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A Novel Quantitative Biochemical and Cellular Target Engagement Assay Platform for Drug Discovery

Abstract

It is well established that ligand binding protects proteins from thermal denaturation; however, the broad application of this concept to drug discovery has been hindered by a lack of simple platforms with sensitive and quantitative readouts. Current denaturation-based approaches, including Thermal Shift Assays (TSA) and Cellular Thermal Shift Assays (CETSA), are proven valuable technologies for particular applications, including family-wide selectivity profil-ing and cellular target engagement, respectively. However, each method has limitations. For example CETSA is a cumbersome low-throughput method and TSA lacks quantitative power. It is important that denaturation-based methods continue to evolve, as they are generic across target classes, can be applied to both biochemical and cellular target engagement, and can circumvent problematic requirements of other common biochemical target engagement formats, including the need for antibodies, immobilized capture ligands, purified proteins, and fluorescent labels. Here we describe HitHunter® Pulse, which capitalizes on two novel DiscoveRx technologies, Enzyme Fragment Complementation (EFC) and Pulse Denaturation, to overcome limitations of current denaturation- based methods. We have successfully applied HitHunter Pulse in both biochemical and cellular settings to rapidly measure quantitative and accurate target engagement potency values for inhibitors of diverse protein classes, including kinases and pseudokinases, bromodomains, methyltransferases, and hydro-lases. These data and results demonstrating that HitHunter Pulse can be applied to elucidate inhibitor mechanism of action shall be described.

Pulse Target Engagement Platform

Simple quantitative measurement of ligand-protein interactions in both cellular and biochemical milieus for diverse targets.

Quantitative denaturation-based ligand binding assays

• Measure quantitative cellular and biochemical inhibitor target-engagementpotencies

• Quantitative biochemical alternative to TSA; facile cellular alternative toCETSA

• Simple one-step readout: no wash, centrifugation, or immunoassay steps

• No protein purification, fluorescent tags, antibodies, or capture ligands

• Exploits DiscoveRx’s proprietary EFC and Pulse technologies

Biochemical and cellular assays validated for diverse targets

• Kinases, hydrolases, methyltransferases, bromodomains

• Accurate potency rank order for known inhibitors

Applications

• Potency rank ordering during lead optimization; HTS hit validation

• Cellular target engagement potency – do compounds enter cell and engagetarget?

• Ideal for targets difficult to assay in cellular milieu

• High throughput cellular and biochemical screens – development in progress

Split β-galactosidase Enzyme System Underpins Pulse Technology

Enzyme Fragment Complementation (EFC)

++

ProLabel™ (PL) ProLabel tagged native protein Enzyme Acceptor (EA)

Inactive Fragments Active Enzyme

EA

Substrate

Light

ProLabel (PL) is a dual-purpose peptide tag ideally suited for Pulse

• PL tag (~40 aa) allows for sensitive detection of properly folded “native” target protein (fM)

• PL tag senses folded state of protein: denatured/aggregated proteins poorly complement EA resulting in reduced luminescence

Pulse: How It Works

Biochemical target engagement Cellular target engagement

EA

No LigandBinding

LigandBinding

Pulse Denaturation

Protein Aggregate

Substrate

Light

Ligand

Protein Target

PLNo Ligand

Binding

LigandBinding

Pulse Denaturation

Protein Aggregate

Substrate

Stable Ligand-Protein Complex

Light

LigandPL TaggedProtein Target

EA

Ligand binding protects protein from thermal denaturation and increases EFC in a dose-dependent manner for the measurement of inhibitor EC50 values

• Similar protocols for live cells and extracted proteins

• PL-tagged target protein expressed in transiently transfected cells

– Cell extract prepared and treated with compound for biochemical studies

– Live cells treated directly with compound for cellular target engagement studies

• Compound-treated cells or extracted protein subjected to Pulse Denaturationin thermocycler

– Gentle Pulse Denaturation protocol does not compromise cell viability or membrane integrity

• For live cells EFC is performed in conjunction with cell lysis post-PulseDenaturation

• Gentle lysis conditions do not refold denatured/aggregated proteins

• Proprietary Pulse Denaturation step improves assay metrics, including assaywindow and EC50 accuracy relative to single step denaturation protocols

Pulse: Rapid and Simple Protocol

Add compounds & DMSO control to

PCR plate

Add PL-tagged target protein or

live cells expressing PL-tagged protein

Incubate Perform PulseDenaturation

Add EFC workingdetection solution

Incubate for 30 min.at room temperature

Read chemiluminescent signal

Overcoming Key Limitations of Denaturation-based Methods

EFC overcomes key limitations of existing denaturation-based methods for biochemical and cellular target engagement

Process bottlenecks

• No sample processing required (e.g. centrifugation or immunoassay)

• No protein purification required

Quantitative power and simplicity of readout

• Superior to immunoassay or TSA

• Sensitive, precise, simple, and quantitative with broad dynamic range

Pulse denaturation improves EC50 accuracy

EC50 values right shift during aggressive denaturation protocols

• Reduced denaturation times and temperatures improve EC50 accuracy

• Pulse denaturation addresses this challenge

Pulse Assay Status

HitHunter Pulse

• 10 validated assays covering 5 target classes

• 8 assay ready kits covering 5 target classes

InCELL Pulse

• 5 validated assays covering 4 target classes

Target Target Class HitHunter Pulse Assay Status InCELL Pulse Assay StatusBRD4(1)

Bromodomain

CREBBP EP300 ABL1

Kinase

ABL1(T315I) BUB1 BRAF MLKL Pseudokinase G9a Methyltransferase MTH1 Hydrolase

Assay ready kits Validated assay In progress Not determined

Pulse Validation

HitHunter Pulse InCELL Pulse

BRD4(1) bromodomain

1.0

0.8

0.6

0.4

0.2

0.0

100

101

102

103

104

Ass

ay S

igna

l (no

rm.)

