Mr. Marc Hixson Hixson@progenra.com

www.progenra.com

Progenra’s UbiPro™ Drug Discovery Platform:

Deubiquitylase 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

•���DUBSelect™,�a�proven�and�patent-pending� technology�for�precluding�the�identification�of� reactive “nuisance” screening hits

•���Large�panel�of�DUBs�to�be�used�for� profiling�and�selectivity

•���Selective�and�semi-selective�DUB�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 DEUBIQUITYLASE SCREENING TECHNOLOGIESTraditionally, high throughput screening of Ub/Ubl isopepti-dases has been performed using Ub-AMC and Ub-Rh110 substrates consisting of a small fluorophore attached to the C-terminus of ubiquitin which results in quenching of fluorescence. Cleavage of this substrate by an ubiquitin isopeptidase leads to a loss of quenching and an increase in fluorescence. The utility of these substrates is limited by several factors including the fact that they do not represent a physiological substrate of the enzymes. Additionally the substrates used in the past are poorly cleaved by several ubiquitin isopeptidases limiting their utility. To address these concerns Progenra has developed several proprietary screening technologies which enable the discovery of selective isopeptidase inhibitors (Table 1).

Assay Format Benefits

Ub-AMC, Ub-Rh110 Traditional Assay, Good�Substrate�for�UCH�enzymes

Ubiquitin-Luciferin Luminescence�based�assay Most sensitive substrate available

Ub-CHOP1, CHOP2, CHOP3

Robust assay, especially for USP�enzymes Proven multiplexing ability

DUBSelect™ Precludes�the�identification�of� reactive “nuisance” screening hits

DiUbiquitin Substrates

K48-linked

K63-linked

Isopeptide�bond�between� two ubiquitin molecules

Multiple linkages available

Profile�linkage�specificity�of�DUBs

Alternative linkages

Table 1: DUB Assays Available from Progenra

Figure 1: Ubiquitin-luciferin assay platform offers higher sensitivity than alternative assay technologies.�A)�The�DUB�cleaves�precisely�at�the�C�terminal�glycine of the ubiquitin-luciferin molecule. The released molecule then serves as a substrate for luciferase, resulting in an increase in luminescence. B)�The�activity�of�many�DUBs�is�not�readily�detected�with�other�DUB�assays� such as Ub-Rh110. The robustness of the Ub-luciferin assay allows one to generate�S:B�ratios�greater�than�500�when�no�significant�activity�is�detected�with other assays.

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

Ub-Luciferin Substrate

Progenra has developed a novel deu-biquitylase substrate which is used in a coupled assay in which luciferin, a small molecule, is cleaved from the C-terminus of ubiquitin (ubiquitin-lucifer-in). This small molecule then serves as a substrate for an enzyme that gener-ates an increase in luminescence (Fig-ure 1). The Ub-luciferin assay is a more sensitive and robust assay than other DUB�assays�such�as�Ub-AMC,�Ub-Rh110,�and�DUB-Glo.�This�increase�in�sensitivity�allows users to measure the activity of DUBs� that� cannot� be� detected� with�any other system (Figure 1).

Otub2

JosD1

JosD2

AMSH

Ataxin3

Ataxin3-like

UCHL5

USP20

USP14

1

10

100

1000

10000250 nM Ub-Luciferin

40 uM DUB-Glo

250 nM Ub-AMC

250 nM Ub-Rh110

S/B

Otub2

JosD1

JosD2

AMSH

Ataxin3

Ataxin3-like

UCHL5

USP20

USP14

1

10

100

1000

10000250 nM Ub-Luciferin

40 uM DUB-Glo

250 nM Ub-AMC

250 nM Ub-Rh110

S/B

A. B.

Otub2

JosD1

JosD2

AMSH

Ataxin3

Ataxin3-like

UCHL5

USP20

USP14

1

10

100

1000

10000250 nM Ub-Luciferin

40 uM DUB-Glo

250 nM Ub-AMC

250 nM Ub-Rh110

S/B

HTS-compatible DiUbiquitin Substrates

In�a�physiological� setting,�deubiquitylases�cleave� the� iso-peptide bond between the C-terminal glycine of ubiquitin and the amine side chain of a lysine residue in either a substrate protein or in another ubiquitin molecule. To date the cleavage of this bond could be detected only by� LC-MS� or� by� SDS-PAGE.� Progenra� has� developed� and�now offers diubiquitin substrates linked by an isopeptide bond between ubiquitin and either lysine 48 or 63 of a second ubiquitin molecule (Figure 2).

Ub-CHOP Substrate

Progenra has developed a suite of isopeptidase assays that utilize a common concept -- fusion of ubiquitin or a� UBL� to� the� amino� terminus� of� the� reporter,� masking� the catalytic activity of the reporter enzyme. Enzymatic activity� is� restored� following�cleavage�of�ubiquitin�or�UBL� by an isopeptidase and is monitored by the release of a fluorescent leaving group from a quenched fluorogenic reporter enzyme substrate (Figure 3). The best evidence of the superiority of the Ub-CHOP platform to the com-monly used Ub-Fluorophore substrates is that several companies� have� failed� to� identify� viable� USP7� inhibitors� using� these�assays�while�Progenra�has� identified�multiple�USP7�inhibitors�from�a�relatively�small� library�using�the�Ub-CHOP platform.

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

Figure 2: Development of DiUbiquitin HTS-compatible isopeptide assay. A)�The�DUB�cleaves�the�isopeptide�bond between the C-terminal glycine of Ubiquitin and the lysine side chain of a second ubiquitin molecule. B)�This�cleavage�increases�fluorescence.� Figure 3: Ub-CHOP reporter Assay. A)�The�DUB�cleaves�

precisely at the C terminal glycine of the ubiquitin (Ub) CHOP reporter fusion protein, releasing catalytically active CHOP reporter enzyme, which is then able to cleave its substrate, resulting in increased fluorescence intensity. Analogous�assay�platforms�have�been�developed�for�UBL�isopeptidases.�B)�USP7�cleaves�Ub-CHOP�resulting�in�a�dose�dependent�increase�in�NBD�fluorescence.�Varying�concentrations�of�USP7�were�incubated�with�30nM�Ub-CHOP and 20μM CHOP reporter substrate.

Figure 2: Development of DiUbiquitin HT-compatible isopeptide assay. A) The DUB cleaves the isopeptide bond between the C-terminal glycine of Ubiquitin and the lysine side chain of a second ubiquitin molecule. B) This cleavage increases fluorescence.

0 10 20 300

100

200

300

400

10nM USP2core No Isopeptidase

Time (min)

RF

U

A B

Ubiquitin

Ubiquitin

DUB

HN

NH

O

G76

HN

OH

O

G76

A B

Ubiquitin

Ubiquitin

DUB

HN

NH

O

G76

HN

OH

O

G76

A B

Figure 2: Development of DiUbiquitin HT-compatible isopeptide assay. A) The DUB cleaves the isopeptide bond between the C-terminal glycine of Ubiquitin and the lysine side chain of a second ubiquitin molecule. B) This cleavage increases fluorescence.

0 10 20 300

100

200

300

400

10nM USP2core No Isopeptidase

Time (min)

RF

U

A B

A.

A.

B.

B.

THE UBIPRO™ DRUG DISCOVERY PLATFORM’S PROVEN ADVANTAGEProgenra has proven that standard off-the-shelf assays and reagents often miss opportunities that the company’s UbiPro™ Platform technology can identify. (Figure 4)� shows� the� identification� of� additional� inhibitors� over� the� standard� Ub-Rh110�assay.�The� identification�of�alkylating�and�reactive�molecules� in�primary�screens�have�been�a�major�hurdle� in�cysteine protease research. (Figure 5) shows the elimination of alkylating and reactive molecules (compounds with inter-mediate�%�inhibition�values)�from�the�same�basic�screen�of�5,000�compounds�utilizing�Progenra’s�DUBSelect™�Technology.

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

Figure 5: Elimination of alkylating and reactive molecules using Progenra’s UbiPro™ Platform

Figure 4: Identification�of�additional�inhibitors�using�Progenra’s�UbiPro™�Platform USP2: Ub-Rh110 vs. DiUb63-4

16 compounds were identified as significant USP2core inhibitors with the DiUb63-4 assay that were not identified using the Ub-Rh110 assay

12 compounds were identified as significant USP2core inhibitors with the Ub-Rh110 assay that were not identified using DiUb63-4 assay

16 compounds were�identified� as�significant�USP2core�inhibitors�with the DiUb63-4 assay that were not�identified�using�the Ub-Rh110 assay

12 compounds were�identified�as�significant�USP2core�inhibitors with the Ub-Rh110 assay that were�not�identified�using DiUb63-4 assay

Standard Assay (Ub-Rh110) (30!M) DUBSelect™ Technology (80!M)

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�preclinical�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 our partners to overcome the greatest barrier to working in the ubiquitin field,�i.e.,�access�to�Progenra’s�intellectual�property,�know-how,�and�technologies.