Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-Å resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-Å crystal structure of c-Kit in complex with STI-571 (Imatinib® or Gleevec®) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.The stem cell factor receptor c-Kit is a receptor proteintyrosine kinase (RPTK) 1 that initiates cell growth and proliferation signal transduction cascades in response to stem cell factor binding (1). c-Kit, named after its viral homolog v-Kit (2), is a member of the Type III transmembrane RPTK subfamily, which includes the colony-stimulating factor-1 receptor (3), also known as the FMS receptor, the related Flt-3 receptor (4), and the platelet-derived growth factor ␣-and ␤-receptors (5, 6), as well as c-Kit (7). The Type III RPTK family is characterized by five extracellular immunoglobulin (Ig) domains, a single transmembrane helix, an autoinhibitory juxtamembrane domain, and a cytoplasmic kinase domain that is split by a kinase insertion domain (KID) (see Fig. 1A) (6,8).The binding of a stem cell factor dimer to the extracellular Ig domains of c-Kit causes two c-Kit RPTKs to dimerize and permits the kinase domains to act in trans as a substrate and enzyme for one another. The result of stem cell factor binding is the phosphorylation of specific tyrosine residues located in c-Kit juxtamembrane regions (9 -12). Tyrosine residue 568 is the primary site of in vivo autophosphorylation (see Fig. 1B). Phosphorylation of the tyrosine initiates a cytoplasmic serine/ threonine phosphorylation cascade that promotes cell growth and proliferation (12). Mutations that cause constitutive activation of c-Kit kinase activity in the absence of stem cell factor binding are implicated in highly malignant human cancers, including gastrointestinal stromal tumors (13, 14), germ cell tumors (15), mast cell and myeloid leukemias (16), and in mastocytosis (17). Moreover, activating c-Kit mutations that occur in the kinase domain are resistant to many kinase inhibitors currently in use as chemotherapy treatments (18 -21).The kinase activity of c-Kit is tightly regulated throughout its signaling cycle. Binding of the protein-tyrosine phosphatase S...

Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.Post-translational modification of C-terminal CAAX sequences by covalent attachment of isoprenyl chains is crucial for intracellular localization and proper function of small GTPases such as Ras, Rac, Rho, and CDC42 (1, 2). The substrates for these modifications are the 15-carbon isoprenoid farnesyl pyrophosphate (FPP) 1 or the 20-carbon isoprenoid geranyl-geranyl pyrophosphate synthesized by enzymes of the mevalonate pathway (3) (Fig. 1A). A key branch point enzyme of the mevalonate pathway is farnesyl pyrophosphate synthetase (FPPS), a ϳ30-kDa Mg 2ϩ -dependent homodimeric enzyme that synthesizes (E,E)-FPP from isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (4, 5) (Fig. 1B). Interest in understanding FPPS activity stems from the recent discovery that FPPS is the molecular target of nitrogencontaining bisphosphonates (6,7,31,32). Bisphophonates are non-cleavable pyrophosphate (P-O-P) analogues in which the central oxygen is replaced by a carbon (P-C-P) with various side chains (Fig. 1C). Against parasitic organisms (8, 9) these agents have been shown in vitro to disrupt cell growth through FPPS inhibition. In people, bisphosphonates are targeted to bone tissue (10) where FPPS inhibition in bone-resorbing osteoclasts is a current therapeutic approach for treating postmenopausal osteoporosis (11,12). Because of their bone-targeting properties, bisphosphonates have also found use as agents to treat tumor-induced hypercalcemia (13), Paget's disease (14), and osteolytic metastases (15).Although structures of apo-and ligand-bound avian FPPS have been solved (16,17), the active sites are unassembled and do not provide substantial information concerning catalysis. Thus, to resolve the molecular basis of catalysis, and also to understand the structural features governing bisphosphonate recognition, we determined the structures of unliganded Staphylococcus aureus FPPS (FPPS-Sa), as well as two Escherichia coli FPPS (FPPS-Ec) ternary complexes. These ternary complexes include a 2.4-Å "substrate-bound" structure containing IPP and the noncleavable DMAPP analogue dimethyla...

No abstract

No abstract

No abstract

The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton's tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

Glycogen synthase kinase 3beta (GSK-3beta) inhibition is expected to be a promising therapeutic approach for treating Alzheimer's disease. Previously we reported a series of 1,3,4-oxadiazole derivatives as potent and highly selective GSK-3beta inhibitors, however, the representative compounds 1a,b showed poor pharmacokinetic profiles. Efforts were made to address this issue by reducing molecular weight and lipophilicity, leading to the identification of oxadiazole derivatives containing a sulfinyl group, (S)-9b and (S)-9c. These compounds exhibited not only highly selective and potent inhibitory activity against GSK-3beta but also showed good pharmacokinetic profiles including favorable BBB penetration. In addition, (S)-9b and (S)-9c given orally to mice significantly inhibited cold water stress-induced tau hyperphosphorylation in mouse brain.