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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)
0
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