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
USP14 Program
Targeting the Ubiquitin
Pathway for the
treatment of
neurodegenerative
disease
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 THE UBIQUITIN PATHWAY &
Progenra is a biotechnology company focused on NEURODEGENERATIVE DISEASES
exploiting ubiquitin and ubiquitin-like protein pathways The ubiquitin proteasome pathway plays a multitude
to develop medicines to treat a wide range of diseases. of roles in human physiology, including neuronal protein
Over the past nine years, Progenra has developed its homeostasis. In fact, all the major chronic human
UbiPro™ Drug Discovery Platform, a suite of assays for neurodegenerative diseases are characterized by an
quantifying and characterizing the activity of ubiquitin accumulation of ubiquitylated proteins in abnormal
pathway enzymes. This platform is amenable to high intraneuronal inclusions. These observations support the
throughput screening and has been employed successfully hypothesis that the intracellular accumulation of
by Progenra to identify inhibitors of both deubiquitylating ubiquitylated proteins is a pathological event in
enzymes and E3 ligases. Benefits of Progenra’s UbiPro™ neurodegeneration. Additional evidence of this notion
Drug Discovery Program include: comes from the findings that the proteasome function is
impaired in the affected brain areas of patients with AD
• Identification of compounds not detectable using and PD and declines with age and oxidative stress.
traditional “off-the-shelf” assays and reagents Furthermore, ~36% of cancer patients treated with the
proteasome inhibitor VELCADE develop peripheral
• A proven patent-pending technology for precluding
neuropathy, clearly implicating proteasomal dysfunction
the identification of reactive “nuisance” screening hits
in neurological impairment. Therefore, therapeutic
• Large panel of DUBs to be used for profiling strategies that promote degradation of the accumulated
and selectivity proteins would be very relevant for the prevention and
• Selective and semi-selective DUB inhibitors as treatment of neurodegenerative diseases. In addition to
molecular probes proteasomal dysfunction, changes in other ubiquitin
• Use of homogeneous assays that closely replicate proteasome pathway components such as E3 ligases and
physiological milieux deubiquitylating enzymes (DUBs) are directly associated
with neurodegenerative diseases (Table 1).
• 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
Table 1: Examples of ubiquitin pathway components involved in neurodegenerative diseases.
USP= ubiquitin specific protease; UCH=Ubiquitin c-terminal hydrolase; SUMO= Small ubiquitin like modifier.
Target Type Indication(s) Function Therapeutic Approach
Machado- Recruit HHR23A to intranuclear Inhibit caspase cleavage
Ataxin3 DUB
Joseph disease, inclusions, cleaves Lys63-linkages. of Ataxin-3
Contribute to the regulation
Increase activity
UCH-L1 DUB PD of the pattern, activity and
of UCH-L1
plasticity of synaptic connectivity.
