1. We develop results
PORTRAIT OF BIOPRA ACTIVITIES
Mathieu Arcand, Nathalie Rouleau,
Stéphane Parent
Co-founders Biopra
2014-05-09
2. MISSION
The masters of Alpha technologies
deliver simple and effective tools to
tackle cutting edge biological
challenges.
With the aim of globally serving the
life sciences industry, we forge the
path to tomorrow’s discoveries.
2
3. THE NEEDS
• A year ago, PerkinElmer closed its Montreal
facility
• Internal worldwide restructuration
• Montreal site primary source of expertise
for Alpha technologies
• Needs for Alpha technology experts post
closure are recognized, even by
PerkinElmer
• Customers mentioned on several occasions
the need for a contract research
organization serving the early stage of the
drug discovery pipeline
3
4. THE BIOPRA SOLUTION
Experience
• Developped, commercialized and
supported Alpha for years
• Helped most large pharmas and
biotechs to develop their Alpha assays
• Involved in more than 200 new
products based on drug discovery
technologies
• Alpha
• TR-FRET
• Others
Approach
• Iterative assay development process
• Tight knowledge-sharing
• Working hand-in-hand with clients
• scientist to scientist
• Flexible & Collaborative
For pharma and
biotech
• Time is big money
• New R&D approach
to rely on experts
• Biopra has an
incredible assay
development
expert team
4
5. BIOPRA ADVANTAGE
• Work executed by Alpha
technology pioneers
• Privileged relationship with
PerkinElmer
• owners of Alpha
• Quick turnaround
• Direct interactions
• scientist to scientist
• Phase-gated process
• Strong understanding of
product development
processes
• Comprehensive reports
• Dry and wet lab services
• Consultation
• Assay development
• Biochemical
• Cell-based
• Assay miniaturization
• Pre-HTS optimization
• Expertise in growing
research segment
• Epigenetics
• Immunogenicity
• Biotherapeutics
• Superior client service
5
10. BIOMARKER EXPERTISE
10
A Luminescent Oxygen Channeling Immunoassay for the
Determination of Insulin in Human Plasma
FRITZ POULSEN and KIRSTEN BORRES JENSEN
The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for
quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of
biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incuba-
tion with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemilumi-
nescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 µL. The
analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for
various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no
interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up
to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did
affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated)
and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R2
= 0.98). This
method is convenient and represents an alternative to ELISA. (Journal of Biomolecular Screening 2007:240-247)
Key words: sandwich, high throughput, biomarker, LOCI™, homogenous, chemiluminescent
INTRODUCTION
PHARMACOKINETIC (PK) AND PHARMACODYNAMIC (PD) stud-
ies are a crucial part of drug discovery and development.
These studies require analysis of a high number of plasma and
serum samples from animals and humans to measure concentra-
tions of drugs and biomarkers.1-4
Demands to speed up the drug discovery process have
resulted in increasing demands for assays that possess faster
sample throughput for PK or PD studies in animal models in
discovery and in clinical development stages.
There is at the same time a need for very sensitive assays and
for reducing assay sample volumes to reduce the volume of blood
to be drawn, especially in testing small animals such as rats
and mice.
Insulin is a drug as well as a biomarker, and the insulin con-
centrations in a high number of samples have to be tested in rela-
tion to drug discovery. Furthermore, insulin concentrations may
vary from very low and to very high. Insulin measurements thus
require assays with high capacity, high sensitivity, and, at th
same time, a broad analytical range.
Presently, insulin assays are frequently performed as enzyme
linked immunosorbent assays (ELISAs) or radio immunoassay
(RIAs) in discovery and clinical development stages. Thes
assays require repeated wash cycles and large sample volume
and often do not have adequate sensitivity and working range.
Using high-throughput screening (HTS) technology an
procedures seems to be an obvious answer to the capacity an
effectiveness needs. The HTS approach will usually includ
assays that are homogenous, easy to automate, and robust i
routine testing. Furthermore, these assays can be run in minia
turized formats. However, they also need to fulfill requirement
of clinical biochemistry assays such as sensitivity, precision
and robustness to plasma interference.
Homogenous insulin sandwich immunoassays, such a
FMAT5
and HTRF,6
have been described for serum and cultur
medium measurements. Heterogeneous assays with a built-i
automated wash procedure, such as electrochemiluminescenc
(ECL),7
the Gyrolab Bioaffi™system,8
and the Luminex system,
with many of the advantages of homogenous assays, have als
been developed.
