Mr. Marc Hixson Hixson@progenra.com

www.progenra.com

Progenra’s UbiPro™ Drug Discovery Platform:

Ubiquitin Ligase Technologies

for Drug Discovery

Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com Property of Progenra, Inc.

CONTACT:

COMPANY SUMMARYProgenra is a biotechnology company focused on exploiting ubiquitin and ubiquitin-like protein pathways to develop medicines to treat a wide range of diseases. Over the past nine years, Progenra has developed its UbiPro™ Drug Discovery Platform for quantifying and characterizing the activity of these enzymes. This platform is amenable to high throughput screening and has been employed successfully by Progenra to identify inhibitors of both deubiquitylating enzymes and E3 ligases.

BENFITS OF PROGENRA’S UBIPRO™ DRUG DISCOVERY PLATFORMOur partners will obtain several key advantages by using Progenra’s UbiPro™ Drug Discovery Platform. They include but are not limited to:

• Identification of compounds not detectable using traditional “off-the-shelf” assays and reagents

• A proven and patent-pending technology for precluding the identification of reactive “nuisance” screening hits

• Large panel of E3 ligases to be used for profiling and selectivity

• Selective and semi-selective E3 ligase inhibitors as molecular probes

• Use of homogeneous assays that closely replicate physiological milieux

• A comprehensive vs. singular approach to inhibitor identification

• Best commercial platform available that covers both DUB and E3 Ligase drug discovery

• Ability to instantly advance a partner’s DUB or E3 Ligase discovery program

AN EXAMPLE OF PROGENRA’S UBIQUITIN E3 LIGASE SCREENING TECHNOLOGIESTraditionally, the study of ubiquitin ligases has been performed using a combination of SDS-PAGE and Western blotting. These assays are limited by their laborious and low throughput nature. Some high throughput assays have been developed but are limited in utility because of their use of non-native ubiquitin. Addition of protein or chemical moieties to ubiquitin has been shown to have a negative impact on ubiquitin function. To address these concerns Progenra has developed several proprietary screening technologies to enable the discovery of selective ubiquitin ligase inhibitors (Table 1).

Assay Format Benefits and Applications

SDS-PAGE/ Western Blotting

Low Throughput

E3LITE Assay Broad Utility for all E3 ligase families

Homogenous E3 Ligase Assay

Homogenous format

Scalable to 384-well & 1536-well format

Amenable to measuring substrate ubiquitylation

E3-Substrate Binding Assay

Measures the selective binding of a substrate to a particular E3 ligase

Table 1: Current Suite of E3 Ligase Assays Available from Progenra

Figure 1: E3LITE Assay Platform for Measuring E3 Ligase Activity. A) Schematic of the E3LITE assay platform. B) The E3LITE assay generates a dose dependent signal with increasing concentrations of MuRF1. C) The E3LITE assay platform exhibits broad utility with different classes of E3s.

Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

E3LITE Assay Format

Progenra has developed a novel proprietary ligase assay platform that takes advantage of ubiquitin binding domains’ inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase dependent manner (Figure 1A). The immobilization of ubiquitin binding domains on microtiter plates allows the capture and isolation of polyubiquitylated proteins from in vitro ubiquitylation reactions. Subsequently, the relative amounts of polyubiquitylated pro-teins can be measured, enabling quantification of E3 ligase activity (Figure 1B). This assay has been used successfully with members of both simple and multimeric RING and HECT families, demonstrating the platform’s broad utility for analyzing the activity of a wide range of E3 ligases (Figure 1C).

01.7 3.4

6.75

12.5 25 50

100

0

5.0!104

1.0!105

1.5!105

2.0!105

2.5!105

[MuRF1], nM

RL

U

MuR

F1

Atrogin

1Hrd

1

Parki

n

CARP2

Praja

1

0

1.0!105

2.0!105

3.0!105

4.0!105

5.0!105

E3 Ligases

RF

U

A.

B. C.

01.7 3.4

6.75

12.5 25 50

100

0

5.0!104

1.0!105

1.5!105

2.0!105

2.5!105

[MuRF1], nM

RL

U

MuR

F1

Atrogin

1Hrd

1

Parki

n

CARP2

Praja

1

0

1.0!105

2.0!105

3.0!105

4.0!105

5.0!105

E3 Ligases

RF

U

01.7 3.4

6.75

12.5 25 50

100

0

5.0!104

1.0!105

1.5!105

2.0!105

2.5!105

[MuRF1], nM

RL

U

MuR

F1

Atrogin

1Hrd

1

Parki

n

CARP2

Praja

1

0

1.0!105

2.0!105

3.0!105

4.0!105

5.0!105

E3 Ligases

RF

U

Homogenous E3 Ligase Assay

Recently Progenra converted the E3LITE assay into a homogenous assay format consisting of an epitope tagged E3 ligase and a biotin tagged TUBE (Figure 2A). Following E3 autoubiquitylation, the TUBE binds to the polyubiquitylated E3 and a signal is generated using a TR-FRET detection format. The assay requires all the components of the ubiquitylation reaction to generate a positive signal (Figure 2B) and is able to report dose dependent increases in signal (Figure 2C). Simply by switching to an epitope tagged substrate instead of a tagged E3, this assay format can also be configured to measure substrate ubiquitylation directly.