Likely involved in diseases Inhibition of USP14 results
USP14 DUB caused by proteotoxic stress Inhibit proteasomes in the activation of the
such as AD and ALS proteasome
Degradation of
CHIP E3 ligase AD misfolded proteins, slows Increase activity
neurodegenerative process
Parkin E3 ligase PD Neuroprotective function Increase activity
Attenuates aggregation and Increase activation of
SUMO Ubiquitin like AD, PD, HD
proteotoxicity SUMOylation enzymes
Down’s Syndrome, Progressive
Forms non-cleavable, non-
UBB+1 Mutant Ubiquitin supranuclear palsy, Pick’s disease, Inhibit processing
degradable PolyUb chains
Frontotemporal dementia, and HD
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

3. THERAPEUTIC POTENTIAL PROGENRA’S USP14 PROGRAM OVERVIEW
OF USP14 INHIBITORS While there are many neurodegenerative therapeutic
The reduced proteolytic clearance reported in target options Progenra believes that prior work and
neurodegenerative diseases is exemplified by a hallmark validation of USP14 makes it an ideal target. Its technology
of Alzheimer’s disease, the presence of neurofibrillary and expertise in the ubiquitin pathway gives Progenra a
tangles composed of hyperphosphorylated, ubiquitylated, tremendous advantage in the identification and
insoluble, filamentous tau protein. Like many other cellular development of USP14 inhibitors for the treatment of
proteins, tau is polyubiquitylated and degraded by the neurodegenerative conditions. Screening for USP14
proteasome. To facilitate the efficient recycling of ubiquitin enzymatic activity is complicated by the fact that
three DUBs are associated with the proteasome; of these, recombinant USP14 exhibits little enzymatic activity when
USP14 is of particular interest given its ability to deubiquitylate tested with standard DUB assay substrates such as ubiquitin-
substrates prior to their degradation by the proteasome. AMC (Ub-AMC). To effectively monitor USP14 enzymatic
Thus, inhibition of USP14 enhances the degradation of activity, it was necessary to mix recombinant USP14 with
proteasomal substrates such as tau (Figure 1) and may be purified proteasomes which had been pretreated with
an effective therapeutic strategy for the treatment of irreversible DUB inhibitors to deplete their associated DUB
Alzheimer’s disease and other tauopathies such as Pick’s activity. Using this strategy, Lee et al reported the first
disease, some prion diseases, amyotrophic lateral sclerosis specific inhibitor of USP14, IU1. IU1 has an IC50 of ~5uM
(ALS) and progressive supranuclear palsy. Furthermore, versus USP14 and displays selective inhibition of USP14
aggregates of an additional substrate of USP14, transactive within the small panel of DUBs tested. Consistent with the
response DNA-binding protein of 43kDa (TDP-43) have role of USP14 in deubiquitylating substrates prior to
been reported to accumulate in subtypes of amyotrophic degradation of the proteasome, treatment of cells with
lateral sclerosis and frontotemporal lobar degeneration, high concentrations (50-75uM) of IU1 enhanced
increasing the therapeutic value of USP14 inhibitors to treat proteasomal degradation of tau, TDP-43 and other proteins
neurodegenerative conditions. implicated in neuronal proteopathies. Unfortunately
exploration of the chemical space surrounding IU1 did not
lead to significant enhancement of the activity of IU1. In
fact few analogs retain activity and these are primarily
limited to substitution of fluorine with other halogens.
Although IU1 has utility as a tool compound for research
there is a clear need to identify and develop additional
inhibitors of USP 14. Progenra has begun applying its
extensive suite of technologies to initiate a drug discovery
program focused on USP14.
Figure 1: USP14 rescues polyubiquitylated proteins such as tau from degradation by the proteasome.
A) USP14 deubiquitylates tau prior to engagement by the proteasome enabling the levels of tau
protein to increase and ultimately resulting in neuronal cell death. B) In the presence of a USP14
inhibitor, polyubiquitylated tau is degraded by the proteasome resulting in a decrease in tau.
A: No inhibitor No inhibitor
A: B: USP14 inhibitor
B: USP14 inhibitor
Tau Poly Ub
Tau Poly Ub
USP14 AND/OR
USP14 AND/OR
USP14 USP14 Inhibitor Inhibitor
Tau Tau
26S 26S aggregates aggregates
proteasome proteasome
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com

4. PROGENRA’s USP14 INHIBITOR PROGRAM: One example of Progenra’s abilities is its identification and
development of the P005091 series of USP7 inhibitors.