All these assays are easy to perform but have limited sensi
tivity (except for ECL), have a relatively long assay time, hav
a limited dynamic range, or do not allow manual assay set u
in an open system.
Biology, Diabetes Research Unit, Novo Nordisk, Bagsvaerd, Denmark.
Received Aug 18, 2006, and in revised form Oct 18, 2006. Accepted for publi-
cation Nov 14, 2006.
Journal of Biomolecular Screening 12(2); 2007
DOI:10.1177/1087057106297566
11. PROTEIN INTERACTIONS EXPERTISE
11
Biochemistry 2010, 49, 3213–3215 3213
DOI: 10.1021/bi100253p
Single-Well Monitoring of Protein-Protein Interaction and
Phosphorylation-Dephosphorylation Events†
Mathieu Arcand,* Philippe Roby, Roger Bosse, Francesco Lipari, Jaime Padros, Lucille Beaudet,
Alexandre Marcil, and Sophie Dahan
PerkinElmer BioSignal Inc., 1744 William Street, suite 600, Montreal, Quebec, Canada H3J 1R4
Received February 18, 2010; Revised Manuscript Received March 15, 2010
ABSTRACT: We combined oxygen channeling assays with
two distinct chemiluminescent beads to detect simulta-
neously protein phosphorylation and interaction events
that are usually monitored separately. This novel method
was tested in the ERK1/2 MAP kinase pathway. It was first
used to directly monitor dissociation of MAP kinase ERK2
from MEK1 upon phosphorylation and to evaluate MAP
kinase phosphatase (MKP) selectivity and mechanism of
action. In addition, MEK1 and ERK2 were probed with an
ATP competitor and an allosteric MEK1 inhibitor, which
generated distinct phosphorylation-interaction patterns.
Simultaneous monitoring of protein-protein interactions
and substrate phosphorylation can provide significant
mechanistic insight into enzyme activity and small mole-
cule action.
Throughout biology, protein structure and function are affec-
ted by post-translational modifications (PTMs) and interactions
with diverse partners. Specifically, phosphorylation and protein-
protein interaction (PPI) events ensure tight regulation and
specific outputs in signal transduction (1). For phosphorylation
and PPIs, multiplexing can be used to detect similar biomolecular
events such as numerous phosphorylated molecules (2) or multi-
ple interacting partners (3). Although the roles of protein
phosphorylation and interaction are well-established in regulat-
ing signaling events, their interdependence, though often correla-
ted, has not yet been directly measured.
harbor rubrene which emits broadly in the wavelength range of
560-620 nm and has an emission peak around 560 nm, while
AlphaLISA beads are dyed with a europium chelate with a sharp
emission peak at 615 nm. We have taken advantage of these
different light emission properties to monitor substrate phos-
phorylation and PPIs in a single well. We used ERK2 as a model
because this essential MAP kinase is tightly regulated by the
phosphorylation of the threonine and tyrosine residues in its
activation loop, as well as by docking interactions with upstream
kinases such as MEK1, downstream effectors, scaffolding pro-
teins, and negative regulators such as MKPs (10).
Tosetupassay conditionsfor the dual detectionofphosphory-
lation and PPI, we first optimized detection of the two biomole-
cular events separately. We assayed PPIs using GSH Donor and
nickel chelate AlphaLISA Acceptor beads. Hence, active histidine-
tagged MEK1 (His-MEK1) was incubated with increasing concen-
trations of glutathione S-transferase-tagged unphosphorylated
ERK2 [GST-ERK2 (Figure 1c of the Supporting Information)].
Complexed MEK1 and ERK2 proteins bridged GSH-coated
Donor and nickel chelate-coated AlphaLISA Acceptor beads to
allow signal generation and thus binding detection (Figure 1b of
the Supporting Information). Maximal interaction was observed
with His-MEK1 and GST-ERK2, each at an assay concentration
of 100 nM, whereas no signal could be detected in absence of
either counterpart.
We then monitored ERK2 phosphorylation by MEK1 in a
Novel Multiplexed Assay for Identifying SH2 Domain
Antagonists of STAT Family Proteins
Kazuyuki Takakuma, Naohisa Ogo, Yutaka Uehara, Susumu Takahashi, Nao Miyoshi, Akira Asai*
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan
Abstract
Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide
12. KINASE EXPERTISE
12
Current Chemical Genomics, 2011, 5, 115-121 115
Open Access
Catalytic Specificity of Human Protein Tyrosine Kinases Revealed by
Peptide Substrate Profiling
Julie Blouin, Philippe Roby, Mathieu Arcand, Lucille Beaudet and Francesco Lipari*
PerkinElmer Bio-discovery, 1744 William Street, Montreal, Quebec, H3J 1R4, Canada
Abstract: Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-
characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-
Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand,
some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the
CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein
tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.
