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Loss of β-cell mass and function can lead to insufficient insulin levels and ultimately to hyperglycemia and diabetes mellitus. The mainstream treatment approach involves regulation of insulin levels; however, approaches intended to increase β-cell mass are less developed. Promoting β-cell proliferation with low-molecular-weight inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) offers the potential to treat diabetes with oral therapies by restoring β-cell mass, insulin content and glycemic control. GNF4877, a potent dual inhibitor of DYRK1A and glycogen synthase kinase 3β (GSK3β) was previously reported to induce primary human β-cell proliferation in vitro and in vivo. Herein, we describe the lead optimization that lead to the identification of GNF4877 from an aminopyrazine hit identified in a phenotypic high-throughput screening campaign measuring β-cell proliferation.

Autoimmune deficiency and destruction in either β-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting β-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human β-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.

Our research groups recently described a series of small-molecule inducers of β-cell proliferation that could be used to increase β-cell mass. To mitigate the risk of nonspecific proliferation of other cell types, we devised a delivery strategy built on the tissue specificity observed in the experimental β-cell imaging agent (+)-dihydrotetrabenazine (DTBZ). The β-cell proliferator agent aminopyrazine (AP) was covalently linked with (+)-DTBZ to afford conjugates that retain both the proliferation activity and binding affinity for vesicular monoamine transporter-2 (VMAT2). In vivo mouse tissue distribution studies of a prototypical AP-DTBZ conjugate showed 15-fold pancreas exposure over plasma. Tissue-to-plasma ratios in liver and kidneys were two- and five-fold, respectively. This work is the first demonstration of enhanced delivery of β-cell-proliferating molecules to the pancreas by leveraging the intrinsic tissue specificity of a β-cell imaging agent.

SummarySeveral small molecule antagonists for Smoothened (Smo) have been developed, and achieved promising preclinical efficacy in cancers that are dependent on Hedgehog (Hh) signaling. However, in a recent clinical study, a drug-resistant D473H SMO mutant was identified that is thought to be responsible for cancer relapse in a patient with medulloblastoma. Here, we report two Smo antagonists that bind to distinct sites, as compared to known antagonists and agonists, and inhibit both wild-type and mutant Smo. These findings provide an insight of the ligand-binding sites of Smo and a basis for the development of potential therapeutics for tumors with drug-resistant Smo mutations.

Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.

Liver sinusoidal endothelial cells (LSEC), the predominant nonparenchyma cells in liver, play critical roles in many important physiological and pathological processes by virtue of their unique location at the blood-tissue interface. To uncover the protein composition of LSEC plasma membrane (PM) comprehensively and give implications for the tissue microenvironment heterogeneity, we have developed an in vivo modified membrane density perturbation method for purification of the PM fraction. The proteins were separated and identified by SDS-PAGE combined with LC-MS/MS (GeLC-MS/MS). A total of 837 nonredundant proteins were identified, including a number of proteins previously reported to be localized to the PM of LSEC, as well as others not described. A diversity of membrane proteins involved in signaling, traffic, transporting and adhesion functions were identified. Our results demonstrated that the in vivo membrane density perturbation was an effective strategy to purify LSEC PM.

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS–PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS–PAGE before nano-Liquid Chromatography-Electrospray Ionization—tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS–PAGE and 2-D 16-BAC/SDS–PAGE on the separation of integral membrane proteins. J. Cell. Biochem. 104: 965–984, 2008. © 2008 Wiley-Liss, Inc.

The bird spider Ornithoctonus huwena Wang is a very venomous spider in China. Several compounds with different types of biological activities have been identified previously from the venom of this spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was preseparated into two parts: the venom proteins with molecular weight (MW) higher than 10,000 and the venom peptides with MW lower than 10 000. Using one-dimensional gel electrophoresis (1-DE), two-dimensional gel electrophoresis (2-DE), and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. More than 100 components were detected by mass spectrometry, and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Also it is revealed that gene duplication and focal hypermutation have taken place during the evolution of the spider toxins.

[reaction: see text] Dirhodium tetrakis((S)-N-dodecylbenzenesulfonyl)prolinate) (Rh2(S-DOSP)4) catalyzed reaction of 1,2-dihydronaphthalenes with an excess of methyl vinyldiazoacetates results in a formal double C-H activation, generating four new stereogenic centers with very high stereoselectivity. The mechanism of the C-H activation is complex, involving a combined C-H activation/Cope rearrangement followed by a retro-Cope rearrangement.

The development of new catalytic asymmetric reactions can lead to exciting new strategies for organic synthesis. This article describes the synthetic utility of the combined C—H activation/Cope rearrangement, achieved by dirhodium tetraprolinate-catalyzed reaction of vinyldiazoacetates with compounds containing allylic C—H bonds. The transformation is highly diastereoselective and enantioselective. The product distribution, however, is highly substrate dependent, the major side products being either direct C—H activation or cyclopropanation.

