Page 1
CONFIDENTIALRESEARCH
Points to Consider When Interpreting SPR Data: An
Insider’s Perspective
Joe Papalia, Gilead Sciences
Page 2
CONFIDENTIALRESEARCH2
• The basics of the SPR response
Overview
• How do we describe avidity?
• What is mass transport?
• How reproducible is binding data?
Page 3
CONFIDENTIALRESEARCH
Biosensors: The Basics
Page 4
CONFIDENTIALRESEARCH
Biosensors: The Basics
Page 5
CONFIDENTIALRESEARCH
Flow Flow
Biosensors: The Basics
Page 6
CONFIDENTIALRESEARCH
FlowFlow
Biosensors: The Basics
Page 7
CONFIDENTIALRESEARCH
Flow Flow
Ligand
Biosensors: The Basics
Page 8
CONFIDENTIALRESEARCH
Flow Flow
LigandAnalyte
Biosensors: The Basics
Page 9
CONFIDENTIALRESEARCH
Flow Flow
LigandAnalyte
θ
Biosensors: The Basics
Page 10
CONFIDENTIALRESEARCH
Flow
θ
LigandAnalyte
Flow
Biosensors: The Basics
Page 11
CONFIDENTIALRESEARCH11
Association, kon, koff
Processed Data:Sensorgrams
Page 12
CONFIDENTIALRESEARCH12
Equilibrium/Steady State
Processed Data:Sensorgrams
Page 13
CONFIDENTIALRESEARCH13
Dissociation, koff
Processed Data:Sensorgrams
Page 14
CONFIDENTIALRESEARCH14
Processed Data:Sensorgrams
Page 15
CONFIDENTIALRESEARCH15
Concentration
Processed Data:Sensorgrams
Page 16
CONFIDENTIALRESEARCH16
Global Fitting
Page 17
CONFIDENTIALRESEARCH17
Global Fitting
Page 18
CONFIDENTIALRESEARCH18
Global Fitting
Page 19
CONFIDENTIALRESEARCH19
Global Fitting
“Simple kinetic model”
Page 20
CONFIDENTIALRESEARCH20
Global Fitting
a) one kon
b) one koff
c) one Rmax
“Simple kinetic model”
Page 21
CONFIDENTIALRESEARCH21
Dose Response
Page 22
CONFIDENTIALRESEARCH22
Dose Response
Page 23
CONFIDENTIALRESEARCH23
Dose Response
Page 24
CONFIDENTIALRESEARCH24
Dose Response
Page 25
CONFIDENTIALRESEARCH25
Dose Response
KD, Rmax
Page 26
CONFIDENTIALRESEARCH26
• How do we describe avidity?
Overview
Page 27
CONFIDENTIALRESEARCH27
Experimental DesignAvidity
Not that straightforward to interpret
Page 28
CONFIDENTIALRESEARCH28
ΔG°FabΔG°Fab
Single-Site BindingExperimental Design
Page 29
CONFIDENTIALRESEARCH29
Anti-Fc
ΔG°FabΔG°Fab
Experimental DesignSingle-Site Binding
Page 30
CONFIDENTIALRESEARCH30
Anti-Fc
ΔG°FabΔG°Fab
Experimental DesignSingle-Site Binding
a) one kon
b) one koff
c) one Rmax
“Simple kinetic model”
Page 31
CONFIDENTIALRESEARCH31
Experimental Design
a) 2 kon’s
b) 2 koff’s
c) one Rmax
Not a “Simple kinetic model”
Avidity
Avoid this if you can
Page 32
CONFIDENTIALRESEARCH32
• What is mass transport?
