Our initial focus is to develop innovative strategies for directly measuring the activity of protein kinases, which transfer a phosphate group from ATP to serine, threonine or tyrosine residues within protein or peptide substrates. There are over 500 protein kinases and this group of enzymes is intensely studied since dysregulation of kinase activities lies at the center of many human diseases. Generation of protein kinase inhibitors and the need to accurately measure kinase activities in basic research, drug development and diagnostic applications is an active area that spans many markets including pharmaceutical, biotechnology, diagnostic and contract research companies, and academic and Government institutions. There has been a surge in the generation of non-ATP-competitive kinase inhibitors, with an increased need for better assay tools to effectively characterize their mechanism of action and drive decisions earlier in the drug development process.

1. AssayQuant Technologies, Inc.
Accelerating progress through innovation at
the interface of Chemistry and Biology
About Us:
We are a Life Science company founded by scientists
that develops innovative enzyme assay formats for a
quantum improvement in performance and productivity
to accelerate discovery and drug development. We
apply rigorous development, validation and
manufacturing practices to create products that deliver
exceptional value (easier, faster, better, cheaper) that
are backed by outstanding customer support.
Mg2+
Ex
Em
1 AssayQuant Technologies
Erik Schaefer, Ph.D., President, CEO & CSO
Barbara Imperiali, Ph.D., Chief Technology
Officer, Secretary & Treasurer

2. Real-Time Sensors of Protein Kinase Activity
Our initial focus is to develop improved formats for measuring the activity of the >500 protein kinases, which are
dysregulated in many disease states and comprise >30% of all drug development. Pioneering efforts by the Imperiali
laboratory at MIT harnessed chelation-enhanced fluorescence (CHEF), by incorporating the sulfonamido-oxine (Sox)
chromophore into peptide or protein substrates, to create real-time sensors of phosphorylation. The result is a
simple yet powerful method to measure the activity of protein kinases using a continuous kinetic read, ​where the
level of fluorescence is directly proportional to the amount of phosphorylated substrate. ​This assay format is ideal
for elucidating drug mechanism of action and is increasingly being applied earlier in the drug development
workflow to address the challenges and opportunities for developing next generation protein kinase inhibitors.
Analysis of Wild-type and Cancer
mutations for the Abl Tyrosine kinase
Mg2+
KINASE, ATP
N- and C- kinase
recognition determinants
Mg2+
S/T/Y
Ex 360 nm
Em 485 nm
2
Sox technology is covered by several patents and has
been exclusively licensed by MIT to AssayQuant
AssayQuant Technologies

3. Workflow for Homogeneous and Continuous Detection
of Protein Kinase Activity Using the Sox Technology
Mg2+
KINASE, ATP
N- and C- kinase
recognition determinants
Mg2+
S/T/Y
Ex 360 nm
Em 485 nm
96-, 384-
or 1536-
well plate
Any Fluorescence
microplate reader
(Kinetic Read)
Immediate progress curve visualization Quantitative outputSimply add & Read
Fluorescence
increase
[KM (μM)] Vmax
(μmol mg-1 min-1)
3.5 0.10 1.8
CONH2 3.9 0.69 2.5
4.4 1.2 1.3
Enzyme Kinetics
(Km, Vmax, Kcat, IC50, Ki, Residence
times, MOA, etc.) with purified enzymes
Activity in Crude Lysates
3
This assay format delivers a quantum improvement in performance and productivity through increased ease of
use/simplicity, reproducibility, and accurate and precise quantitation to enable discovery, accelerate drug
development, reduce costs and improve drug efficacy by making better decisions earlier in the process.
AssayQuant Technologies