There are several aspects of USP14 that can be exploited Progenra identified P005091 as an inhibitor of USP7 using
for drug discovery, including catalytic activity when bound the UbiProTM Drug Discovery Platform and utilized medicinal
to proteasome or subunits thereof, recruitment of USP14 to chemistry to improve potency by >10-fold while also
the proteasome, and binding of small molecules to USP14 in improving drug-like characteristics of the compound class.
the absence of the proteasome. Initial biophysical studies Progenra has demonstrated efficacy of USP7 inhibitors in a
demonstrated that IU1 binds to USP14 in the absence of the variety of cell models and in multiple tumor models, and
proteasome (Figure 2A), providing evidence that the can apply this experience to advance a partner’s USP14
inclusion of proteasome is not necessary for the identification drug discovery program quite rapidly.
of USP14 inhibitors. Progenra has applied two high
throughput screening assay formats from its UbiProTM Drug Figure 2: Initial exploration of USP14. A) SPR plot of binding
Discovery Platform to study recombinant USP14 in the and dissociation of IU1 to USP14, but not the unrelated DUB
absence of purified proteasomes. The first assay is a non- USP47. B) Dose response analysis of the binding of USP14 to
catalytic assay that monitors the binding of a fluorescently
DUBTracker™. C) Dose response analysis of USP14 activity
labeled small molecule probe, DUBTracker™, using a
as measured by cleavage of Ub-luc. D)PROGRAM: Representative
fluorescence polarization readout (Figure 2B). The second PROGENRA’s USP14 INHIBITOR
USP14 EC50 inhibition curves aspects of USP14 thatwith the
There are several as determined can be exploited for drug discovery, in
assay measures the catalytic activity of USP14 in the when bound to proteasome or subunits thereof, recruitment of USP14 to the pro
absence of proteasome. As stated above, USP14 has Ub-luc assay format. small molecules to USP14 in the absence of the proteasome. Initial biophysica
minimal enzymatic activity in the absence of proteasome that IU1 binds to USP14 in the absence of the proteasome (Figure 2A), prov
Binding of DUB Probe
and to overcome this hindrance Progenra employed A: IU1 binds USP14 B: USP14: DUBTracker™ do
40
Ubiquitin-luciferin (Ub-luc) as a substrate as it is the most USP14 200
sensitive DUB assay available and relies on the fact that USP47
SPR Signal (RU)
Polarization, mP
cleavage of ubiquitin from luciferin yields the native luciferin 20 175
substrate, which then reports a luminescence signal in the 150
presence of luciferase (Figures 2C, 2D). These assays 125
0
enabled Progenra to monitor the function of USP14 in two
PROGENRA’s USP14 INHIBITOR PROGRAM: PROGENRA’s USP14 INHIBITOR PROGRAM: 100
orthogonal assays without the complex and time consuming of USP14 that can be exploited are drug discovery, including catalytic activity
There are several aspects There for several aspects of USP14 that can be exploited for drug discovery, in
prior preparation of DUB inactivated proteasomes. proteasome or subunits thereof, recruitment of 100to proteasome or subunits400binding of
when bound to when bound
0 USP14 to200 proteasome, and
the 300 thereof, recruitment -9 USP14 to the pro
of -6
small molecules to USP14 in the absence of the proteasome. Initialto USP14 in studies demonstrated
small molecules biophysical the absence of the proteasome. Initial biophysica
Time, seconds [USP14], Log (M
that IU1 binds to USP14 in the absence of the proteasome (Figure 2A), prov
Progenra is equally capable, however,that IU1 binds to USP14 in the absence of the proteasome (Figure 2A), providing evidence that the Binding of DUB Probe
of employing a
USP14
C: USP14: Ub-luc dose response to USP14
Binding of DUB Probe 1
A: IU1 USP14: DUBTracker™ dose response
B: binds USP14 USP14
D: USP14: Ub-luc inhibitor p
B: DUBTracker™ do
variety of HT-compatible substrates to IU1 binds USP14 activity
A: monitor the
40 40
2500
of USP14 in the presence of proteasome or subunits thereof USP14 200 USP14 100
200
when these assays are appropriate. Additionally Progenra USP47 2000 USP47
Polarization,(RU)
Polarization, mP
SPR Signal (RU)
SPR Signal mP
175 175
can monitor the binding of USP14 to the proteasome or
% Inhibition
20 20
1500
RLU
proteasomal subunits using HT-compatible assays. By utilizing 150 150
50
1000
a suite of assays to monitor USP14 activity and function
0
125
0
125
Progenra will be able to identify USP14 inhibitorsUSP14 INHIBITOR PROGRAM: 500
PROGENRA’s
with a 100 100
0
variety of modes of action and explore chemical space of USP14 that can be exploited for drug discovery, including catalytic activity
There are several aspects 0
that is inaccessible using standard approaches. Through or subunits400
when bound to proteasome 300
0 100 200 thereof, recruitment of 100
0 -9 USP14 to200 proteasome, and binding of
the -6 300 400 -9 -6 -6
1
5
5
25
25
1
03
12
0.
