Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body

Utilising Cyclopropane Scaffolds in Synthetic Methodology and Syntheses of Prodrugs

The ring-opening process of cyclopropane derivatives is prominent in biological processes and synthetic methodology. The reactivity of bifunctional three-membered rings is attributed to the ring-strain and the attached functional groups which can stabilise their ring-opened counterparts. This thesis reports the use of cyclopropane derivatives in synthetic methodology and syntheses of prodrugs

Synthetic methods: part (ii) oxidation and reduction methods

This report highlights selected advances in some of the most commonly used oxidation and reduction reactions, focusing on the literature from 2012. The structure of this review follows that of the previous years report (J. K. Howard and C. J. T. Hyland, Annu. Rep. Prog. Chem., Sect. B: Org. Chem., 2012, 108, 29-52 (). In the field of oxidation a number of important advances have been made. Notably, the group of Miller has reported a peptide-based catalyst that allows the selective epoxidation of polyenes (P. A. Lichtor and S. J. Miller, Nat. Chem., 2012, 4, 990). Meanwhile, the Tomkinson group has reported osmium-free alkene dihydroxylation reactions with cyclopropyl-derived malonoyl peroxides (S. Picon, M. Rawling, M. Campbell and N. C. O. Tomkinson, Org. Lett., 2012, 14, 6250-6253). In the area of reduction methods the Chirik group has made an exciting breakthrough in the use of non-precious metal enantiopure C1-symmetric bis(imino)pyridine cobalt complexes for asymmetric hydrogenation of a range of styrene derivatives in up to 96% ee (S. Monfette, Z. R. Turner, S. P. Semproni and P. J. Chirik, J. Am. Chem. Soc., 2012, 134, 4561-4564). Meanwhile Kuwano et al. have reported the first PhTrap-ruthenium catalysed asymmetric hydrogenation of naphthalenes (R. Kuwano, R. Morioka, M. Kashiwabara and N. Kameyama, Angew. Chem., Int. Ed., 2012, 51, 4136-4139)

Oxidative ring-opening of ferrocenylcyclopropylamines to N-ferrocenylmethyl β-hydroxyamides

The in situ reduction of ferrocenyl cyclopropylimines to the corresponding amines triggers a facile oxidative ring-opening to yield the formal four-electron oxidation products: N-ferrocenylmethyl β-hydroxyamides. This process is believed to proceed via generation of a ferrocinium ion in the presence of air, leading to facile formation of a distonic radical cation that is ultimately trapped by oxygen

The Diastereoselective Synthesis of Pyrroloindolines by Pd-Catalyzed Dearomative Cycloaddition of 1-Tosyl-2-vinylaziridine to 3‑Nitroindoles

An efficient, diastereoselective synthesis of densely functionalized pyrroloindolines is reported. The reaction proceeds via cycloaddition of a vinylaziridine-derived Pd-stabilized 1,3-dipole to electron-deficient 3-nitroindoles. The reactions give the <i>trans</i> diastereoisomer with high selectivity; however, when a 4-substituent is present on the indole ring, a reversal of diastereoselectivity is observed

Pd-Catalyzed Dearomative [3 + 2] Cycloaddition of 3‑Nitroindoles with 2‑Vinylcyclopropane-1,1-dicarboxylates

A <i>trans</i>-diastereoselective Pd-catalyzed dearomative [3 + 2] cycloaddition between vinylcyclopropane dicarboxylates and 3-nitroindoles has been developed. The reaction provides densely functionalized cyclopenta­[<i>b</i>]­indolines with versatile vinyl and nitro-groups. The addition of a halide additive was found to be critical for the diastereoselectivity of the reaction, which is proposed to be a result of a rapid π-σ-π interconversion between the intermediates allowing for Curtin–Hammett control. A switch in diastereoselectivity to afford products with the vinyl and nitro groups <i>cis</i> to each other is observed with a 4-substituted 3-nitroindole

Surface α-Enolase Promotes Extracellular Matrix Degradation and Tumor Metastasis and Represents a New Therapeutic Target

<div><p>In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.</p></div

Ab against ENO1 delayed tumor lung metastasis after s.c. implantation of tumor cells.