Keywords: Peptide substrates, protein-tyrosine kinases.
INTRODUCTION
PTKs are involved in many cell signaling pathways.
The IRS1 (Tyr983) peptide was chosen to represent a
YMXM peptide. Peptides containing the YMXM motif were
shown as effective substrates for receptor tyrosine kinases
INTRODUCTION
LARGE-SCALE TECHNOLOGIES USED WITHIN THE LIFE SCI-
ENCE INDUSTRY for drug discovery have recently proven
useful in academic research laboratories, facilitating chemical
genomics and the investigation of specific biological functions.
These technologies, including homogeneous luminescence (e.g.,
AlphaScreen®
and Aequorin), fluorescence intensity (FI) or
polarization (FP), time-resolved fluorescence (TRF), laser scan-
ning cytometry, or microscopy-based approaches, have become
enabling tools to characterize complex functions of biological
targets.
In the present study, we used AlphaScreen®
to investigate
cell signaling pathways1
and in particular those emanating from
the endoplasmic reticulum (ER) under stress conditions. The
ER ensures proper protein folding and export to later compart-
ments of the secretory pathway. This is achieved through com-
plex machineries, including protein synthesis, translocation/
folding, quality control, ER-associated degradation (ERAD),
and export.2
In addition to these functional attributes, the ER
has evolved a highly conserved adaptive signaling pathway,
referred to as the unfolded protein response (UPR), whose acti-
vation occurs upon accumulation of improperly folded proteins
in the ER.3
UPR signaling is mediated by 3 ER resident transmembrane
proteins—the PKR-like ER kinase (PERK), the activating tran-
scription factor 6 (ATF6), and the inositol requiring enzyme 1
alpha (IRE1α).3,4
Our work has focused on IRE1α, which is a
transmembrane sensor of ER stress. The luminal domain of
IRE1α contains binding sites for the chaperone BiP, whereas the
cytosolic region has 2 main catalytic elements: a serine/threonine
kinase and an endoribonuclease domain.4-6
Under basal condi-
tions, IRE1α is thought to exist as a monomer, and in response
to the accumulation of misfolded proteins in the ER, IRE1
proteins oligomerize, resulting in its trans-autophosphorylation
and triggering of its endoribonuclease activity.4
However,
structural studies in Saccharomyces cerevisiae showed that the
luminal domain of IRE1 exists as a dimer/oligomer and sug-
gested that it could potentially bind directly to unfolded pep-
tides similar to MHC class I molecules.7
This was confirmed by
structural studies on the cytosolic domain of IRE1 in S. cerevi-
siae, which revealed a dimer conformation that is subjected to
1
Inserm, U889, Avenir, Bordeaux, France.
2
Inserm, U920, Bordeaux, France.
3
PerkinElmer Biosignal, Inc., Montréal, Quebec, Canada.
4
Present address: PerkinElmer Biosignal, Inc., Montreal, Quebec, Canada.
AlphaScreen®
-Based Characterization of the Bifunctional Kinase/
RNase IRE1α: A Novel and Atypical Drug Target
MARION BOUCHECAREILH,1,2
MARIE-ELAINE CARUSO,1,4
PHILIPPE ROBY,3
STÉPHANE PARENT,3
NATHALIE ROULEAU,3
SAID TAOUJI,1
OLIVIER PLUQUET,1
ROGER BOSSÉ,3
MICHEL MOENNER,2
and ERIC CHEVET1
Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug
discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the
authors investigated and characterized the biological properties of a novel target, IRE1α, a bifunctional kinase/RNase stress
sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology
AlphaScreen®
to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1α.
They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphoryla-
tion ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based
approaches, the authors show that the results obtained using AlphaScreen®
were biologically relevant. Preliminary charac-
terization of assay robustness indicates that both AlphaScreen®
assays should be useful in HTS for the identification of IRE1
activity modulators. (Journal of Biomolecular Screening 2010:406-417)
Key words: endoplasmic reticulum, stress, IRE1, AlphaScreen®
13. A COMBINATION OF GREATNESS
ALPHA ALPHA + BIOPRA
• Complete instructions
• Simple solutions
• Great achievements
• Simple
• Versatile
• Powerful
13
