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

Biophysical Characterization, Including Disulfide Bond Assignments, of the Anti-Angiogenic Type 1 Domains of Human Thrombospondin-1 †

ABSTRACT: 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. Thrombospondin-1 (TSP1) 1 belongs to a family of large oligomeric proteins consisting of multiple modules (1). Analysis of the number and type of modules indicates there are two sub-families: TSP1 and TSP2 form the first branch; TSP3, TSP4, and TSP5/COMP (cartilage oligomeric matrix protein) form the second branch (1). TSP1 and TSP2 contain one procollagen and three type 1 domains (TSR) that are missing from the other TSPs. Numerous studies have shown that TSP1 and TSP2 can serve as anti-angiogenic agents by their ability to specifically inhibit endothelial cell proliferation, migration, and tubulogenesis in vitro and to inhibit new blood vessel growth in vivo (2). In addition, overexpression of TSP1 in several carcinomas decreases the in vivo growth of these tumors and decreases the vascularization of these tumors (3-6). As a result, TSP1 is recognized as a potent and relevant angiogenesis inhibitor. Attempts to localize the anti-angiogenic region(s) of TSP1 using in vitro as well as in vivo assays have implicated the TSRs of TSP1 (2). Specifically, with regard to an ability to inhibit stimulant-induced endothelial cell (EC) migration, synthetic peptides based on TSP1 TSRs and containing the three conserved tryptophans ( TSP TSRs are found alone and in multiple arrays within numerous extracellular receptors and secreted proteins in diverse species, from flies and round worms to humans (14)