Custom-tailored assay examples
Surface Plasmon Resonance Assay (SPR)
SPR is commonly used to determine the kinetics of target-analyte binding kinetics. The assay detects changes in the molecular mass of a target after binding of the analyte. The target is immobilized to a sensor chip and the analyte flows to the target. Target binding is monitored in real-time for both: association and dissociation.
Isothermal Titration Calorimetry (ITC)
ITC is used to determine the binding affinity, stoichiometry, and enthalpy as well as entropy of the binding event of an agent to a target. The assay measures temperature changes when the agent binds to a target determining the mechanism of action on the molecular level.
An ATP competition study can determine whether a compound is competitive with ATP or not. The results can include kinetic constants, Km for ATP, and Ki for the inhibitor, as well as the mechanism of inhibition at a constant substrate concentration.
Example of dependency of the IC50 values on ATP concentration of the MEK1 inhibitor Selumetinib compared to ATP-competitive reference inhibitor Staurosporine
The Substrate Competition Study tests whether the customer’s compound is competitive concerning the substrate. The results include kinetic constants, Km for substrate, and Ki for the inhibitor, as well as the mechanism of inhibition at a constant ATP concentration.
The kinase assay will be performed at room temperature. Compounds will be added as 10-dose IC50 mode into the enzyme/substrate mixture at 5 substrate concentrations, and pre-incubated for 20 min to ensure compounds are equilibrated and bound to the enzyme. Then a constant concentration of ATP will be added to initiate the reaction. The activity will be monitored every 5-15 min for a time-course study.
The Reversibility Study tests whether the customer’s compound inhibits enzyme reversibly or not. The assay is designed for a non-tight binding inhibitor. The protocol in brief is; the enzyme at 100x concentration is incubated with compound (1:1 molar ratio), and then diluted 100-fold into the reaction buffer to initiate the reaction. The activity will be monitored every 5-15 min for a time-course study.
The Time-Dependent Inhibition Study tests the on-rate of an inhibitor which is irreversible and ATP competitive.
The kinase assays will be performed at room temperature. The compound with serial dilution in DMSO will be added at 10-dose IC50 mode into the enzyme/substrate mixture, and 5 concentrations of ATP will be added immediately without pre-incubation to initiate the reaction. The activity will be monitored every 5-15 min for the time course.
The Kinase Activation Assay is suitable for the discovery of allosteric compounds that inhibit the activation of a target kinase by an upstream kinase.
Example of 33PanQinaseTM based ERK2 activation assay. Non-activated ERK2 was incubated with active MEK1 (D) in the presence of various amounts of a generic ERK2 substrate (RBER- CHKtide) and 5 μM ATP. Neither non-activated ERK2 (C) nor active MEK1(B) on their own could phosphorylate RBERCHKtide above substrate background signal (A). Activation of non-active ERK2 by catalytic amounts of active MEK1 resulted in ERK2 activity comparable to that of pre-activated ERK2 (compare D and E).
A. Substrate (RBER-CHKtide) background signal.
B. MEK1 was incubated with a substrate to ensure MEK1 does not directly phosphorylate the substrate.
C. Non-activated ERK2 was incubated with a substrate.
D. Non-activated ERK2 was incubated with MEK1 and substrate.
E. Recombinantly activated ERK2 was incubated with a substrate for positive control.
Reaction Biology has the following compound libraries available for high-throughput screening. We can also use other compound libraries provided by the client or third companies.
Drug Like Diversified Small Molecule Libraries: >110,000 Compounds
(Hand-picked chemically diverse and pure libraries)
Purity - >90%
General Molecular Weight 200 - 500
CLogP < 5
H donors 0-5
H acceptors 0-10
Rotatable bonds 0-10
Caco-2 >= 100 nm/sec
LogS -6 to .5
Focused small libraries
Natural Products: 600 compounds
Kinase Inhibitors: 355 compounds
FDA-approved: 2,701 compounds
FDA-approved mini library (compiled by us): 50 compounds
Epigenetic Inhibitor: 120 compounds
Fragment Libraries: ~5,800 Fragments
Ultimate Fragment Library: ~1,000 compounds
Fsp3 (Fsp3 = ratio of sp3 hybridized carbons vs total carbon count) enriched Fragment Library: 250 compounds
3D Fragment Library: 200 compounds
Covalent Fragment Library: 1,500 compounds
Protein-Protein-Inhibition Fragment Library: 3,873 compounds
Our experienced assay development team will realize all steps necessary for your customized screening project including
- Construct selection
- Molecular cloning
- Protein production with a variety of tags in insect or bacterial expression systems
- Protein purification via affinity chromatography
- Substrate identification
- Assay development and optimization
- Primary, secondary and counter screenings