ARC-based Protein Kinase Assays are homogenous, high-throughput binding assays designed to identify inhibitors of basophilic protein kinases from the AGC group by competitive displacement of an innovative fluorescent probe (labeled-ARC probe) from its complex with a protein kinase^[1-3].

Labeled-ARC probes originate from novel bisubstrate-type protein kinase inhibitors (ARCs: conjugates of adenosine mimics and D-arginine-rich peptides) that possess a high affinity (K_d = 0.01 ... 3 nM) towards basophilic kinases^[1]. The unique bisubstrate character of ARCs (simultaneous association with both binding sites of the kinase) enables the characterization of both ATP- and substrate competitive inhibitors.

There are two assay formats available:

• Fluorescence anisotropy-based (ARC-Fluo Kinase Assay) (Fig. 1, Tab. 1)
• Time-gated luminescence-based (ARC-Lum Kinase Assay) (Fig. 2, Tab. 1)

Figure 1: Binding of the ARC-Fluo probe to a protein kinase (PK) leads to a high fluorescence anisotropy value as the rotation of the probe is restricted by the high molecular weight of the kinase (emission of polarized light). Displacement of the probe from the complex by a competitive inhibitor (C) results in a decrease of fluorescence anisotropy (emission of depolarized light)^[5].

Figure 2: Binding of the ARC-Lum probe to a protein kinase (PK) leads to a high emission of time-gated luminescence (TGL) upon excitation. Displacement of the probe from the complex by a competitive inhibitor (C) results in a loss of the luminescence signal that is not affected by the presence of unbound ARC-Lum probe^[6].

ARC-based Protein Kinase Assays are amenable to automation 384-well microtiter plate format) and ideally suited for HTS experiments since they are

• reliable: Z-factor > 0.8 as determined with a PheraStar (BMG) platereader
• simple: 1 step mix & set up (homogenous assay)
• quick: 15 minutes incubation time
• cost-sensitive: no lanthanides, no antibodies, no substrates required

	ARC-Lum Protein Kinase Assay Kit	ARC-Fluo Protein Kinase Assay Kit
Application	Kd determination of both ATP- and substrate-competitive protein kinase inhibitors
Targets	Basophilic protein kinases (AGC group)
Currently tested for	PKA, AKT3, ROCK I, ROCK II, PKG I, PKC, MSK1, Pim1 → Easy assay adaption for the analysis of other basophilic kinases possible
Assay setup	Homogeneous, 1-step mix & read; 384-well format
Assay volume	10 - 25 µl
Detection principle	Time-gated luminescence (TGL) Exc: 337 nm/ Em: 590 nm[3]	Fluorescence anisotropy Exc: 540 nm/ Em: 590 nm[2]
Ligand	ARC-Lum590 probe	ARC-Fluo4 probe
Main benefits	Amenable to automation (384-well format) No requirement of substrates or antibodies Generation of IC50 displacement curves within 1 h Analysis of fluorescent inhibitors due to TGL-based detection	Amenable to automation (384-well format) No requirement of substrates or antibodies Generation of IC50 displacement curves within 1 h Ratiometric Less demanding equipment

[1] Uri et al. (2012) Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors. Biochimica et Biophysica Acta 1804:541.
[2] Vaasa et al. (2009) High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK. Analytical Biochemistry 385(1):85.
[3] Enkvist et al. (2012) Protein-Induced Long Lifetime Luminescence of Nonmetal Probes. ACS chemical biology 6:1052.

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