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CONFIDENTIALRESEARCH
Points to Consider When Interpreting SPR Data: An
Insider’s Perspective
Joe Papalia, Gilead Sciences
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• The basics of the SPR response
Overview
• How do we describe avidity?
• What is mass transport?
• How reproducible is binding data?
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Biosensors: The Basics
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Biosensors: The Basics
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Flow Flow
Biosensors: The Basics
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FlowFlow
Biosensors: The Basics
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Flow Flow
Ligand
Biosensors: The Basics
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Flow Flow
LigandAnalyte
Biosensors: The Basics
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Flow Flow
LigandAnalyte
θ
Biosensors: The Basics
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Flow
θ
LigandAnalyte
Flow
Biosensors: The Basics
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Association, kon, koff
Processed Data:Sensorgrams
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Equilibrium/Steady State
Processed Data:Sensorgrams
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Dissociation, koff
Processed Data:Sensorgrams
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Processed Data:Sensorgrams
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Concentration
Processed Data:Sensorgrams
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Global Fitting
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Global Fitting
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Global Fitting
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Global Fitting
“Simple kinetic model”
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Global Fitting
a) one kon
b) one koff
c) one Rmax
“Simple kinetic model”
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Dose Response
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Dose Response
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Dose Response
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Dose Response
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Dose Response
KD, Rmax
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• How do we describe avidity?
Overview
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Experimental DesignAvidity
Not that straightforward to interpret
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ΔG°FabΔG°Fab
Single-Site BindingExperimental Design
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Anti-Fc
ΔG°FabΔG°Fab
Experimental DesignSingle-Site Binding
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Anti-Fc
ΔG°FabΔG°Fab
Experimental DesignSingle-Site Binding
a) one kon
b) one koff
c) one Rmax
“Simple kinetic model”
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Experimental Design
a) 2 kon’s
b) 2 koff’s
c) one Rmax
Not a “Simple kinetic model”
Avidity
Avoid this if you can
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• What is mass transport?
Overview
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Mass Transport
When ligand (surface) demand > analyte supply
A Description
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Flow Flow
LigandAnalyte
Biosensors: The Basics
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Under MTL…
Mass Transport
Some Math
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•J Biol Chem 2015 Mar 27;290(13):8439-46
Mass Transport: Idelalisb Binding
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Mass Transport Limited KineticsCompound “B” binding to PI3Kδ
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Actual KineticsCompound “B” binding to PI3Kδ
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Sometimes difficult to ascribe a unique exponential to such
linear data
Actual Kinetics
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• How reproducible is biosensor data?
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Experimental Error
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Benchmark Study
Limit of off-rate detection
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When data are good
Benchmark Study
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When data are not so good
Benchmark Study
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Benchmark Study
CV-kon = 15% CV-koff = 13% CV-KD = 18%
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Summary• The basics of the SPR response
• Avidity can be difficult to deal with
• MT; ligand demand > analyte supply
• Good CV’s; but understand limitations
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Back-Ups
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Flow •FlowFlow
The Bulk Response
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Flow •FlowFlow
The Bulk Response
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Flow •FlowFlow
The Bulk Response
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Flow •FlowFlow
Bulk And Binding Responses
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Flow •FlowFlow
Bulk And Binding Responses
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Flow •FlowFlow
Binding
Bulk And Binding Responses
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Experimental DesignImmobilize Antigen or ‘Antigen Down’
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Experimental DesignFirst Step: Single Site Binding
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Experimental DesignAvidity
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Experimental DesignAvidity
Adapted from W.P. Jencks (1981) , PNAS Vol. 78, No.7, pp 4046-4050 *
ΔG° = + + = RTln(KD-Overall)Overall ΔGiFab ΔGi
Fab* ΔGs
ΔG° = + + + (i-1) + + - RTln(Ωi / Ωo)N iΔH°affinity ** (i) i TΔS°
affinity ΔH°trans+rotTΔS°(i-1) linker (i-1)TΔS°
conf ΔG°coop
From V.M. Krishnamurthy et al. from “Multivalency in Drug Design” in Fragment-based Approaches in Drug Discovery **
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Experimental DesignAvidity
*
ΔG° = + + = RTln(KD-Overall)Overall ΔGiFab ΔGi
Fab* ΔGs
ΔG° = + + + (i-1) + + - RTln(Ωi / Ωo)N iΔH°affinity ** (i) i TΔS°
affinity ΔH°trans+rotTΔS°(i-1) linker (i-1)TΔS°
conf ΔG°coop
From V.M. Krishnamurthy et al. from “Multivalency in Drug Design” in Fragment-based Approaches in Drug Discovery **Adapted from W.P. Jencks (1981) , PNAS Vol. 78, No.7, pp 4046-4050
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Experimental DesignAvidity
*
ΔG° = + + = RTln(KD-Overall)Overall ΔGiFab ΔGi
Fab* ΔGs
ΔG° = + + + (i-1) + + - RTln(Ωi / Ωo)N iΔH°affinity ** (i) i TΔS°
affinity ΔH°trans+rotTΔS°(i-1) linker (i-1)TΔS°
conf ΔG°coop
From V.M. Krishnamurthy et al. from “Multivalency in Drug Design” in Fragment-based Approaches in Drug Discovery **Adapted from W.P. Jencks (1981) , PNAS Vol. 78, No.7, pp 4046-4050
Try to avoid this
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Wortmannin: Irreversible T100 Data
Idelalisb Binding to PI3Kδ
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Idelalisib
kon = 5.1 x 106 M-1s-1
koff = 5.8 x 10-3 s-1
KD = 1.1 nM
Wortmannin: Irreversible T100 Data
Idelalisb Binding to PI3Kδ
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Idelalisib
Wortmannin: Irreversible T100 Data
Idelalisb Binding to PI3Kδ
kon = 5.1 x 106 M-1s-1
koff = 5.8 x 10-3 s-1
KD = 1.1 nM
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Correlation Matrix & kon x Rmax / kt
kon* Rmax / kt ≤ 5
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Cmpd “B” Binding to PI3Kδ
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Target
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6 74
32 8 9
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compound KI (µM) potency1 50 very low2 10 medium3 4 low4 2 medium5 1 high6 1 low7 1 low8 1 low9 0.05 high10 0.05 high
Benchmark Study
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•4-(aminomethyl) benzene • sulfonamide
•sulpiride
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sulpiride
Benchmark Study
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4-carboxy benzene sulfonamide
Benchmark Study
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Benchmark Study
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acetozoalmide
Benchmark Study
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7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide
Benchmark Study
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Benchmark Study
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Experimental DesignAvidity
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Experimental Error
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