Development of homogeneous nonradioactive methyltransferase and demethylase assays targeting histone H3 lysine 4.

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Catalytic specificity of human protein tyrosine kinases revealed by Peptide substrate profiling.

Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand, some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.

Development of a high-throughput AlphaScreen assay measuring full-length LRRK2(G2019S) kinase activity using moesin protein substrate.

Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson's disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75nM. The estimated apparent K(m) value for adenosine triphosphate (6 microM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z' factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Expression and purification of PI3 kinase alpha and development of an ATP depletion and an alphascreen PI3 kinase activity assay.

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal His-tag and the p85alpha regulatory domain in Sf9 insect cells. The complex consisting of p110alpha and p85alpha was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110alpha/p85alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110alpha/p85alpha and determined the IC(50) for wortmannin, a known PI3 kinase inhibitor. The IC(50) for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 microM. p110alpha/p85alpha underwent autophosphorylation most prominently at the p85alpha subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors.