points to consider when interpreting spr data: an insider ...research confidential points to...

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

“Simple kinetic model”


Global Fitting

a) one kon

b) one koff

c) one Rmax



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


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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

Bulk And Binding Responses


Flow •FlowFlow

Binding

Bulk And Binding Responses


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

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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


•4-(aminomethyl) benzene • sulfonamide

•sulpiride

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sulpiride

Benchmark Study


4-carboxy benzene sulfonamide

Benchmark Study

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Benchmark Study


acetozoalmide

Benchmark Study

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

Benchmark Study

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Benchmark Study


Experimental Error

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