<p>(A) Serum level of Ab against ENO1 in mice adoptively transferred with an isotype-control or mENO1-specific Ab (mENO1 Ab) throughout the experimental period was determined by an ELISA. (B) Tumor growth in mice after s.c. injection of LLC/luc cells and adoptive transfer of an isotype-control Ab or mENO1 Ab was measured every 2 days. To prevent overgrowth of the primary tumor, all mice received 3 Gy of local irradiation on day 18 (indicated by the arrow). (C) The percentage of mice (n = 5) without lung metastasis after adoptive transfer of an isotype-control or mENO1 Ab was determined. The arrows indicated the period of Ab injection. (D) LLC/luc was injected s.c. into C57BL/6 mice. After tumor volume had reached 200 mm³, 100 µg Alexa Fluor 488 labeled mENO1 Ab or isotype control Ab (control Ab) was injected i.v. into mice. The fluorescent (indicating Ab) and luminescent (indicating tumor) intensity were detected 4 h later by the IVIS System. The location of tumor was indicated by dotted circle. **<i>p</i><0.01. The error bars were defined as mean±SD.</p

ENO1 accumulated at the pericellular proteolytic site and Ab against ENO1 suppressed invasion and lung metastasis of human A549 lung cancer cells.

<p>(A) A549/luc cells were embedded in matrigel containing fluorescence DQ-collagen to allow the growth of cells in a 3D fashion, and then further stained with a PE-labeled Ab against ENO1 to detect the presence of ENO1. The cleaved product of DQ-collagen (green fluorescence) and the presence of ENO1 (red fluorescence) was observed under a confocal microscope. Cells were counterstained with DAPI. (B) In matrigel invasion assay, A549/luc cells in the upper chamber were suspended in serum free medium containing ENO1-specific IgY (ENO1 IgY) or control IgY (10 µg/ml) and medium containing 5% FBS was used as a chemoattractant in the bottom chamber. The number of invaded cells was determined after 24 h. (C) NOD-SCID mice (n = 5 in both experiments 1 and 2) were i.v. injected with A549/luc cells and adoptively transferred with control IgY or ENO1 IgY. Lung metastasis of A549/luc cells was determined by the presence of luminescence through the IVIS System. Results obtained at day 30 of experiment 1 were shown. (D) The percentage of mice without lung metastasis after adoptive transfer of control IgY or ENO1 IgY was determined in two independent experiments (experiments 1 and 2). The arrows indicated the period of Ab injection. *<i>p</i><0.05. The error bars were defined as mean±SD.</p

Ab against ENO1 delayed tumor lung metastasis after i.v. implantation of tumor cells.

<p>(A) Serum level of Ab against ENO1 in mice adoptively transferred with an isotype-control or mENO1-specific Ab (mENO1 Ab) throughout the experimental period was determined by an ELISA. (B) C57BL/6 mice (n = 10 for experiment 1 and n = 5 for experiment 2) were i.v. injected with LLC/luc cells and adoptively transferred with an isotype-control or mENO1 Ab. Lung metastasis of LLC/luc cells was determined by the presence of luminescence through the IVIS System. Results were obtained from experiment 1 and representative pictures of mice at days 0 and 32 were shown. (C) Macroscopic view of the 5 lobes of lung (left) and the presence of luminescence in the lungs (middle) of two representative mice (experiment 1) treated with an isotype-control (top) and mENO1 Ab (bottom) were shown. Establishment of lung metastasis of LLC/luc cells was confirmed by H&E staining (right). Scale bar = 1mm. The arrows indicated the location of tumor from mice sacrificed on day 32. (D) The percentage of mice without lung metastasis after adoptive transfer of an isotype-control or mENO1 Ab was determined in two independent experiments (experiments 1 and 2). The arrows indicated the period of Ab injection. **<i>p</i><0.01 and ***<i>p</i><0.001. The error bars were defined as mean±SD.</p