Evaluating PI3 Kinase Isoforms Using Transcreener™ ADP Assays

Abstract

Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Transcreener ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP--the invariant product of all kinase reactions--provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance (r(2)=0.93) in the IC(50) values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3Kalpha isoform, and inhibitors affected PI3Kalpha intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.

Sensitized Bioassays

“…Kinase assay applications vary from membrane-bound phosphoinositide-3-kinase (PI3K) done with europium [75] or terbium [76], to MAP kinase family [77][78][79][80], to Tie and AbI kinases [81][82][83][84] and VEGFR autophosphorylation [85][86][87][88], just to give some examples. Transcreener 1 assay of BellBrooks measures kinase activities by quantitation of created ADP [89], and the Invitrogens kinase-binding assays and enzymes are measured as ATP-binding proteins using ATP competing tracer and kinase proteins expressed with suitable tags [90].…”

Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

“…Signal is generated when nucleotides produced in an enzyme reaction displace a fluorescent tracer from antibody resulting in a change in its fluorescent properties. The Transcreener assay for ADP has been formatted for ratiometric fluorescent readouts, including fluorescence polarization (FP) 2 and time-resolved Föster energy transfer (TR-FRET) 3,4 that minimize compound interference, and has been broadly applied for diverse ATP-utilizing enzymes. [4][5][6][7] Here, we describe…”

Regulators of G-Protein Signaling and Their Gα Substrates: Promises and Challenges in Their Use as Drug Discovery Targets

“…9). Measuring GTPase activity through the detection of generated GDP product using this "mix and measure" FP assay overcomes the low signal-to-noise ratio and other limitations of inorganic phosphate detection methods [e.g., malachite green and phosphate-binding protein assays (Van Veldhoven and Mannaerts, 1987;Geladopoulos et al, 1991;Willard and Siderovski, 2004;Shutes and Der, 2005)] and has previously been validated as a robust HTS method in the case of ADP detection for kinases and ATPases (Huss et al, 2007;Liu et al, 2007;Klink et al, 2008). Moreover, because this Transcreener GDP-based assay for the G␣/ RGS domain interaction is one based on enzymatic activity, it should enable detection of all types of modulators of RGS domain GAP activity, including those that FIG.…”