PresentationMicrocalorimetry – Guiding the SAR Process

Agenda

• Principles of microcalorimetry• Factors involved in ligand/macromolecule binding• Application examples of Calorimetry

•

SAR•

Profiling existing drug molecules•

Confirming binding mechanisms•

Optimizing crystallization potential• Instrument overview

Microcalorimetry Offers Enhanced Information Content

•

Experimental biological relevance •

Label-free•

True in solution •

No immobilization requirement •

No molecular weight limitations •

Optical clarity unimportant•

Non-destructive•

Minimal to no assay development saves time and conserves sample

Heat is a Fundamental Natural Property…

•

Heat absorption or release are universal properties of all chemical reactions

•

Calorimetry measures heat directly•

No reporters required•

A single reaction can yield •

Overall binding affinity •

Attractive hydrogen and van der Waals forces

•

Hydrophobic and conformational effects •

Stoichiometry

calorimetry is a direct readout

Microcalorimetry Applications in the Drug Discovery Process

Target Identification

Target Validation Robust Assay Development

High Throughput Screening

Secondary Screening

Lead Optimization Candidate Selection

Identify Unknown Targets

Elucidate Mechanism Of Action

Evaluate Expressed Proteins

QC AssayTargets and Conditions

Binding Affinity, Rank Order

SAR/QSAR QC the Candidate Leads

Confirm Targets ID/Confirm Pathways

Evaluate TargetCo-factors

Eliminate False Positives, Confirm Hits

Simultaneous Affinity of Enatiomers/Isomers

Evaluate Back-up Candidates

Deorphan

Receptors

Test Effects of “Reporters" for Biological Relevance

Enzyme/Substrate Interactions and Kinetics

Counter Screening for Selectivity

Optimize OrthostericAllosteric

Effects For Affinity and Selectivity "High Ligand Efficiency"

Establish a Thermodynamic Profile on Competitive Drugs

Confirm Analogues to Endogenous Ligands

Test Binding Affinity/IC50 Against Model Ligands

Confirm Mechanism Of Action

Optimize Protein Crystallization Potential

Drug Rescue

Fragment Based Screening

Confirm Binding Mechanisms

Topic of Discussion

Optimization Goals

Efficacy

Binding Affinity

Selectivity

Adaptability

Microcalorimetry Provides a Total Picture of Binding Energetics

SARS Coronavirus main protease

with an inhibitor docked into the binding pocket

Green dashes hydrogen bonds

Orange area are hydrophobic interactions

ITC Measures Overall Binding Affinity: The First Dimension of Binding Quality

Usually expressed as Kd

which typically correlates with IC50

or EC50

This is directly related to ∆G the total free binding energy

Binding affinity can give a general picture, but by itself lacks predictive

power and can result in false positives and

negatives

ITC Measures Enthalpy (∆H) : The Second Dimension of Binding Quality

∆H is a measure of hydrogen and van der

Waals bonding

between the surface groups on a ligand and the binding

pocket

These bonds are very specific to the binding

pocket and add selectivity to the drug

design process

ITC Determines Entropy (∆S) : The Third Dimension of Binding Quality

∆S is a measure of hydrophobic interaction between the ligand and

the macromolecule and conformational changes to the

ligand

Binding is due to repulsion from the solvent rather than specific attraction to the macromolecule

These bonds add to the affinity but are

very non-specific to the binding pocket

ITC Measures Stoichiometry (n): The Fourth Dimension of Binding Quality

n = Stoichiometry indicates the ratio of ligand molecules that bind each

macromolecule

The stoichiometry can provide insights into the mechanism

of action, non-specific binding, or indicate protein

degradation

n = 1:1

Protein

Ligand

Total Binding is Just Part of the Picture

Total “binding constant”

Contents of Binding SignatureWithout ITC

Indinavir Nelfinavir Saquinavir Ritonavir DrugYour

ΔG

-12

-16

-20

4

0

-4

-8

kca

l/m

ol

Mechanism of Binding Remains Unknown

ITC Provides Unique Information

Total “binding constant”(tightness of binding)

Identifies binding mechanism

Lock & key binding

Identifies opportunities to optimize selectivity

Contents of Binding SignatureWith ITC Results

Nelfinavir Saquinavir Ritonavir Drug

-8

-12

-16

-20

ΔG

ΔH

0

-4

TΔS

4

YourIndinavir

kca

l/m

ol

Mechanism of Binding Is Clearly Revealed

-

Optimizing Diaminopyrimidine Renin Inhibitors

Aided by ITC and structural data

Abstracted from Sarver, et al, Anal. Biochem. 2007

-12-10

-8-6-4-2024

1

Kca

l/mol

∆G ∆H -T∆S

The Starting Point – A “Weak” Binder

How can this information help us design a better molecule?

