Synthesis and reactivity of alpha-thio-beta-chloroacrylamides and alpha-thio-beta-chloroenones

Development of novel synthetic methodology for selective transformation of organic compounds is a central element underpinning organic synthesis with control of chemo-, regio- and stereoselectivity a very high priority. Reactions which can be conducted under mild reaction conditions and, ideally in an environmentally attractive manner, are particularly advantageous. The principal objective of this thesis was to explore the synthesis, reactivity and synthetic utility of a series of α,β-thio-β-chloroenones. The stereochemical features of these transformations and the potential of this novel series of compounds in the synthesis of bioactive compounds were of particular interest. In exploring the reactivity of these compounds, the key transformations included nucleophilic additions and Stille cross-coupling at the β-carbon. Chapter 1 reviews the literature relevant to the research conducted, and focuses in particular on the synthesis of β-chloroenones and related unsaturated carbonyl compounds. The synthesis of chalcone compounds from various precursors is also discussed, with particular emphasis on the use of palladium cross-coupling reactions in the preparation of these compounds. The biological activity of chalcones is also summarised in this chapter. The second chapter delineates the stereoselective synthesis of the novel α-thio-β-chloroenones from the corresponding α-thioketones in a multistep reaction cascade initiated by a NCS-mediated chlorination. A range of both alkyl and aryl β-chloroenones were prepared in this work and the oxidation of these compounds to the corresponding sulfoxides and sulfones is also outlined. The electrophilicity of the β-carbon of the enones was examined in nucleophilic addition/substitution reactions with successful access to a variety of synthetically useful novel adducts including acetals and enaminoketones. Investigation of the synthetic potential of the Stille cross-coupling reaction with the novel α-thio-β-chloroenones was explored and provided an efficient route for the synthesis of a novel series of chalcones. Most importantly this new methodology provided a new and synthetically powerful approach for carbon-carbon bond formation at the β-carbon under mild neutral conditions. A preliminary investigation into the use of these β-chloroenones as dienophiles in Diels-Alder cycloaddition reactions is also discussed in this chapter. Chapter 2 also reports the nucleophilic addition of N, O, S and C nucleophiles to previously described β-chloroacrylamides and their corresponding sulfoxide derivatives. This work builds on previous research carried out in this programme and the reactivity of these β-chloroacrylamides at the sulfide and sulfoxide level is compared. Comparison of the reactivity of the β-chloroacrylamides, in nucleophilic substitution and Stille-coupling, with that of the novel β-chloroenones is of interest. Finally, the biological activity of both the β-chloroenones and the β-chloroacrylamides in terms of cytotoxicity is summarised in Chapter 2. The final chapter, Chapter 3, details the full experimental procedures, including spectroscopic and analytical data for the compounds prepared during this research.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.

Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.

A prochelator activated by beta-secretase inhibits Abeta aggregation and suppresses copper-induced reactive oxygen species formation.

The intersection of the amyloid cascade hypothesis and the implication of metal ions in Alzheimer's disease progression has sparked an interest in using metal-binding compounds as potential therapeutic agents. In the present work, we describe a prochelator SWH that is enzymatically activated by beta-secretase to produce a high affinity copper chelator CP. Because beta-secretase is responsible for the amyloidogenic processing of the amyloid precursor protein, this prochelator strategy imparts disease specificity toward copper chelation not possible with general metal chelators. Furthermore, once activated, CP efficiently sequesters copper from amyloid-beta, prevents and disassembles copper-induced amyloid-beta aggregation, and diminishes copper-promoted reactive oxygen species formation.

Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture.

BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Beta-arrestin-2 regulates the development of allergic asthma.

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels.

Epithelial Na(+) channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel beta subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na(+) channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gbeta1gamma2.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates activated G protein-coupled receptors (GPCRs), which ultimately leads to their desensitization and/or downregulation. The enzyme is recruited to the plasma membrane via the interaction of its carboxyl-terminal pleckstrin-homology (PH) domain with the beta and gamma subunits of heterotrimeric G proteins (Gbetagamma). An improved purification scheme for GRK2 has been developed, conditions under which GRK2 forms a complex with Gbeta(1)gamma(2) have been determined and the complex has been crystallized in CHAPS detergent micelles. Crystals of the GRK2-Gbetagamma complex belong to space group C2 and have unit-cell parameters a = 187.0, b = 72.1, c = 122.0 A, beta = 115.2 degrees. A complete data set has been collected to 3.2 A resolution with Cu Kalpha radiation.