Time, seconds Time, seconds
[USP14], Log (M)
small molecules to USP14 in the absence of the proteasome. Initial biophysical studies demonstrated [USP14], Log (M
0.
06
[Compound], Log
the use of its UbiPro Drug Discovery Platform, Progenrain the absence of the proteasome (Figure 2A), providing evidence that the
0.
0.
TM
0.
that IU1 binds to USP14 [USP14] (M)
can initiate a screening campaign to identify USP14USP14
novel USP14 USP14
C: USP14: Ub-luc dose response USP14
D: USP14: Ub-luc inhibitor p
C: USP14: Ub-luc dose response
A: IU1 binds
USP14
Figure B: USP14: exploration of doseto USP14 plot of binding and dissociation of IU
2: Binding of DUB Probe 1 response
D: USP14: Ub-luc inhibitor profiling
Initial DUBTracker™ USP14. A) SPR
2500
inhibitors from its own or a partner’s diverse collection of
2500
40 unrelated DUB USP47. B) Dose response analysis of the binding of USP14 to DUBTra
100 PR-619 100
analysis of USP14 activity as measured by cleavage of Ub-luc. D) Representative USP1
small molecules 2000 USP14 200
2000 IU1
USP47 determined with the Ub-luc assay format.
SPR Signal (RU)
Polarization, mP
NEM
% Inhibition
% Inhibition
Hit identification using the UbiProTM Drug 20 Discovery Platform
1500 175
1500
inclusion of proteasome is not necessary for the identification of USP14 inhibitors
RLU
RLU
50 50
is the starting point for initiation of hit-to-lead optimization.
1000
150
1000
two high throughput screening assay formats from its UbiPro
TM
Drug Disco
recombinant USP14 in the absence of purified proteasomes. The first assay is a
Progenra has identified several selective inhibitors of DUBs 125monitors the binding of a fluorescently labeled small molecule probe, D
0
500 500
and utilized its expertise in this field and internal medicinal 100
fluorescence polarization readout (Figure 2B). The second assay measures
0 0
USP14 in the absence of proteasome. As stated above, USP14 has minimal e
chemistry to improve the potency and drug-like 0 0
absence of proteasome and to overcome this hindrance Progenra employed Ub
characteristics of several of these small molecules. Following 300
0 100 200 400 as a substrate as it is the -6 -3 sensitive DUB assay available -6 relies on th
-9 -6 most and
1
5
5
25
25
1
1
5
5
25
25
1
03
12
0.
03
12
0.
0.
06
0.
06
[Compound], Log (M) [Compound], Log
0.
0.
Time, seconds ubiquitin from luciferinLog (M)the native luciferin substrate, which then reports a
[USP14], yields
0.
0.
initial hit identification Progenra assays a large panel
0.
0.
