Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses (23). It is responsible for persistent infections in humans, with associated risk of chronic liver diseases, including cirrhosis and hepatocellular carcinoma. Nearly 3% of the global population is chronically infected with HCV, and there are no clinically proven vaccines. Antiviral therapeutic agents are at an early stage of clinical evaluation, and standard treatments (interferon and ribavirin combinations) are associated with suboptimal response rates and/or high incidence of side effects. Complicating the discovery of new therapies is the highly complex and incompletely understood nature of the viral life cycle. The HCV genome consists of a single strand of RNA of about 9,600 nucleotides encoding a polypeptide precursor of about 3,000 amino acids (26). Co-and posttranslational proteolytic cleavage of this precursor by cellular and viral enzymes yields structural proteins involved in viral assembly, along with nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which are required for membrane-associated RNA replication (14).Nonstructural protein NS5A is a critical component of HCV replication and is involved in several cellular processes, such as interferon resistance (3, 13) and apoptotic regulation (9). It is a phosphoprotein of 447 residues with three domains (35), and while no clear enzymatic functions have been assigned, it appears to function through interactions with other HCV proteins and host cell factors (17). Domain I (residues 1 to 213) contains a zinc-binding motif (35) and an amphipathic N-terminal helix which promotes membrane association (4, 12, 30), possibly through specific interaction of the helix with target membrane proteins (8). Domain II (residues 250 to 342) has regulatory functions, such as interactions with protein kinase PKR and PI3K (13), as well as NS5B (32); contains the interferon sensitivity-determining region (13); and appears to lack major elements of secondary structure (22). Recent studies have demonstrated that domain III (residues 356 to 447) plays a critical role in infectious virion assembly but not in RNA replication (1,34) and that the former role is modulated by phosphorylation within the domain (33). High-throughput screening of small-molecule inhibitors using HCV replicon cell systems has identified NS5A as a promising therapeutic target (31).A crystal structure of domain I lacking the amphipathic helix and spanning residues 25 to 215 showed two subdomains and a homodimeric association and was interpreted as having a potential role in RNA binding (36). Although specific binding to domain I was not described, RNA binding to full-length NS5A has been reported, using, for example, the 3Ј nontranslated region of HCV (15). Efforts in our laboratory to study the structure of NS5A have yielded an alternative arrangement of the domain I homodimer (residues 33 to 202) that differs substantially from that previously described. The observation that the NS5A do...

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.Receptor protein-tyrosine kinases (RPTKs) 1 regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1, 2). The type III RPTK family includes c-Kit (3), the colonystimulating factor-1 (formerly FMS) (4), the platelet-derived growth factor ␣ and ␤ receptors (1, 5), and the FMS-related receptor FLT-3 (6). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7,8). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9), including the proteintyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1).The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12-15). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal t...

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.