Points to Consider When Interpreting SPR Data: An … · RESEARCH CONFIDENTIAL Points to Consider When Interpreting SPR Data: An Insider’s Perspective . Joe Papalia, Gilead Sciences

CONFIDENTIALRESEARCH

Points to Consider When Interpreting SPR Data: An

Insider’s Perspective

Joe Papalia, Gilead Sciences

CONFIDENTIALRESEARCH2

• The basics of the SPR response

Overview

• How do we describe avidity?

• What is mass transport?

• How reproducible is binding data?


Biosensors: The Basics




Flow Flow



FlowFlow



Flow Flow

Ligand



Flow Flow

LigandAnalyte



Flow Flow

LigandAnalyte

θ



Flow

θ

LigandAnalyte

Flow



Association, kon, koff

Processed Data:Sensorgrams


Equilibrium/Steady State



Dissociation, koff





Concentration



Global Fitting


Global Fitting


Global Fitting


Global Fitting

“Simple kinetic model”


Global Fitting

a) one kon

b) one koff

c) one Rmax



Dose Response


Dose Response


Dose Response


Dose Response


Dose Response

KD, Rmax


• How do we describe avidity?

Overview


Experimental DesignAvidity

Not that straightforward to interpret


ΔG°FabΔG°Fab

Single-Site BindingExperimental Design


Anti-Fc

ΔG°FabΔG°Fab

Experimental DesignSingle-Site Binding


Anti-Fc

ΔG°FabΔG°Fab

Experimental DesignSingle-Site Binding

a) one kon

b) one koff

c) one Rmax



Experimental Design

a) 2 kon’s

b) 2 koff’s

c) one Rmax

Not a “Simple kinetic model”

Avidity

Avoid this if you can


• What is mass transport?

Overview


Mass Transport

When ligand (surface) demand > analyte supply

A Description


Flow Flow

LigandAnalyte



Under MTL…

Mass Transport

Some Math


•J Biol Chem 2015 Mar 27;290(13):8439-46

Mass Transport: Idelalisb Binding


Mass Transport Limited KineticsCompound “B” binding to PI3Kδ


Actual KineticsCompound “B” binding to PI3Kδ


Sometimes difficult to ascribe a unique exponential to such

linear data

Actual Kinetics


• How reproducible is biosensor data?


Experimental Error


Benchmark Study

Limit of off-rate detection


•a

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User

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When data are good

Benchmark Study


When data are not so good

Benchmark Study


Benchmark Study

CV-kon = 15% CV-koff = 13% CV-KD = 18%


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


Back-Ups


Flow •FlowFlow

The Bulk Response


Flow •FlowFlow

The Bulk Response


Flow •FlowFlow

The Bulk Response


Flow •FlowFlow

Bulk And Binding Responses


Flow •FlowFlow



Flow •FlowFlow

Binding



Experimental DesignImmobilize Antigen or ‘Antigen Down’


Experimental DesignFirst Step: Single Site Binding





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 **



*


Fab* ΔGs



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



*


Fab* ΔGs



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


Wortmannin: Irreversible T100 Data

Idelalisb Binding to PI3Kδ


Idelalisib

kon = 5.1 x 106 M-1s-1

koff = 5.8 x 10-3 s-1

KD = 1.1 nM




Idelalisib



kon = 5.1 x 106 M-1s-1

koff = 5.8 x 10-3 s-1

KD = 1.1 nM


Correlation Matrix & kon x Rmax / kt

kon* Rmax / kt ≤ 5


Cmpd “B” Binding to PI3Kδ


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


•4-(aminomethyl) benzene • sulfonamide

•sulpiride

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User

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sulpiride

Benchmark Study


4-carboxy benzene sulfonamide

Benchmark Study

User

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Benchmark Study


acetozoalmide

Benchmark Study

User

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7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide

Benchmark Study

User

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Benchmark Study




Experimental Error

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Points to Consider When Interpreting SPR Data: An … · RESEARCH CONFIDENTIAL Points to Consider When Interpreting SPR Data: An Insider’s Perspective . Joe Papalia, Gilead Sciences