[USP14] (M) [USP14] (M)
the presence of luciferase (Figures 2C, 2D). These assays enabled Progenra to
(>30) of DUBs to determine the selectivity profiledose response
C: USP14: Ub-luc USP14 of initial
Figure 2: Initial exploration of USP14. A) SPR plot of D:USP14 in two orthogonal profilingwithout plot not binding and dissociation of pr
Figure binding and dissociation of IU1 to A) SPR but complex and time consuming IU
2:USP14: exploration of USP14. USP14, the of the
Initial Ub-luc inhibitor assays
USP14
unrelated hit-to-lead Dose response analysis unrelatedinactivated proteasomes.response analysis ofresponse however, of employing a v
hits. This profiling is utilized throughout the DUB USP47. B) stage
2500 of the binding of USP14 toDose Progenra is equally capable,
DUB USP47. B) DUBTracker™. C) Dose the binding of USP14 to DUBTra
analysis of USP14 activity as measured by cleavage of Ub-luc. USP14 activity as measured by cleavage of Ub-luc. D) Representative or subu
analysis of D) Representative the activity of USP14 PR-619 as
substrates to monitor USP14 EC inhibition curves in the presence of proteasome USP1
to ensure the development of potent, selective inhibitors.
determined with the Ub-luc assay format.
2000
100 50
assays are appropriate. Additionally Progenra can monitor the binding of USP1
determined with the Ub-luc assay format. IU1
proteasomal subunits using HT-compatible assays. By utilizing a suite of ass
Progenra uses a combination of biophysical and function Progenra will be able NEM
% Inhibition
activity and USP14 inhibitors. Progenra for the identification inhibitors with a va
inclusion of proteasome is not necessary to identify USP14 of USP14 inhibitors
biochemical techniques, monitoring such high of proteasome is not necessaryformats identification ofthroughput screening assay hastoapplied its UbiProTM Drug Disco
inclusion
1500
two characteristics
for the
RLU
throughput screening assay two UbiProTM Drug Discovery Platform
from itshigh
50 formats from
study
Progenra, Inc. 277 Great Valley Parkw ay, Malvern PA 19355 (p) 610.644.6974 (f) 610.644.8616
as reversibility, binding kinetics, metabolic liabilities, and absence of purified proteasomes. The USP14 in theaabsence of purified proteasomes. The first assay is a
recombinant USP14 in the
1000 recombinant first assay is non-catalytic assay that
monitors the binding of a fluorescently labeled monitors the binding of a fluorescently using a small molecule probe, D
labeled
formulation, to characterize inhibitors further. Progenra hasreadout (Figure 2B). The small molecule measuresDUBTracker™,activity The second assay measures
fluorescence polarization
500 second assay
probe,
fluorescence polarization readout (Figure 2B). of
the catalytic
extensive experience in establishing and utilizing a variety proteasome. As stated above, USP14 has minimalproteasome. As stated above, USP14 has minimal e
USP14 in the absence of USP14 in the absence of enzymatic activity in the
0
of cellular models for confirming the cellular activityas it DUB mostovercome this hindrance Progenra employedand tofact that cleavageassay available and relies onUb
absence of proteasome and to
0
as a substrate
of is the absence of proteasome Ubiquitin-luciferinthis hindrance Progenra employed
sensitive DUB assay available -6 as it is on the
overcome (Ub-luc)
as a substrate relies the most sensitive DUB of
and th
-3
1
5
5
25
25
1
inhibitors. In the case of USP14, Progenra will performluciferin yields the native luciferin substrate, which then reports a (M) native luciferin substrate, which then reports a
ubiquitin from cellular
03
12
0.
ubiquitin from luciferin yields luminescence signal in
[Compound], Log the
0.
06
0.
0.
0.
assays enabled luciferase to monitor2C, 2D). These
Progenra (Figures the function of
assays measuring the degradation of presence [USP14] (M)(Figureswithout the complex presence oforthogonal prior preparation the DUB assays enabled Progenra pr
the
model luciferase
of
USP14
USP14 in two orthogonal assays
2C, 2D). These the
USP14 in twoconsuming assays without of complex and time consuming
and time
to
substrates such as tau and TDP-43. Figure 2: Initial exploration of USP14. A)isSPR plot of binding and dissociation ofaIU1 to USP14, butcapable, however, of employing a v
inactivated proteasomes. Progenra equally capable, however,proteasomes. Progenra isHT-compatible
inactivated of employing variety of equally not the
DUB USP47. B) Dose activity of USP14 of the binding of USP14 to DUBTracker™. C) Dose response
unrelatedsubstrates to monitor the response analysisin the presence of proteasome or subunits of USP14 in the presence of proteasome or subu
substrates to monitor the activity thereof when these
Progenra, Inc • 277 Great Valley Parkway, are appropriate. 19355 by(p) Progenra Ub-luc. (f) Representative USP14 EC50 inhibition curves monitor the binding of USP1
Malvern PA Additionally 610.644.6974 D) 610.647.8616 • www.progenra.com
analysis of USP14 activity as measured • cleavage of can monitor are binding of USP14 to the proteasome or as
assays assays the appropriate. Additionally Progenra can
determined with the Ub-luc assay format.
