Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor.

The activation of G-protein-coupled receptors (GPCRs) can result in the stimulation of numerous signaling networks that extend beyond canonical secondary messenger-dependent pathways. It is well-established that many of these diverse networks converge on the MAPK pathway, resulting in the activation of extracellular-signal regulated kinase 1/2 (ERK). Since the link between GPCRs and ERK can be modulated via both G-protein-dependent and -independent mechanisms, measurement of ERK phosphorylation may serve as an ideal surrogate for GPCR activation. We have combined BacMam-mediated gene delivery of the GFP-ERK2 with a time-resolved Foerster resonance energy transfer (TR-FRET) immunoassay for the measurement of intracellular phospho-ERK2 levels. Together these technologies enable a flexible platform for measuring GPCR and MAPK activation in the cell line of interest. This technology has been applied to the measurement of activation of the serotonin 5-hydroxytryptamine-1A (5-HT(1A)) receptor expressed in CHO-K1 cells. In addition to demonstrating the flexibility of this assay platform, we provide the first reported profile for 5-HT(1A) receptor-mediated ERK activation using a panel of known Parkinson's disease drugs. Our results demonstrate the value of using ERK activation as a downstream sensor for GPCR function, providing an attractive complement to upstream endpoints such as ligand occupancy and binding of GTPgammaS.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

Optimizing the MALDI-TOF-MS observation of peptides containing disulfide bonds.

The observation of peaks corresponding to both disulfide-bonded and reduced peptides in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of disulfides could suggest that the samples are either mixtures prior to analysis or that the measurement process has converted single compounds into mixtures. This is an important distinction when characterizing potentially disulfide-bonded peptides obtained from proteolyzed proteins or from oxidized synthetic peptides. It is well documented that disulfides can undergo in-source decay (ISD) when using a 337-nm laser. However, the mixed matrix 2-(4-hydroxyphenylazo)benzoic acid:alpha-cyano-4-hydroxycinnamic acid (1:10) not only suppresses the ISD reduction of disulfides to thiols but allows the same low threshold laser power generally used with alpha-cyano-4-hydroxycinnamic acid to be applied.

A fluorescence anisotropy assay for the muscarinic M1 G-protein-coupled receptor.

In the search for new chemical entities that interact with G-proteincoupled receptors (GPCRs), assays that quantify efficacy and affinity are employed. Traditional methods for measuring affinity involve radiolabeled ligands. To address the need for homogeneous biochemical fluorescent assays to characterize orthosteric ligand affinity and dissociation rates, we have developed a fluorescence anisotropy (FA) assay for the muscarinic M1 receptor that can be conducted in a 384-well plate. We used membranes from a muscarinic M1 cell line optimized for high-throughput functional assays and the previously characterized fluorescent antagonist BODIPY FL pirenzepine. The affinities of reference compounds were determined in the competitive FA assay and compared with those obtained with a competitive filter-based radioligand-binding assay using [(3)H] N-methylscopolamine. The IC(50) values produced from the FA assay were well-correlated with the radioligand-binding K(i) values (R(2) = 0.98). The dissociation of the BODIPY FL pirenzepine was readily monitored in real time using the FA assay and was sensitive to the presence of the allosteric modulator gallamine. This M1 FA assay offers advantages over traditional radioligandbinding assays as it eliminates radioactivity while allowing investigation of orthosteric or allosteric muscarinic M1 ligands in a homogeneous format.

Fluorescent cascade and direct assays for characterization of RAF signaling pathway inhibitors.

RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.

Homogenous fluorescent assays for characterizing small-molecule activators of AMP-activated protein kinase (AMPK).

AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the gamma-subunit of the kinase, which increases the activity of the catalytic alpha-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK alpha(1)beta(1)gamma(1) and alpha(2)beta(1)gamma(1) isoforms in response to small molecule activators and inhibitors.

Biophysical characterization, including disulfide bond assignments, of the anti-angiogenic type 1 domains of human thrombospondin-1.

Thrombospondin-1 (TSP1), a modular secreted glycoprotein, possesses anti-angiogenic activity both in vitro and in vivo. This activity has been localized to the thrombospondin type 1 repeats/domains (TSR). A TSP1 monomer contains three TSRs, each with a hydrophobic cluster with three conserved tryptophans (WxxWxxW), a basic cluster with two conserved arginines (RxR), and six conserved cysteines. Using the baculovirus system, we expressed TSRs of human TSP1 as either the three domains in tandem (P123) or the third domain alone (P3) and demonstrated that both P123 and P3 at nanomolar concentrations inhibit either basic fibroblast-growth-factor or sphingosine-1-phosphate induced endothelial cell migration. Far-UV circular dichroism (CD) indicated that P123 and P3 have a common global fold that is very similar to properdin, a protein with six TSRs. Near-UV CD and fluorescence quenching studies indicated the conserved tryptophans are in a structured, partially solvent-accessible, positively charged environment. N-terminal sequence and mass spectrometry analysis of trypsin-digested TSRs indicated that the RFK linker sequence between P1 and P2 is readily proteolyzed and the conserved arginines are solvent accessible. By a combination of proteolysis and mass spectrometry, the recombinant TSRs were determined to be fully disulfide bonded with a connectivity of 1-5, 2-6, and 3-4 (cysteines are numbered sequentially from N- to C-terminus). TSRs are found in numerous extracellular proteins. These TSRs share the hydrophobic and basic clusters of the TSP TSRs but some have quite different placement of cysteine residues. We propose a sorting of TSRs into six groups that reconciles our results with information about other TSRs.

Increased plasma thrombospondin-1 (TSP-1) levels are associated with the TNF alpha-308A allele in children with juvenile dermatomyositis.

Vascular occlusion is more frequent in children with juvenile dermatomyositis (JDM) who have the TNF alpha-308A allele. One of the potent anti-angiogenic factors is thrombospondin-1 (TSP-1). This study investigated the association of the TNF alpha-308A allele with circulating levels of angiogenic mediators, TSP-1, and platelet factor 4 (PF4) using fresh, platelet-poor plasma (PPP). The TNF alpha-308A allele was characterized by PCR amplification and NcoI digestion. Concentrations of TSP-1 and PF4 in PPP from 31 JDM patients and 25 matched pediatric controls were determined by ELISA. The majority of the JDM children with the TNF alpha-308A allele (7/12) produced more TSP-1 than their TNF alpha-308G counterparts (P < 0.05), and their TSP-1 values were inversely related to those for PF4 (P < 0.0006). We conclude that the increased circulating concentrations of TSP-1 associated with the TNF alpha-308A allele suggest that this anti-angiogenic regulator may play a significant role in the augmented vascular occlusion observed in JDM children with this genetic marker.