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.

1. Progenra’s UbiPro™
Drug Discovery Platform:
Deubiquitylase
Technologies
for Drug Discovery
CONTACT:
Mr. Marc Hixson
Hixson@progenra.com
www.progenra.com
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com
Property of Progenra, Inc.

2. COMPANY SUMMARY AN EXAMPLE OF PROGENRA’S
Progenra is a biotechnology company focused on DEUBIQUITYLASE SCREENING TECHNOLOGIES
exploiting ubiquitin and ubiquitin-like protein pathways Traditionally, high throughput screening of Ub/Ubl isopepti-
to develop medicines to treat a wide range of diseases. dases has been performed using Ub-AMC and Ub-Rh110
Over the past nine years, Progenra has developed its substrates consisting of a small fluorophore attached to
UbiPro™ Drug Discovery Platform for quantifying and the C-terminus of ubiquitin which results in quenching of
characterizing the activity of these enzymes. This platform fluorescence. Cleavage of this substrate by an ubiquitin
is amenable to high throughput screening and has been isopeptidase leads to a loss of quenching and an increase
employed successfully by Progenra to identify inhibitors in fluorescence. The utility of these substrates is limited by
of both deubiquitylating enzymes and E3 ligases. several factors including the fact that they do not
represent a physiological substrate of the enzymes.
BENFITS OF PROGENRA’S UBIPRO™ Additionally the substrates used in the past are poorly
DRUG DISCOVERY PLATFORM cleaved by several ubiquitin isopeptidases limiting their
utility. To address these concerns Progenra has developed
Our partners will obtain several key advantages by using several proprietary screening technologies which enable
Progenra’s UbiPro™ Drug Discovery Platform. They include the discovery of selective isopeptidase inhibitors (Table 1).
but are not limited to:
Table 1: DUB Assays Available from Progenra
•dentificationofcompoundsnotdetectableusing
I
traditional “off-the-shelf” assays and reagents Assay Format Benefits
• UBSelect™,aprovenandpatent-pending
D Ub-AMC, Ub-Rh110 Traditional Assay,
technologyforprecludingtheidentificationof GoodSubstrateforUCHenzymes
reactive “nuisance” screening hits
Ubiquitin-Luciferin Luminescencebasedassay
• argepanelofDUBstobeusedfor
L Most sensitive substrate available
profilingandselectivity
Ub-CHOP1, CHOP2, Robust assay, especially for
• electiveandsemi-selectiveDUBinhibitors
S CHOP3 USPenzymes
as molecular probes Proven multiplexing ability
• seofhomogeneousassaysthatclosely
U DUBSelect™ Precludestheidentificationof
replicate physiological milieux reactive “nuisance” screening hits
• comprehensivevs.singularapproach
A DiUbiquitin Substrates
toinhibitoridentification
Isopeptidebondbetween
• estcommercialplatformavailablethatcovers
B K48-linked two ubiquitin molecules
bothDUBandE3Ligasedrugdiscovery Multiple linkages available
K63-linked
• bilitytoinstantlyadvanceapartner’sDUB
A ProfilelinkagespecificityofDUBs
orE3Ligasediscoveryprogram Alternative linkages
10000
250 nM Ub-Luciferin
250 nM Ub-AMC
1000
250 nM Ub-Rh110
Ub-Luciferin Substrate
10000
40 uM DUB-Glo 250 nM Ub-Luciferin
Progenra has developed a novel deu-
S/B
100
250 nM Ub-AMC
biquitylase substrate which is used in a
1000
250 nM Ub-Rh110
10
coupled assay in which luciferin, a
40 uM DUB-Glo
S/B
100
1 small molecule, is cleaved from the C-
terminus of ubiquitin (ubiquitin-lucifer-
b2
1
2
L5
SH
n3
ke
U 20
14
10
sD
sD
-li
SP
SP
tu
H
xi
M
Jo
Jo
C
ta
n3
O
A
U
U
in). This small molecule then serves as a
A
xi
ta
A
1
substrate for an enzyme that gener-
2
1
2
L5
SH
n3
ke
U 20
14
b
sD
sD
-li
SP
SP
tu
H
xi
M
Jo
Jo
C
ta
n3
ates an increase in luminescence (Fig-
O
A
U
U
A
xi
A. B.
ta
A
ure 1). The Ub-luciferin assay is a more
Figure 1: Ubiquitin-luciferin assay platform offers higher sensitivity than
10000 sensitive and robust assay than other
250 nM Ub-Luciferin
alternative assay technologies.A)TheDUBcleavespreciselyattheCterminal
250 nM Ub-AMC DUBassayssuchasUb-AMC,Ub-Rh110,
1000
250 nM Ub-Rh110
glycine of the ubiquitin-luciferin DUB-Glo
40 uM molecule. The released molecule then serves andDUB-Glo.Thisincreaseinsensitivity
S/B
as a substrate for luciferase, resulting in an increase in luminescence.
100
allows users to measure the activity of
B)TheactivityofmanyDUBsisnotreadilydetectedwithotherDUBassays
10 DUBs that cannot be detected with
such as Ub-Rh110. The robustness of the Ub-luciferin assay allows one to any other system (Figure 1).
1
generateS:Bratiosgreaterthan500whennosignificantactivityisdetected
b2
1
2
L5
SH
n3
e
U 20
14
sD
sD
lik
SP
SP
tu
H
xi
M
-
Jo
Jo
C
ta
n3
O
A
U
U
A
with other assays.
xi
ta
A
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