E3 Ligase-Substrate Binding Assay

One of the primary functions of E3 ligases is to coordinate the interaction of E2 conjugating enzymes and proteins targeted for ubiquitylation. It can be advantageous to measure the direct binding of E3 ligases to their substrate proteins. In certain cases the region of the substrate that binds E3 ligase has been identified and a number of investigators have shown that its binding to E3 ligase can measured using fluorescence polarization (Figure 3). By utilizing a fluorescence polarization format, Progenra has developed an E3-ligase substrate binding assay, which can be used either in initial HTS or as a secondary assay to assist in the determination of the mechanism of action of an identified inhibitor. If the substrate-E3 Ligase interaction has not been mapped or consists of large protein domains unsuitable for use with FP it is possible to establish alternative fluorescence based assays for measuring substrate-E3 ligase interactions.

Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

Figure 2: Homogenous Assay for Measuring E3 Ligase Activity. A) Diagram of homogeneous assay format. B) MuRF1 autoubuiqtylation requires all the components of the ubiquitylation reaction. C) The homogeneous assay format generates a dose dependent increase in signal with increasing concentrations of MuRF1.

Figure 3: Measurement of Substrate Binding to E3 Ligase. A small peptide which binds to an E3 ligase is labeled with a fluorophore (i.e. Fluorescein). Binding of this labeled peptide to the E3 ligase can be monitored by a change in the fluorescence polarization. Inhibition of this binding event by a small molecule or unlabeled peptide will prevent this change in fluorescence polarization.

01.5

6

3.125

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[GST-MuRF1], nM

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GST-MuRF1 + + + - + +

ATP + + + + + -

E2 + + - + + +

Ubiquitin + + + + - +

E1 + - + + + +

Ratio

A.

B. C.

Progenra, Inc. 277 Great Valley Parkway, Malvern PA 19355 (p) 610.644.6974 (f) 610.644.8616 www.progenra.com

Progenra, Inc. 277 Great Valley Parkway, Malvern PA 19355 (p) 610.644.6974 (f) 610.644.8616 www.progenra.com

Inhibition by Small Molecule or Unlabeled Competitive

Peptide; Low FP

Increased Fluorescence Polarization

Low Fluorescence Polarization

TARGET IDENTIFICATION AND VALIDATION

Available Reagents

Progenra has amassed a large collection of ubiquitin conjugating enzymes and validated many of the ligases for high throughput screening. Progenra has utilized a plethora of expression systems including bacterial, insect, yeast, mammalian and cell-free systems to produce high quality enzymes for functional studies. Progenra performs extensive biochemical characterization of these enzymes using a variety of substrates to identify the optimal assay conditions for a given enzyme. Proper selection of assay conditions is essential to successful drug discovery. Previous screening efforts have yielded a variety of useful tool compounds that can be used during the optimization process.

Substrate Identification Technology

One of the major limitations in the relatively new field of the ubiquitin-proteasome pathway is the identification of substrates of E3 ubiquitin ligases. Working in collabora-tion with LifeSensors, Progenra has developed array technology enabling the detection of substrates for different E3 ligases (Figure 4). Figure 4 demonstrates that using microarray technology we can identify selective and non-selective substrates of E3 ligases. We are currently in the process of confirming these results using additional in vitro assays.

BIOCHEMICAL CHARACTERIZATION OF E2-E3 INTERACTIONSOne generally overlooked issue of working with E3 ubiquitin ligases is the use of the appropriate cognate E2 enzyme. To address this, Progenra uses the E3LITE assay platform to identify the appropriate E2 enzyme(s) for each E3 enzyme it studies (Figure 5A). Furthermore, using the same platform it is possible to determine apparent affinities of the different E2s for E6AP (Figure 5B).

Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

Figure 4: Identification of E3 ligase Substrates using Protein Microarrays. Arrays of immobilized proteins were incubated with Ubiquitin E1, UBE2D3, Ubiquitin, and ATP in the presence or absence of the indicated E3 ligass. The ubiquitylation components were removed and protein ubiquitylation was detected using an antibody to ubiquitin.