proteasomal subunits using HT-compatible assays. proteasomal subunits of assays to monitor assays. By utilizing a suite of ass
By utilizing a suite using HT-compatible USP14
activity and function Progenra will be able to identifyactivity and function Progenra will be able to identify USP14 inhibitors with a va
USP14 inhibitors with a variety of modes of action
inclusion of proteasome is not necessary for the identification of USP14 inhibitors. Progenra has applied
TM
two high throughput Great Valley Parkw ay, Malvern PAfrom its 610.644.6974 Drug Great Valley ww .progenra.com study
Progenra, Inc. 277 screening assay formats 19355 (p) UbiPro Inc. 277 Discovery Platform to PA 19355 (p) 610.644.6974 (f) 610.644.8616
Progenra, (f) 610.644.8616 w Parkw ay, Malvern

5. THE PROGENRA ADVANTAGE SELECTED PROGENRA PUBLICATIONS AND
Progenra has the singular focus of developing technologies RECOMMENDED READING
and strategies for identifying and developing selective Lee BH, Lee MJ, Park S, Oh DC, Elsasser S, Chen PC, Gartner
inhibitors of enzymes in the ubiquitin pathway for the C, Dimova N, Hanna J, Gygi SP Wilson SM, King RW, Finley D
,
treatment of disease. To this end we have developed, and “Enhancement of proteasome activity by a small-molecule
continue to develop, several proprietary and patented inhibitor of USP14.” Nature. 2010, Sep:467(7312):179-84
screening technologies in addition to performing extensive
biochemical characterization of this enzyme class. Tian X, Isamiddinova NS, Peroutka RJ, Goldenberg SJ,
Mattern MR, Nicholson B, Leach C. “Characterization of
selective ubiquitin and ubiquitin-like protease inhibitors
using a fluorescence-based multiplex assay format.”
PARTNERSHIP OPPORTUNITIES
Assay Drug Dev Technol. 2011 Apr;9(2):165-73.
If you are interested in jumpstarting a novel drug
Edelmann, MJ, Nicholson, B, Kessler, BM “Pharmacological
discovery program for a validated CNS target, Progenra is
targets in the ubiquitin system offer new ways of treating
an ideal partner that can bring your organization to the
cancer, neurodegenerative disorders and infectious
forefront of this field. Progenra is open to discussing
diseases” Expert Review Mol Med. in press
mutually beneficial licensing and risk sharing agreements
for the screening platforms and/or the early stage Altun M, Kramer HB., Willems LI, McDermott JL, Leach CA,
inhibitor leads the company has discovered. Research Goldenberg SJ, Suresh Kumar KG, Konietzny R, Fischer R,
collaborations involving one or more targets are of Kogan E, Mackeen MM, McGouran J, Khoronenkova SV,
high interest to Progenra. Parsons JL, Dianov GL, Nicholson B and Kessler BM.“Activity-
based chemical proteomics accelerates inhibitor
development for deubiquitylating enzymes.” Chem and
PARTNERING WITH PROGENRA INCLUDES: Biol. in press
• 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,
knowhow, and technologies.
Progenra, Inc • 277 Great Valley Parkway, Malvern PA 19355 • (p) 610.644.6974 (f) 610.647.8616 • www.progenra.com