bonding coupled to a conformational change

B is entropicallydriven. Hydrophobic Interactions and possibly ‘rigid body’

C is mildly enthalpic and entropic -100

-80

-60

-40

-20

0

20

40

60

ΔHΔG

-TΔS

kJ mol-1

A B C

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Drug Discovery – Binding of Inhibitors to HIV-1 Protease

KNI-764KNI-272LopinavirAmprenavirRitonavirSaquinavirNelfinavirIndinavir

-20

-15

-10

-5

0

5

kcal

/mol

e

ΔG

ΔH

−TΔS

Ohtaka, et al. Protein Sci. 11, 1908-1916 (2002)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Enzyme kinetics and ITC

ITC measures thermal power (dQ/dt)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Enzyme kineticsRate =

Vo·

dQ/dt

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

ITC and Binding - Summary

Quick and Easy AffinitiesMechanism of ActionSAR-Structure Activity RelationshipsDrug designMulti-probe technique detects contributions that ‘affinity only’ methods may missEnzyme kinetics

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Differential Scanning CalorimetryTypical Data

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Minimum Protein for DSC - Lysozyme

Kholodenko and Freire, Anal. Biochem. 270, 336-338 (1999)

25 μg/ml

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Data Interpretation

30 40 50 60 70 80 90

0

2

4

6

8

10

12

14

Cp

(kca

l/mol

e/o C)

Temperature ( oC)

TM Shelf-Life/ Stability

Oligomers

Binding

ΔCp}

ΔH Stabilizing Forces/ Energetics

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Data Interpretation

30 40 50 60 70 80 90

0

2

4

6

8

10

12

14

Cp

(kca

l/mol

e/o C)

Temperature ( oC)

TM

ΔCp}

ΔH

Shelf-Life/ Stability

Oligomers

Binding

Stabilizing Forces/ Energetics

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Stability Intrinsic molecular stability

Thermodynamic characterization of macromolecular unfolding- proteins, nucleic acids, lipids, Domain structure identificationAssessment of viability and/or ‘crystallization potential’ of protein constructs and mutants

Extrinsic molecular stabilityFormulation studies – effect of different excipients, preservatives BindingMembrane and lipid studies

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

DSC –Protein Stability and Mutant Characterization

Sot, et al, J. Biol. Chem. 278, 32083 - 32090 (2003)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Domain Identification

Structural organization of biomolecules

Protein has a least two structural domains

O’Brien, et al, Biophys J. 70, 2403-2407 (1996)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Formulations/Protein Storage

CD40 ligand has a longer shelf-life at pH 6-7.DSC experiment could be completed in 1 day as opposed to 8 days by size exclusion chromatography

Remmele and Gombotz, BioPharm 13, 36-46 (2000)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Binding

20 40 60 80 100 120-0.00135

-0.00130

-0.00125

-0.00120

-0.00115

-0.00110

-0.00105

-0.00100

Cp(

cal/o C

)

Temperature (oC)

FBS at 60 μM Plus VAF955 and 1827 (at 1:1 and 1:20 [Ratios])

No ligand

1827(1:1)

1827 (1:20)

VAF955 (1:20)

VAF (1:1)

HTS validation OR Ligand Identification

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Antibody StabilityEffects of Glycosylation

Native Partially Deglycosylated Deglycosylated

Mimura, et al, J. Biol. Chem. 276, 45539-45547 ( 2001)

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Lipid/Membrane systems

Uptake of proteins into lipid membrane causes peak broadening

Lipid phase transitions

Broad Narrow

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Microcalorimetry Summary

Affinities and Binding EnergeticsMechanisms of BindingStoichiometryDetermination of Active ConcentrationEnzyme KineticsThermodynamic StabilityFormulations and Drug delivery

Ultrasensitive Calorimetry for the Life SciencesTM

kineticsstabilitybinding

Conclusion

Microcalorimetry is one of the most versatile technologies available for

characterization and analysis of biological molecules