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+

OEx

OEm

1 AssayQuant Technologies

Erik Schaefer, Ph.D., President, CEO & CSO Barbara Imperiali, Ph.D., Chief Technology

Officer, Secretary & Treasurer

Real-Time Sensors of Protein Kinase ActivityOur 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

OEx 360 nm

OEm 485 nm

2

Sox technology is covered by several patents and has been exclusively licensed by MIT to AssayQuant AssayQuant Technologies

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

OEx 360 nm

OEm 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

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

1Real-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.

2Minimal 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 32P-ATP format, which is the Gold standard.

3Modified 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.

4Direct, 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.

5High Accuracy, Precision and Lot-

to-Lot ConsistencyAssay 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.

8No Sophisticated Equipment

RequiredAssays 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

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)

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2000

4000

6000

8000

10000

12000

ALK

AXL

EPHA

1EP

HA2

EPHA

4EP

HA7

EPHA

8EP

HB1

EPHB

2EP

HB3

EPHB

4FG

FR1

FGFR

2FL

T1FL

T4HE

R4IG

F1R

INSR KD

RKI

TLT

KM

ETPD

GFRα

PDGF

Rβ RET

RON

TIE2

TRKB

Activ

ity (R

FU)

5

0

5000

10000

15000

20000AL

KAX

LEG

FREP

HA1

EPHA

2EP

HA4

EPHA

7EP

HA8

EPHB

1EP

HB2

EPHB

3EP

HB4

FGFR

1FG

FR2

FLT1

FLT4

HER2

HER4

IGF1

RIN

SRKD

RKI

TLT

KM

ETM

USK

PDG…

PDG…

RET

RON

TIE2

TRKB

Activ

ity (R

FU)

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