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.

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.