[Inhibitor], nM

JQ1BromosporineSGC-CBP30

Biochemical target engagement assay

Inhibitor EC50 (nM)JQ1 220Bromosporine 700SGC-CBP30 3,000

• Robust assay window (4.1); JQ1 EC50 in agree-ment with published ITC KD value (50 nM)

• Diverse BRD4(1) inhibitors give expectedpotency rank order

3.0

2.5

2.0

1.5

1.0

100

101

102

103

104

Ass

ay S

igna

l (R

LU x

106

)

[JQ1], nM

Cellular target engagement assay

Inhibitor EC50 (nM)JQ1 390

• Robust assay window (3.5); good agreementwith published cellular IC50 from c-Myc expres-sion assay (100 nM)

ABL1 tyrosine kinase

1.0

0.8

0.6

0.4

0.2

0.0

10-2

10-1

100

101

102

103

104

Ass

ay S

igna

l (no

rm.)

[Inhibitor], nM

Biochemical target engagement assay

Dasatinib: EC50 = 0.57 nM Nilotinib: EC50 = 0.81 nM Ponatinib: EC50 = 1.3 nM Imatinib: EC50 = 6.1 nM VX-680: EC50 = 48 nM Staurosporine: EC50 = 320 nM SU-14813: EC50 = 650 nM Purvalanol B: EC50 = 3400 nM

• Robust assay window (3.2)• Diverse ABL1 inhibitors

including both approved drugsand tool compounds giveexpected potency and rankorder

• Companion ABL1(T315I) resis-tance mutant assay showsselective sensitivity to Pona-tinib as expected (data notshown)

500

400

300

200

10-1

100

101

102

103

104

Ass

ay S

igna

l (R

LU x

103

)

[Inhibitor], nM

Cellular target engagement assay

DasatinibVX-680

Inhibitor EC50 (nM)Dasatinib 2.8VX-680 570

• Robust assay windows (4-5 fold)• Good agreement with published dasatinib

cellular potency data (IC50 = 1 nM)

MTH1 hydrolase

600

500

400

300

200

100

101

102

103

104

105

Ass

ay S

igna

l (R

LU x

103

)

[SCH 51344], nM

TH588(S)-CrizotinibSCH 51344

Biochemical target engagement assay

Inhibitor EC50 (nM) Published IC50 (nM)TH588 93 15(S)-Crizotinib 2,600 70SCH 51344 3,000 50

• Robust assay windows (~4-fold)• Diverse inhibitors give correct potency rank order• TH588 EC50 offset from published value only

6-fold; larger offsets for other two inhibitors

750

700

650

600

550

500

400

Ass

ay S

igna

l (R

LU x

103

)

[SCH 51344], nM

Cellular target engagement assay

100

101

102

103

104

Inhibitor EC50 (nM) Published IC50 (nM)SCH 51344 370 < 20,000

• Simple solution for target difficult to query in cells• Published SCH 51344 cellular IC50 < 20 µM• Optimized assay window >3-fold

G9a methyltransferase

1.0

0.8

0.6

0.4

0.2

0.0

Ass

ay S

igna

l (no

rm.)

[UNC0638], nMPublished UNC0638 biochemical IC50 = 10 nM

Biochemical target engagement assay

100

101

102

103

104

Plus SAMMinus SAM

Inhibitor EC50 (nM)UNCO638 (+SAM) 22

UNCO638 (-SAM) 480

• Cooperative binding of UNC0638 and SAMcofactor reveals uncompetitive inhibitionmechanism

• Robust assay window (3.5) and accurate inhibitorpotency data

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

1.0 1 10 100 1000

Ass

ay S

igna

l (R

LU x

106

)

[UNC0638], nMPublished UNC0638 cellular IC50 = 80 nM

Cellular target engagement assay

EC50 (nM) Assay WindowInhibitor UNCO638 13 5.0

• Robust assay window and good agreement withpublished UNC0638 cell potency data

• Much simpler solution than measuring cellularhistone methylation levels

Summary

HitHunter Pulse Pulse InCELL PulseBiochemical target engagement Cellular target engagementTechnology

Novel biochemical and cellular ligand binding assay platform

• Improves upon and complements existing denaturation-based methodsincluding TSA and CETSA

• Simple, quantitative method with minimal reagent requirements

• Proof of concept demonstrated for five diverse target classes

• Serves major unmet need for cellular target engagement assays

Applications

• Biochemical and cellular target affinities to support lead optimization

• HTS hit validation and proof of cellular target engagement

• Inhibitor mechanism of action

• Biochemical and cellular HTS applications in development

• Applications for integral membrane proteins in development

Elena Menichelli, Pietro Ciceri, Alex Lun, Mark Floyd, and Daniel K. Treiber

DiscoveRx Corporation, Fremont, CA 94538

HitHunter Pulse InCELL Pulse

Daniel K. Treiber and Elena Menichelli are the co-inventors on a pending patent application covering aspects of the Pulse method, to be assigned to DiscoveRx Corporation.