Discovery of C-imidazole azaheptapyridine FPT inhibitors

The discovery of C-linked imidazole azaheptapyridine bridgehead FPT inhibitors is described. This novel class of compounds are sub nM FPT enzyme inhibitors with potent cellular inhibitory activities. This series also has reduced hERG activity versus previous N-linked imidazole series. X-ray of compound 10a bound to FTase revealed strong interaction between bridgehead imidazole 3N with catalytic zinc atom.     

Actions of vasopressin and the Ca(2+)-ATPase inhibitor, thapsigargin, on Ca2+ signaling in hepatocytes.

When hepatocytes were loaded with fura-2 by incubation with the acetoxymethyl ester (fura-2/AM), addition of Mn2+ resulted in a rapid quench of a fraction of cellular fura-2 fluorescence. Addition of vasopressin caused a second, rapid quench of cellular fura-2, whereas the addition of thapsigargin had no effect. When hepatocytes were loaded by microinjection of fura-2 acid, addition of Mn2+ caused a slower, sustained rate of quench, and both vasopressin and thapsigargin increased this rate of quench. When Mn2+ was removed from the medium of fura-2/AM-loaded cells after preincubation with Mn2+, vasopressin still caused quench of cellular fura-2. In contrast, neither vasopressin nor thapsigargin increased fura-2 quench when Mn2+ was removed from fura-2-injected cells. When fura-2/AM-loaded cells were permeabilized with saponin, only a fraction of the cell-associated fura-2 was quenched by addition of Mn2+. A second fraction was then quenched by addition of inositol 1,4,5-trisphosphate. These results indicate that in hepatocytes loaded with the acetoxymethyl ester of fura-2, the increased quench of cellular fura-2 seen with phospholipase C-linked agonists is not due to effects of the agonist on Mn2+ entry across the plasma membrane, but rather is due to agonist activation of Mn2+ penetration into an intracellular organelle, presumably through inositol 1,4,5-trisphosphate-regulated channels. Thus, it appears that compartmentalization of fura-2 accounts for previously reported anomalies in Ca2+ signaling in hepatocytes, such as the apparent failure of Ca(2+)-ATPase inhibition to increase divalent cation entry, as well as the apparent ability of phospholipase C-linked agonists to stimulate efflux of Ca2+.     

The effect of pKa on pyrimidine/pyridine-derived histamine H4 ligands

During the course of our efforts toward the discovery of human histamine H₄ antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H₄ receptor. This observation was further investigated by synthesizing a series of ligands that varied in pK_a of the pyrimidine derived H₄ ligands by over five orders of magnitude and the effect on histamine H₄ affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H₄ receptor antagonists.     

Novel antifungals based on 4-substituted imidazole: a combinatorial chemistry approach to lead discovery and optimization

A series of 4-substituted imidazole sulfonamides has been prepared by solid-phase chemistry. These compounds were found to have good in vitro antifungal activity and constitute the first examples of C-linked azoles with such activity. The most potent inhibitor (30) demonstrated inhibition of key Candida strains at an in vitro concentration of < 100nM and compared favorably with in vitro potency of itraconazole.     

Design, synthesis and biological evaluation of novel lipoamino acid-based glycolipids for oral drug delivery

A series of lipoamino acid-based glycolipids were synthesised. Suitably derivatised lipoamino acid derivatives were prepared and conjugated to monosaccharides (including glycosyl azides, isothiocyanates, thiols and sulphones) to yield novel O-, N-, S- and C-linked glycolipids in good yields. Their potential to improve the oral absorption of piperacillin is reported.     

Multiple Ca2+ signaling pathways converge on CaM kinase in PC12 cells.

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in mediating various Ca2+ signaling pathways was examined in PC12 cells. Conversion of the kinase to a Ca(2+)-independent form was used to monitor which neurotransmitters activate the enzyme in situ. CaM kinase responds to Ca2+ influx elicited by ligand-gated Ca2+ channels for ATP and acetylcholine. It also responds to Ca2+ mobilization of IP3-sensitive stores elicited by phospholipase C-linked receptors for ATP and acetylcholine as well as by caffeine. CaM kinase mediates the actions of many neurotransmitters and Ca2+ signaling pathways.     

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.     

Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. Although NCS-1 is thought to enhance synaptic efficacy or exocytosis mainly by activating ion channel function, the detailed molecular basis for the enhancement is still a matter of debate. Here, mechanisms underlying the NCS-1-evoked enhancement of exocytosis were investigated using PC12 cells overexpressing NCS-1. NCS-1 was found to have a broad distribution in the cells being partially distributed in the cytosol and associated to vesicles and tubular-like structures. Biochemical and immunohistochemical studies indicated that NCS-1 partially colocalized with the light synaptic vesicle marker synaptophysin. When stimulated with UTP or bradykinin, agonists to phospholipase C-linked receptors, NCS-1 enhanced the agonist-mediated elementary and global Ca2+ signaling and increased the levels of downstream signals of phosphatidylinositol 4-kinase. NCS-1 enhanced the UTP-evoked exocytosis but not the depolarization-evoked Ca2+ responses or exocytosis, suggesting that the enhancement by NCS-1 should involve phospholipase C-linked receptor-mediated signals rather than the Ca2+ channels or exocytotic machinery per se. Taken together, NCS-1 enhances phosphoinositide turnover, resulting in enhancement of Ca2+ signaling and exocytosis. This is a novel regulatory mechanism of exocytosis that might involve the activation of phosphatidylinositol 4-kinase.     

