ARC-Photo KINASERA
Fluorescence Polarization-based Kinase-Binding
Assay kits for HTS and characterization of
inhibitors of protein kinases
Principle
Screening and characterization of inhibitors of protein kinases
(PK) is usually performed in the form of kinetic studies where
the retarding effect of the compound on the rate of kinasecatalyzed phosphorylation reaction is assigned.
An alternative approach is used in the present assay format:
kinase inhibitors are characterized on the bases of
measurement of binding of the compound to the kinase
(see Scheme overleaf).
ARC-Photo kits from KINASERA are applicable in competition
displacement assays for identification and characterization of
inhibitors of several protein kinases and for determination of
the active (binding) concentration of the protein kinase. The
fluorescent probe ARC-Photo-1 has very high affinity towards
cAMP-dependent protein kinase, PKA (KD = 0.5 nM) and Rho
kinase ROCK2 (KD = 3.6 nM) [1]. The bisubstrate-analog
character of the probe (the inhibitor ARC simultaneously binds
to both pockets of PK) enables the characterization of inhibitors
(and substrates) targeted to ATP binding site and/or substrate
protein/peptide binding domain of the kinase [2, 3].
Protein kinases for which ARC-Photo
kits are currently available
PKA
ARC-Photo-1
ROCK II
ARC-Photo-1
PKB
ARC-Photo-2
P70S6K
ARC-Photo-2
ARC-Photo-2
PKC
PKG
PKCβ1
ARC-Photo-2
ARC-Photo-4
PKCδ
ARC-Photo-4
PKCε
ARC-Photo-4
MSK1
ARC-Photo-4
Assays will be available for other kinases
soon. Please contact us for any specific
needs.
Probes ARC-Photo-2 and ARC-Photo-4 have wider kinase profile and bind with high affinity (KD < 10 nM) to
several pharmaceutically important PK, e.g., PKB, P70S6K, PKCPKCβ1, PKCδ, PKCε, PKA. As tested in a
panel of more than 50 protein kinases ARC-type compounds are good inhibitors of many basophilic PK (e.g.,
majority of PKs of the AGC group).
Key features of the assay
Fast
Easy to perform
Multi-purpose
Comprehensive
Economic
Productive
Results in 30-60 minutes
Just mix and read fluorescence anisotropy/polarization, no need for substrates,
antibodies or ATP; no separation procedures and radioactive materials used
Can be used for determination of the concentration of the kinase, screening of
inhibitors, the affinity of inhibitors and substrates, and as a cAMP biosensor
Competitive inhibitor binding to kinase active site is measured which enables the
determination of affinities of ATP- and protein-competitive ligands of basophilic
protein kinases
Low concentrations of the probe (< 5 nM) and the kinase (< 5 nM) and minute
reaction volumes (10 - 30 L in the 384-well microplate format) are required
Easy to test with new kinases (incl. mutated and non-active forms of PK) as no
substrates and product-associated antibodies are needed
ARC-Photo fluorescent probes possess excellent characteristics:
1. High chemical and photo-stability, good solubility in water;
2. Wide distribution of appropriate filter sets in fluorescence platereaders (540 nm excitation and 590 nm
emission filters for ARC-Photo-Orange and 485 nm excitation and 520 nm emission filters for ARCPhoto-Green);
3. Large signal window (> 130 mA) ensures values of Z'-factor > 0.75.
KINASERA OÜ
Uus 63-55, Tartu, Estonia
Phone: +372 53 311 111
e-mail: info@kinasera.com
www.kinasera.com
KINASERA is the owner of
the exclusive license for
the fluorescent probe ARC-Photo
Scheme
Protein / peptide binding domain
PROTEIN
KINASE
ATPbinding
pocket
1. ARC-Photo is bound to the protein
kinase with high affinity.
2. Bisubstrate character of ARC means
that ARC-Photo is displaced from its
ARC-Photo, bound complex by both ATP and protein
binding-site targeted inhibitors (and
substrates). Thus ARC-Photo can be
used for HTS and characterization of all
inhibitors that bind to the active cite of
Protein /
the kinase.
peptide
substrate
or B-type
inhibitor
ATP or
A-type
inhibitor
3. The displacement of ARC-Photo
(MW<2000) from
its slow-rotating
complex with the kinase leads to
substantial decrease of the fluorescence
anisotropy/polarization of the solution
which can be measured in a 384-well
microtiter
plate
with
fluorescence
platereader.
ARC-Photo, free
1.0
0.8
NAC
Displacement of fluorescent probe ARC-Photo-1 from its
complex with PKA by various competitors [ARC-902 (●),
H89 (▼), ARC-341(■), ATP (▲) and ATP + 10 mM Mg(OAc)2
(●)] (assay in a 384-well microplate, volume 20 microL). KD
value of 11,4 nM for H89 was calculated from the data
presented in the Figure. (NAC – normalized anisotropy
change).
0.6
0.4
0.2
0.0
-10 -9
-8
-7
-6
-5
-4
-3
-2
log C (competitor)
150
mA
100
50
0
0
25
50
75
Titration of the fluorescent probe ARC-Photo-1 [2 nM (●) and
20 nM (●)] with the kinase (PKA catalytic subunit) enables
the determination of concentration of the active form of the
kinase and the affinity of the probe towards the kinase (KD =
0.5 nM).
Experiments performed:
PHERAstar multi-detection microplate reader (BMG
Labtech), 384-well microtiter plate; detection of fluorescence
anisotropy [excitation 540 (20) nm, emission 590 (20) nm].
C (PKA C ), nM
References:
[1] A.Vaasa et al. Anal Biochem. 2009; 385(1):85-93. High-affinity bisubstrate probe for fluorescence anisotropy
binding/displacement assays with protein kinases PKA and ROCK.
[2] D.Lavogina et al. J Med Chem. 2009; 52(2):308-21. Structural analysis of ARC-type inhibitor (ARC-1034)
binding to protein kinase A catalytic subunit and rational design of bisubstrate analogue inhibitors of basophilic
protein kinases.
[3] A. Uri et al. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, (2010), 1804(3), 541 – 546.
Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors.
KINASERA OÜ
Uus 63-55, Tartu, Estonia
Phone: +372 53 311 111
e-mail: info@kinasera.com
www.kinasera.com
KINASERA is the owner of
the exclusive license for
the fluorescent probe ARC-Photo