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Hypothalamic CAMKK2 represents a potential mechanism for chemically affecting satiety and promoting weight loss in clinically obese patients. Single-digit nanomolar inhibitors of CAMKK2 were identified in three related ATP-competitive series. Limited optimization of kinase selectivity, solubility, and pharmacokinetic properties were undertaken on all three series, as SAR was often transferrable. Ultimately, a 2,4-diaryl 7-azaindole was optimized to afford a tool molecule that potently inhibits AMPK phosphorylation in a hypothalamus-derived cell line, is orally bioavailable, and crosses the blood-brain barrier. When dosed orally in rodents, compound 4 t limited ghrelin-induced food intake., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients. We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure-activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.

The Aurora kinases play critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. Selective inhibitors may provide a new therapy for the treatment of tumors with Aurora kinase amplification. Herein we describe our lead optimization efforts within a 7-azaindole-based series culminating in the identification of GSK1070916 (17k). Key to the advancement of the series was the introduction of a 2-aryl group containing a basic amine onto the azaindole leading to significantly improved cellular activity. Compound 17k is a potent and selective ATP-competitive inhibitor of Aurora B and C with K(i)* values of 0.38 +/- 0.29 and 1.5 +/- 0.4 nM, respectively, and is >250-fold selective over Aurora A. Biochemical characterization revealed that compound 17k has an extremely slow dissociation half-life from Aurora B (>480 min), distinguishing it from clinical compounds 1 and 2. In vitro treatment of A549 human lung cancer cells with compound 17k results in a potent antiproliferative effect (EC(50) = 7 nM). Intraperitoneal administration of 17k in mice bearing human tumor xenografts leads to inhibition of histone H3 phosphorylation at serine 10 in human colon cancer (Colo205) and tumor regression in human leukemia (HL-60). Compound 17k is being progressed to human clinical trials.

The Aurora kinases AurA, B and C are serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. Among them, AurB is required for maintaining proper chromosome alignment, separation and segregation during mitosis, and regulating a number of critical processes involved in cytokinesis. AurB overexpression has been observed in a variety of cancer cell lines, and inhibition of AurB has been shown to induce tumour regression in mouse xenograft models. In the present study we report the enzymatic characterization of a potent and selective AurB/AurC inhibitor. GSK1070916 is a reversible and ATP-competitive inhibitor of the AurB-INCENP (inner centromere protein) enzyme. It selectively inhibits AurB-INCENP (K(i)*=0.38+/-0.29 nM) and AurC-INCENP (K(i)*=1.5+/-0.4 nM) over AurA-TPX2 (target protein for Xenopus kinesin-like protein 2) (K(i)=490+/-60 nM). Inhibition of AurB-INCENP and AurC-INCENP is time-dependent, with an enzyme-inhibitor dissociation half-life of >480 min and 270+/-28 min respectively. The extremely slow rate of dissociation from the AurB and AurC enzymes distinguishes GSK1070916 from two other Aurora inhibitors in the clinic, AZD1152 and VX-680 (also known as MK-0457).

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC(50) values less than 1 microM against human tumor cells in vitro.

Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appended to this ring was favored. Selected molecules in the series demonstrated some activity against human tumor cell lines.

The discovery, synthesis, potential binding mode, and in vitro kinase profile of several pyrido[1',2':1,5]pyrazolo[3,4-d]pyrimidines as potent kinase inhibitors are discussed.

GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.

Earlier we reported potent cRaf1 kinase inhibitors with a key acidic phenol pharmacophore that had, at best, adequate cellular efficacy. To improve the cellular potency, phenol isosteres and prodrugs were investigated. Many phenol isosteres were synthesized and tested, but failed to provide adequate enzyme potency. A prodrug approach resulted in a 2- to 17-fold improvement over the parent compound in cell-based efficacy.

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein., (Copyright 1999 Academic Press.)

Ca2+/calmodulin-dependent protein kinases are implicated in regulating the Ca2+ signaling involved in T cell activation and in thymocyte selection. One of the earliest events in signaling through the T cell antigen receptor is activation of the protein tyrosine kinase p56lck. Following T cell activation or signaling through the IL-2 receptor, Ca(2+)-mediated phosphorylation of p56lck occurs on serine/threonine residues. Isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinases, CaM kinase-II and CaM kinase-Gr are found in human T lymphocytes. CaM kinase-II, but not CaM kinase-Gr, phosphorylates the T cell tyrosine kinase p56lck in vitro. Tryptic phosphopeptide maps indicate that CaM kinase-II phosphorylates p56lck on multiple sites in vitro. Kinase assays of p56lck modified by CaM kinase-II indicate that CaM kinase-II modification does not appreciably affect p56lck phosphotransfer activity.

CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is enriched in brain and thymus. The enzyme was isolated from rat cerebellum, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypeptides, and rat forebrain, which contained only the alpha polypeptide. Both enzyme preparations readily underwent autophosphorylation with dramatic up-regulation of their Ca2+/calmodulin-dependent, as well as-independent, activity. Autophosphorylation also caused a characteristic retardation in the electrophoretic gel mobility of the alpha and beta polypeptides. Treatment of autophosphorylated CaM kinase-Gr with acid phosphatase fully dephosphorylated the enzyme and reversed the changes in electrophoretic migration of both polypeptides. Phosphopeptide mapping indicated that the alpha and beta polypeptides were phosphorylated on identical or homologous sites, which probably induces similar structural and catalytic modifications in the two polypeptides. The actual site(s) of autophosphorylation was determined by the purification and amino acid sequencing of tryptic peptides from 32P-labeled CaM kinase-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The functional and structural studies on CaM kinase-Gr autophosphorylation imply that the enzyme is comprised of two regulatory domains, one on either side of a catalytic domain, followed by a C-terminal, putative association domain. The properties of such a structural model are discussed.

We report the development of a quantitative assay for measuring SH2 domain binding in vitro. Using this assay we have analyzed the binding of purified recombinant SH2 domains from ras GTPase activating protein (GAP) and the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) to proteins from epidermal growth factor-stimulated and v-src-transformed cells. The purified recombinant SH2 domains from GAP and p85 bind to the tyrosine phosphorylated epidermal growth factor receptor with nanomolar affinities. Moreover, competition studies suggest that these two proteins bind to equivalent or overlapping sites on this receptor. In v-src-transformed cells the purified recombinant SH2 domains from GAP and p85 bind to distinct but overlapping sets of proteins.

Sphingosine activates casein kinase II in the presence of endogenous substrates as well as a synthetic peptide substrate. The activation response occurred between 12 and 25 micrograms/ml sphingosine and exhibited positive cooperativity with a Hill coefficient of 3.0. Sphingosine not only increased the Vmax of casein kinase II but decreased the Km(app) for the peptide substrate from 0.5 to 0.08 mM. In contrast, the Km(app) for MgCl2 was increased from 0.12 to 0.7 mM. Consequently, sphingosine altered significantly several parameters which determine casein kinase II activity. The effect of sphingosine was relatively specific, inasmuch as related lipids were less potent activators or largely ineffective in stimulating casein kinase II. On the other hand, the effect of sphingosine itself could be potentiated or inhibited by other lipids. Ceramide and sphingosylphosphorylcholine augmented the sphingosine effect. Phospholipids alone did not alter the activity of casein kinase II significantly, but abolished enzyme activation by sphingosine with different potencies (phosphatidylserine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine). Moreover, the sphingosine effect could be abrogated by KCI and NaCl, which alone are known to induce enzyme activation and dissociation of aggregated casein kinase II protein; LiCl and NH4Cl also inhibited the sphingosine effect. Polyamines, known activators of casein kinase II, partially mimicked the effect of sphingosine on endogenous polypeptide phosphorylation but failed to do so with the peptide substrate. These observations demonstrate that sphingosine is a potent activator of casein kinase II. The potential pharmacological and physiological modulation of casein kinase II by sphingoid bases is discussed.

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.

Triton X-100 extracts of purified rat brain synaptosomes exhibited marked phosphorylation of an endogenous Mr 87,000 polypeptide following chromatography on DEAE-cellulose. The protein kinase catalyzing this reaction was insensitive to cyclic AMP, Ca2+, calmodulin, and phorbol esters. However, phosphatidylinositol 4-phosphate (PIP) proved to be a potent inhibitor of the Mr 87,000 polypeptide phosphorylation at submicromolar concentrations, whereas phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were less potent inhibitors. Unsaturated fatty acids could also mimic the effects of PIP at levels above 4 micrograms/ml. The inhibitory effect of PIP largely reflected a profound increase in the apparent Km for Mg2+ such that increasing Mg2+ levels could partially offset the action of PIP. The PIP-sensitive protein kinase was enriched in hypotonic lysates of synaptosomes from which it was partially purified by DEAE-cellulose, hydroxylapatite, and gel permeation chromatography. This purification separated the enzyme from its Mr 87,000 substrate; however, the presence of this polypeptide in heat-inactivated alkali extracts of rat brain provided an exogenous source of substrate which could be used to assay enzyme activity. The relevance of these data to a possible role for PIP and Mg2+ in cellular signaling is discussed.

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.