Overview
Page 33
CONFIDENTIALRESEARCH
Mass Transport
When ligand (surface) demand > analyte supply
A Description
Page 34
CONFIDENTIALRESEARCH
Flow Flow
LigandAnalyte
Biosensors: The Basics
Page 35
CONFIDENTIALRESEARCH
Under MTL…
Mass Transport
Some Math
Page 36
CONFIDENTIALRESEARCH
•J Biol Chem 2015 Mar 27;290(13):8439-46
Mass Transport: Idelalisb Binding
Page 37
CONFIDENTIALRESEARCH
Mass Transport Limited KineticsCompound “B” binding to PI3Kδ
Page 38
CONFIDENTIALRESEARCH
Actual KineticsCompound “B” binding to PI3Kδ
Page 39
CONFIDENTIALRESEARCH
Sometimes difficult to ascribe a unique exponential to such
linear data
Actual Kinetics
Page 40
CONFIDENTIALRESEARCH40
• How reproducible is biosensor data?
Page 41
CONFIDENTIALRESEARCH41
Experimental Error
Page 42
CONFIDENTIALRESEARCH
Benchmark Study
Limit of off-rate detection
Page 43
CONFIDENTIALRESEARCH
•a
•b
•c
•d
•e
•f
•g
•h
•a
•b
•c
•d
•e
•f
•g
•h
User
1
2
3
4
5
6
7
8
When data are good
Benchmark Study
Page 44
CONFIDENTIALRESEARCH44
When data are not so good
Benchmark Study
Page 45
CONFIDENTIALRESEARCH45
Benchmark Study
CV-kon = 15% CV-koff = 13% CV-KD = 18%
Page 46
CONFIDENTIALRESEARCH46
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
Page 47
CONFIDENTIALRESEARCH47
Back-Ups
Page 48
CONFIDENTIALRESEARCH
Flow •FlowFlow
The Bulk Response
Page 49
CONFIDENTIALRESEARCH
Flow •FlowFlow
The Bulk Response
Page 50
CONFIDENTIALRESEARCH
Flow •FlowFlow
The Bulk Response
Page 51
CONFIDENTIALRESEARCH
Flow •FlowFlow
Bulk And Binding Responses
Page 52
CONFIDENTIALRESEARCH
Flow •FlowFlow
Bulk And Binding Responses
Page 53
CONFIDENTIALRESEARCH
Flow •FlowFlow
Binding
Bulk And Binding Responses
Page 54
CONFIDENTIALRESEARCH54
Experimental DesignImmobilize Antigen or ‘Antigen Down’
Page 55
CONFIDENTIALRESEARCH55
Experimental DesignFirst Step: Single Site Binding
Page 56
CONFIDENTIALRESEARCH56
Experimental DesignAvidity
Page 57
CONFIDENTIALRESEARCH57
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 **
Page 58
CONFIDENTIALRESEARCH58
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
Page 59
CONFIDENTIALRESEARCH59
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
Page 60
CONFIDENTIALRESEARCH
Wortmannin: Irreversible T100 Data
Idelalisb Binding to PI3Kδ
Page 61
CONFIDENTIALRESEARCH
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
Page 62
CONFIDENTIALRESEARCH
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
Page 63
CONFIDENTIALRESEARCH
Correlation Matrix & kon x Rmax / kt
kon* Rmax / kt ≤ 5
Page 64
CONFIDENTIALRESEARCH
Cmpd “B” Binding to PI3Kδ
Page 65
CONFIDENTIALRESEARCH
Target
1
5
6 74
32 8 9
10
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
Page 66
CONFIDENTIALRESEARCH
•4-(aminomethyl) benzene • sulfonamide
•sulpiride
•a
•b
•c
•d
•e
•f
•g
•h
User
1
2
3
4
5
6
7
8
sulpiride
Benchmark Study
Page 67
CONFIDENTIALRESEARCH
4-carboxy benzene sulfonamide
Benchmark Study
User
1
2
3
4
5
6
7
8
Page 68
CONFIDENTIALRESEARCH
Benchmark Study
Page 69
CONFIDENTIALRESEARCH
acetozoalmide
Benchmark Study
User
1
2
3
4
5
6
7
8
Page 70
CONFIDENTIALRESEARCH
7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide
Benchmark Study
User
1
2
3
4
5
6
7
8
Page 71
CONFIDENTIALRESEARCH
Benchmark Study
Page 72
CONFIDENTIALRESEARCH72
Experimental DesignAvidity
Page 73
CONFIDENTIALRESEARCH73
Experimental Error