Spatial and temporal organization of calcium signalling in hepatocytes.

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.     

Synthesis and antimicrobial activity of novel C-linked imidazole glycoconjugates

Novel C-linked imidazole derivatives have been synthesized in good to excellent yields and characterized by analytical and spectral analysis. Four of the newly synthesized compounds exhibited moderate antibacterial activity.     

A Ca(2+)-independent activation of a type IV cytosolic phospholipase A(2) underlies the receptor stimulation of arachidonic acid-dependent noncapacitative calcium entry

The oscillatory [Ca(2+)](i) signals typically seen following physiologically relevant stimulation of phospholipase C-linked receptors are associated with a receptor-activated entry of Ca(2+), which plays a critical role in driving the oscillations and influencing their frequency. We have recently shown that this receptor-activated entry of Ca(2+) does not conform to the widely accepted "capacitative" model and, instead, reflects the activity of a distinct, novel Ca(2+) entry pathway regulated by arachidonic acid (Shuttleworth, T. J., and Thompson, J. L. (1998) J. Biol. Chem. 273, 32636-32643). We now show that the generation of arachidonic acid under these conditions results from the activity of a type IV cytosolic phospholipase A(2) (cPLA(2)). Although cPLA(2) activation commonly involves a Ca(2+)-dependent translocation to the membrane, at these low agonist concentrations cPLA(2) activation was independent of increases in [Ca(2+)](i), and no detectable translocation to the membrane occurs. Nevertheless, stimulation of cPLA(2) activity was confined to the membrane fraction, where an increase in phosphorylation of the enzyme was observed. We suggest that, at the low agonist concentrations associated with oscillatory [Ca(2+)](i) signals, cPLA(2) activation involves an increased phosphorylation of a discrete pool of the total cellular cPLA(2) that is already localized within the membrane fraction at resting [Ca(2+)](i).     

New 1-C-(5-thio-D-xylopyranosyl) derivatives as potential orally active venous antithrombotics

In the search for new orally active antithrombotic drugs that are metabolically stable, we explored the synthesis of 1-C-(5-thio-D-xylosyl) derivatives, examining radical and nucleophilic methods. Thus synthesized were aryl, benzyl, alkylcarboxymethylenyl, arylsulfonylmethylenyl and alkylaminocarboxymethylenyl C-linked analogues of 5-thio-D-xylopyranosides.     

Dehalogenation of Chlorinated Hydroxybiphenyls by Fungal Laccase

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by Pycnoporus cinnabarinus. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C—C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified.     

Mechanisms of phospholipase C-regulated calcium entry

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.     

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.     

Synthesis of C-glycosyl compounds of N-acetylneuraminic acid from D-gluconolactone

A general strategy towards the synthesis of C-glycosyl compounds of N-acetylneuraminic acid (Neu5Ac) has been developed and successfully applied to the synthesis of C-methyl and C-phenyl derivatives. The key strategic elements are (i) chain extension of a D-gluconolactone derivative as C(6)-precursor with an allyl Grignard reagent as C(3)-precursor having in 2 position the C-linked aglycon moiety, (ii) stereoselective C-4/C-5 erythro-diol formation, (iii) 6-exo-trig selective heterocyclization, and (iv) installment of the 5-acetylamino and C-1 carboxylate functionalities. The efficiency and potential versatility of this approach was exemplified in the synthesis of C-methyl derivative 1 as target molecule.     

C-H-deprotonation mediated by a remote syn-axial acetoxy group--an unprecedented double bond formation upon cyanation of the dimer from L-fucal

Replacement of the anomeric acetate by a cyanide group in the dimer of di-O-acetyl-L-fucal by the action of mild Lewis acid [Hg(CN)(2)-HgBr(2)-Me(3)SiCN], resulted not only in the desired transformation but also in the introduction of an additional double bond between C-2A and C-3A. Due to its configuration, the remote C-4A acetoxy group may facilitate the deprotonation by functioning as an internal base. 1H NMR spectroscopy and X-ray crystallography indicate that the conformations of both rings A and B and their relative orientation in the resulting C-linked disaccharidic glycosyl cyanide, 4-O-acetyl-2-C-(4-O-acetyl-2,3-dideoxy-alpha-L-threo-hex-2-enopyranosyl)-2,3-dideoxy-2-eno-alpha-L-fucopyranosyl cyanide, in solution are virtually identical to the crystal structure.     

Synthesis of symmetrical C- and pseudo-symmetrical O-linked disaccharide analogs for arabinosyltransferase inhibitory activity in Mycobacterium tuberculosis

Herein we report the synthesis of symmetrical C-linked and pseudo-symmetrical O-linked disaccharides structurally related to Araf motifs present in the cell wall of MTB. Their activity in a competition-based arabinosyltransferase assay using [14C]-DPA as the glycosyl donor is also presented. In addition, in vitro inhibitory activity for the disaccharides was determined in a colorimetric broth microdilution assay system against MTB H37Ra and Mycobacterium avium.     

Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization

A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.