3. HTS-compatible DiUbiquitin Substrates Ub-CHOP Substrate
In a physiological setting, deubiquitylases cleave the iso- Progenra has developed a suite of isopeptidase assays
peptide bond between the C-terminal glycine of ubiquitin that utilize a common concept -- fusion of ubiquitin or
and the amine side chain of a lysine residue in either a UBL to the amino terminus of the reporter, masking
a substrate protein or in another ubiquitin molecule. the catalytic activity of the reporter enzyme. Enzymatic
To date the cleavage of this bond could be detected only activity is restored following cleavage of ubiquitin or UBL
by LC-MS or by SDS-PAGE. Progenra has developed and by an isopeptidase and is monitored by the release of a
now offers diubiquitin substrates linked by an isopeptide fluorescent leaving group from a quenched fluorogenic
bond between ubiquitin and either lysine 48 or 63 of a reporter enzyme substrate (Figure 3). The best evidence
second ubiquitin molecule (Figure 2). 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
A B
A. using these assays while Progenra has identified multiple
USP7inhibitorsfromarelativelysmalllibraryusingtheUb-
400
CHOP platform.
A B
300
A.
H O
RFU
Ubiquitin N
N
G76 H
200
DUB
100
H O
Ubiquitin
N
0 G76
OH
0 10 20 30
Time (min)
B
10nM USP2core No Isopeptidase
B.
400
Figure 2: Development of DiUbiquitin HT-compatible isopeptide assay. A) The DUB cleaves
the300
isopeptide bond between the C-terminal glycine of Ubiquitin and the lysine side chain of a
A B
second ubiquitin molecule. B) This cleavage increases fluorescence. B.
RFU
200 Ubiquitin H
N
O
N
G76 H
100
DUB
0
0 10 20 30
Ubiquitin Time (min)
H O
N
OH
G76
10nM USP2core No Isopeptidase
tin HT-compatible isopeptide assay. A) The DUB cleaves
C-terminal glycine of Ubiquitin and the lysine side chain of a
cleavage increasesDevelopment of DiUbiquitin HTS-compatible
Figure 2: fluorescence.
isopeptide assay. A)TheDUBcleavestheisopeptide
bond between the C-terminal glycine of Ubiquitin and
the lysine side chain of a second ubiquitin molecule.
B)Thiscleavageincreasesfluorescence. Figure 3: Ub-CHOP reporter Assay. A)TheDUBcleaves
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.
AnalogousassayplatformshavebeendevelopedforUBL
isopeptidases.B)USP7cleavesUb-CHOPresultingina
dosedependentincreaseinNBDfluorescence.Varying
concentrationsofUSP7wereincubatedwith30nM
Ub-CHOP and 20μM CHOP reporter substrate.
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

4. THE UBIPRO™ DRUG DISCOVERY PLATFORM’S PROVEN ADVANTAGE
Progenra 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%inhibitionvalues)fromthesamebasicscreenof5,000compoundsutilizingProgenra’sDUBSelect™Technology.
Figure 4: IdentificationofadditionalinhibitorsusingProgenra’sUbiPro™Platform
USP2: Ub-Rh110 vs. DiUb63-4
16 compounds were identif
16 compounds significant USP2core inhi
wereidentified 12 compounds DiUb63-4 assay
with the
assignificant were not identified using th
wereidentifiedas
Rh110 assay
significantUSP2core
USP2coreinhibitors
with the DiUb63-4 inhibitors with the
assay that were Ub-Rh110 assay that
notidentifiedusing werenotidentified
using DiUb63-4 assay
the Ub-Rh110 assay 12 compounds were identi
significant USP2core inh
with the Ub-Rh110 assa
were not identified using D
4 assay
Figure 5: Elimination of alkylating and reactive molecules using Progenra’s UbiPro™ Platform
Standard Assay (Ub-Rh110) (30!M) DUBSelect™ Technology (80!M)
PARTNERSHIP OPPORTUNITIES
Progenra 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:
• nparalleledexpertiseintheUbiquitinProteasomepathway
U
• ccesstoallthetoolsandtechnologyunderProgenra’sUbiPro™DrugDiscoveryPlatformonanexclusiveornon-
A
exclusivebasistoidentifyanddevelopamodifierofadeubiquitylaseorligaseforuseasatherapeutic
• esearchandearlypreclinicaldevelopmentworktoassistintheidentificationanddevelopmentofaproposed
R
therapeutic, including counterscreening and selectivity screening
• evelopmentofnewtargetswithintheubiquitin-proteasomepathwayfortheidentificationofmodifiersofaspecific
D
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.,accesstoProgenra’sintellectualproperty,know-how,andtechnologies.
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com