∆H is highly favorable

-T∆S is not favorable

∆G is low represented byIC50

=6.6 µM and Kd

=3.6 μM

Favo

rabl

e

The Binding Orientation for Lead Template to Renin was Determined X-ray Crystallography

favorable ∆H is consistent with the strong network of hydrogen bonds.

The unoccupied hydrophobic S2 and S3

pockets are opportunities to increase affinity

Modeling Suggest Addition of Ether Would Extend into S3 Pocket

-20

-15

-10

-5

0

5

10

1 2

kcal

/mol

∆G ∆H -T∆S

Increase in ∆H is consistent with increase in S3

pocket due to van der

Waals bonds

Structural alteration resulted in 7X improvement in affinity

Increase in unfavorable -T∆S

Modeling Suggest Adding Aryl-Benzamide to Extend into S2 Pocket

-20

-15

-10

-5

0

5

10

1 2 3

kcal

/mol

∆G ∆H -T∆S

Loss in ∆H indicating no significant hydrogen

bonds were formed

Another in 2X improvement in affinity

Significant increase in -T∆S

due to hydrophobic binding in S2

pocket

Modeling Suggests Substituting Aryl- Benzamide with Aryl-Sulfonamide to Improve H-bonds

-20

-15

-10

-5

0

5

10

1 2 3 4

kcal

/mol

∆G ∆H -T∆S

Another 3.4X improvement in affinity

Dramatic Increase in ∆H is consistent with increase in S2

pocket H-bonds

Decrease in -T∆S due to conversion

of hydrophobic binding in S2 pocket to H-

bonds

S2 S2

S3 S3

Renin Inhibitor Affinity Improved 45X from Initial 3.6 μM Lead to 79nM

•

S3 Pocket - Improved enthalpy due to van der

Waals bonds

•

S2 Pocket – Improved binding enthalpy while

retaining hydrophobic advantage

Aryl-Sulfonamide

Ether

Statins: Inhibitors of HMG CoA- Reductase

Dramatic increase in ∆H is consistent

improved hydrogen and van der

Waals bonds

Abstracted from Carbonell and Freire, Biochem. 2005

Clinically proven as least potent

statin

kcal

/mol

Statins: Inhibitors of HMG CoA- Reductase

Fluvastatin

PravastatinCerivastatin

Atorvastatin

Rosuvastatin-10

-8-6-4-202

6.5 7 7.5 8 8.5 9

y = 27.343 - 4.1749x R= 0.97679

Δ H k

cal/m

ol

log K a

•

Affinity increase of 300X•

Improvement of in hydrogen and van der Waals

binding (∆H) from 0 to -9 kcal/mole

(Crestor)

(Baycol)(Lipitor)

(Pravacol)

(Lescol)

kcal

/mol

The Evolution HIV-1 Protease Inhibitors Over 12 Years

Abstracted from Ohtaka and Freire, Biophy Mol Bio. 2005

-20000

-15000

-10000

-5000

0

5000

Indi

navi

r

Nel

finav

ir

Saq

uina

vir

Rito

navi

r

Am

pren

avir

Lopi

navi

r

Ata

zana

vir

KN

I-577

KN

I-272

KN

I-764

TMC

-126

TMC

-114

ΔG ΔH -TΔS

cal/m

ol

Unfavorable

Favorable12 YEARS

The Evolution HIV-1 Protease Inhibitors Over 12 Years• Affinity increase of 300X• Improvement in hydrogen and van der Waals binding (∆H) from +2 to -12.5

kcal/mole

Higher potency and better adaptability to protease polymorphisms

• Gold standard “first principles”

data –

reference standard for other

methods• A perfect complement to NMR and X-ray crystallography

Isothermal Titration Calorimetry Method of choice for complete characterization of biomolecular interactions

ITC Instruments

VP-ITC iTC200 Auto-iTC200

Sample Required: 50+ μg ~5-10 μg

~5-10 μg Throughput: 4 - 5 per day 8 - 16 per day 50 –100 +/day

Ease of Use: Good Better Best

Characterize a Broad Range of Interactions• Protein/Protein• Protein/Small

Molecule• Protein/Carbohydrate• Protein/Lipid• Protein/Nucleic Acid• Receptors/Ligand• Non-Biological

Molecules

• Lipid/Lipid Small Molecule

• Nucleic Acid/Small Molecule

• Nucleic Acid/Lipid• Nucleic Acid/Nucleic

Acid• Antibody/Ligand

Over 8,000 Literature Citations Here are some examples:

Applications Area Number of CitationsProtein-Small Molecule Interaction 1002

Protein-Protein Interactions 686

Protein-Carbohydrate Interactions 276

Protein-Lipid Interactions 140

Protein Folding and Structural Studies 1620

Protein Engineering and Mutagenesis 969

Lipid and Membrane Studies 601

Small Molecule-Small Molecule Interactions 211

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• Over 30 years of experience in calorimetry for the life sciences

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