Figure 5: Characterization of E2-E3 Interactions. A) Ubiquitin conjugate formation was determined with a panel of E2s. B) The affinity of various E2s for Praja1 was determined by fitting the reaction rates to the Michaelis-Menten equation.

UBE2K

UBE2A

UBE2G2

UBE2R1

UBE2D2

UBE2D3

UBE2E3

UBE2H

UBE2I

UBE2F

UBE2M

UBE2N/UBE2V2

UBE2S

UBE2C

UBE2W

UBE2Z

UBE2L3

UBE2L6

0

1.0!107

2.0!107

3.0!107No Ligase

With Praja1

RLU

UBE2K

UBE2A

UBE2G2

UBE2R1

UBE2D2

UBE2D3

UBE2E3

UBE2H

UBE2I

UBE2F

UBE2M

UBE2N/UBE2V2

UBE2S

UBE2C

UBE2W

UBE2Z

UBE2L3

UBE2L6

0

1.0!107

2.0!107

3.0!107No Ligase

With Praja1

RLU

A.

B.

INHIBITOR IDENTIFICATION AND PROFILINGA major issue with E3 ligases drug discovery is the dearth of E3 ligase inhibitors in clinical trials. To address this issue, Progenra configured the E3LITE assay platform for use in HTS (Figure 6A) and screened for inhibitors of multiple E3s including MuRF1 (Figure 6B). A hit from the MuRF1 screen, P013222 is currently being optimized by medicinal chemistry, more information about this series is available upon request.

THE UBIPRO™ DRUG DISCOVERY PLATFORM’S PROVEN ADVANTAGEBenefits of Progenra’s novel E3 ligase assay platform developed to date:

• Generates quantitative readouts using standard plate readers

• Exhibits HTS compliance: 96, 384 & 1536 well plate formats

• Possesses a broad utility with multiple families of E3 ligases

• Utilizes native, untagged ubiquitin

• Has been used to successfully identify E3 ligase inhibitors with cellular efficacy

• Is complemented by a large panel of E2 conjugating enzymes and E3 ligases

Furthermore, Progenra continues to innovate and these assays will be complemented by additional substrate specific assays currently in development.

PARTNERSHIP OPPORTUNITIESProgenra is open to discussion of mutually beneficial licensing agreements for the screening platforms and/or the early stage inhibitors Progenra has discovered. Research collaborations on one or more targets are of high interest to Progenra.

PARTNERING WITH PROGENRA INCLUDES:• Unparalleled expertise in the Ubiquitin

Proteasome pathway

• Access to all the tools and technology under Progenra’s UbiPro™ Drug Discovery Platform on an exclusive or non-exclusive basis to identify and develop a modifier of a deubiquitylase or ligase for use as a therapeutic

• Research and early development work to assist in the identification and development of a proposed therapeutic, including counterscreening and selectivity screening

• Development of new targets within the ubiquitin- proteasome pathway for the identification of modifiers of a specific ligase or isopeptidase intended for development as a therapeutic

Research collaborations with Progenra allow partners to overcome a formidable barrier to working in the ubiquitin field, i.e., access to Progenra’s intellectual property, know-how, and technologies.

Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

Figure 6: Identification of E3 ligase Inhibitors. A) The E3LITE assay platform exhibits a Z’ of >0.5 in 384 well plates. B) Dose response curves for P013222 and three analogs against MuRF1.

A.

B.

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5.0!104

1.0!105

1.5!105

2.0!105

2.5!105

-E3

MuRF1

Z': 0.81

S/B: 438

Well

RLU

-8 -7 -6 -5 -40

20

40

60

80

100 P010497

P013222

Various Compounds Vs MuRF1 EC50s

P045188

P045185

[Compound], Log M

% In

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5.0!104

1.0!105

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2.5!105

-E3

MuRF1

Z': 0.81

S/B: 438

Well

RLU

-8 -7 -6 -5 -40

20

40

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80

100 P010497

P013222

Various Compounds Vs MuRF1 EC50s

P045188

P045185

[Compound], Log M

% In

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5.0!104

1.0!105

1.5!105

2.0!105

2.5!105

-E3

MuRF1

Z': 0.81

S/B: 438

Well

RLU

-8 -7 -6 -5 -40

20

40

60

80

100 P010497

P013222

Various Compounds Vs MuRF1 EC50s

P045188

P045185

[Compound], Log M

% In

hib

itio

n

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5.0!104

1.0!105

1.5!105

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2.5!105

-E3

MuRF1

Z': 0.81

S/B: 438

Well

RLU

-8 -7 -6 -5 -40

20

40

60

80

100 P010497

P013222

Various Compounds Vs MuRF1 EC50s

P045188

P045185

[Compound], Log M

% In

hib

itio

n