4. Key Advantages Offered by Sox-based Protein Kinase AssaysAssay Features & Benefits:
Strong set of features and benefits that provide high value to the research community. This is the only assay
format of its kind.
Features Benefits
1
Real-time Continuous Kinetic
Format
1) Ease of use (no time points or secondary steps), 2) Corrects for false positives (compound auto fluorescence)
and false negatives (quenching) thereby minimizing interference, 3) Ideal for analysis of different inhibitor classes
(ATP-competitive, Allosteric, Substrate-competitive, Covalent or non-covalent, Reversible and/or Irreversible), 4)
Can also be performed as an endpoint assay.
2
Minimal Perturbation of
Substrates and Positioned for
Optimimum Performance
Modified peptide substrates are generated using standard chemical synthesis methods. In all cases to date, this
can be done without changing kinetic parameters or specificity. In addition, many labs have shown that results are
equivalent to those obtained with 32
P-ATP format, which is the Gold standard.
3
Modified Substrates Optimized
for Kinetic Parameters and
Selectivity
One can choose physiologially-relevant peptide sequences, use combinatorial peptide libraries and/or combine
distinct regions from a protein substrate (e.g., docking and phosphorylation sequences) to create novel kinase
substrates with improved properties (e.g., improved kinetics or selectivity). Km's typically in the low uM range,
therefore 0.1mg of substrate is good for 100 assays. Kinase detection limits are typically in low ng/mL range.
4
Direct, homogenous and Rapid
Measure of Enzyme Activity
Measurements are performed by continuous monitoring of fluorescence intensity (Simply add and read, e.g.,
every 15-30 seconds for 1–2 h ) to determine initial reaction rates. Progress curves develop where increases in
kinase activity result in a corresponding increase in fluorescence. It is a homogenous assay format with no
additional steps to obtain data.
5
High Accuracy, Precision and Lot-
to-Lot Consistency
Assay exhibits high precision, with Z' values >0.8, even with only 10% of the substrate phosphorylated.
6
Compatible with Wide Range of
ATP Concentrations and Broad
Tolerance to Other Assay
Components
Can run assays with low or high (physiological ~mM) ATP concentrations. Indeed, the assays show minimal
interference with a variety of chemicals. We know what chemicals do interfere with the assay and should not be
used above the indicated concentrations to limit interference to less than 10%, as judged by reduction in signal:
10% dimethyl sulfoxide (DMSO), 20 mM DTT, 1 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM CaCl2, 0.8
mM MnCl2, 0.040 mM Na3VO4, 50 mM NaCl, 0.01% SDS, and 1% Triton X-100.
7 Non-Radioactive
Increased user safety and no additional cost or risk of environmental contamination from using or disposing of
radioactive 32P-ATP.
8
No Sophisticated Equipment
Required
Assays are run in standard 96, 384 or 1536 well plates on a standard plate reader capable of kinetic read.
9 Economical
Each assay well produces a continuous curve telling you much more about the activity of the protein kinase. Sell
as 2 formats: 1) Biochemical kits at $495 for 96 assays using 96-well plate ($5.15/test) or 384 assays if using a
384-well plate ($1.72/test), 2) Crude cell or tissue lysate kits at $595 for 96 assays using 96-well plate ($6.20/test)
or 384 assays if using a 384-well plate ($2.07/test).
10 Stored as Lyophilized Powder Very stable. Can be stored and shipped at room temperature.
AssayQuant Technologies4

5. Our R&D Scientists are actively developing next generation Sox Technology Sensors to meet the needs
of our customers. These efforts incorporate improvements developed by the Imperiali laboratory and
target highly generic substrates for use with many different purified protein kinases and highly-
selective substrates for use with crude cell or tissue lysates (see Below and also Peterson et. al., 2014).
Introducing Next Generation Sox Technology Sensors
Evaluation of a Generic Sox-based Substrate
(AQT0025) with Multiple Receptor Tyrosine Kinases
28 RTKs were assayed at 5 nM in triplicate with 10 μM substrate for 60 minutes at 30˚C
(28 of 28 detected)
0
2000
4000
6000
8000
10000
12000
ALK
AXL
EPHA1
EPHA2
EPHA4
EPHA7
EPHA8
EPHB1
EPHB2
EPHB3
EPHB4
FGFR1
FGFR2
FLT1
FLT4
HER4
IGF1R
INSR
KDR
KIT
LTK
MET
PDGFRα
PDGFRβ
RET
RON
TIE2
TRKB
Activity(RFU)
5
0
5000
10000
15000
20000 ALK
AXL
EGFR
EPHA1
EPHA2
EPHA4
EPHA7
EPHA8
EPHB1
EPHB2
EPHB3
EPHB4
FGFR1
FGFR2
FLT1
FLT4
HER2
HER4
IGF1R
INSR
KDR
KIT
LTK
MET
MUSK
PDG…
PDG…
RET
RON
TIE2
TRKB
Activity(RFU)
High Selectivity Screen - Dramatic
improvement in selectivity after
just 1 round of optimization
Generic Sox-based Substrates (e.g., AQT0025) Selective Sox-based Substrates (e.g., AQT0007)
For a current list of Sox-based protein kinase assays or to talk with us about custom assay development
for your targets, please send us a message using the link below. We look forward to hearing from you.
Note: Web site under development with First Scribe
for Feb 1st, 2016 (www.assayquant.com).
AssayQuant Technologies