The C-H activation of silyl ethers by means of rhodium carbenoid-induced C-H insertion represents a very direct method for the stereoselective synthesis of silyl-protected beta-hydroxy esters. The reaction can proceed with very high regio-, diastereo-, and enantioselectivity and represents a surrogate to the aldol reaction. The reaction is catalyzed by the rhodium prolinate complex Rh(2)(S-DOSP)(4). A critical requirement for the high chemoselectivity is the use of donor/acceptor-substituted carbenoids such as those derived from methyl aryldiazoacetates. A range of silyl ethers may be used such as allyl silyl ethers, tetraalkoxysilanes, and even simple trimethylsilyl alkyl ethers. In general, C-H activation preferentially occurs at methylene sites, as the reactivity is controlled by a delicate balance between steric and electronic effects.

[reaction: see text]. Tetrakis[N-[4-dodecylphenyl)sulfonyl]-(S)-prolinato]-dirhodium [Rh2(S-DOSP)4] catalyzed decomposition of methyl aryldiazoacetates in the presence of alkenes results in allylic C-H activation by means of a rhodium-carbene induced C-H insertion. The resulting gamma,delta-unsaturated esters are equivalent to products that would be traditionally obtained from an asymmetric Claisen rearrangement. Highly regio- and enantioselective C-H insertions can be achieved, and in certain cases, good diastereocontrol is also possible.

Background Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are thought to be associated with increased mortality in elderly patients. Low retinol-binding protein-4 (RBP4) is associated with a high risk of respiratory infections in the general population. Therefore, we hypothesized that low RBP4 levels are associated with an increased risk of AECOPD and can be used as a biomarker for AECOPD in elderly patients. Methods Enzyme-linked immunosorbent assays were used to assess RBP4 levels in elderly with AECOPD within the first 24 hours after intensive care unit admission. Forty-six elderly patients with stable COPD in outpatient clinics and 50 healthy elderly persons who had physical examinations as outpatients were controls. Results In AECOPD patients, RBP4 levels were lower than those in stable COPD patients and healthy controls (59.7 vs 91.2 and 113.6 mg/L, p < 0.001). RBP4 levels were decreased by 30.6% in non-survivors compared with survivors (51.5 vs 74.2 mg/L, p < 0.001). A higher Acute Physiology and Chronic Health Enquiry II (APACHE II) score and Simplified Acute Physiology score (SAPS II) were associated with lower RBP4 levels (r = −0.692, p = 0.024 and r = −0.670, p = 0.015, respectively). RBP4 was positively correlated with creatinine and body mass index, and negatively correlated with C-reactive protein and Global Initiative for Chronic Obstructive Lung Disease stage. Multivariate logistic regression showed that RBP4 was an independent mortality predictor of AECOPD (odds ratio: 0.926, p = 0.007). Analysis of the area under the receiver operating characteristic (AUC) curve showed that RBP4 showed good discrimination (AUC: 0.88; 95% confidence interval: 0.78–0.94; p = 0.008) in predicting mortality. RBP4 improved the prognostic accuracy of mortality for the APACHE II and SAPS II scores. Conclusions Serum RBP4 levels are significantly reduced in elderly AECOPD patients. RBP4 might be a good predictive biomarker for mortality in elderly AECOPD patients in the intensive care unit.

[reaction: see text]. Tetrakis[N-[4-dodecylphenyl)sulfonyl]-(S)-prolinato]-dirhodium [Rh2(S-DOSP)4] catalyzed decomposition of methyl aryldiazoacetates in the presence of alkenes results in allylic C-H activation by means of a rhodium-carbene induced C-H insertion. The resulting gamma,delta-unsaturated esters are equivalent to products that would be traditionally obtained from an asymmetric Claisen rearrangement. Highly regio- and enantioselective C-H insertions can be achieved, and in certain cases, good diastereocontrol is also possible.

An on-line optimization model is established for a large scale coal processing and town gas plant with the application of Input-Output analysis. This paper presents a class of MES based on the concept of Message Bus with the three-layer structure of plant-wide automation and management system. Determining the appropriate values to assign the parameters of the model from MES and ERP on a message bus is a critical character of the study. A lot of obstacles to implementation of the on-line optimization have been overcome and a considerable marginal profit has been achieved.

The hepatocyte is a highly polarized cell with a heterogeneous distribution of plasma-membrane (PM) proteins. To reduce the complexity of the proteome of liver tissue and give a comprehensive profile of hepatocyte PM, two PM purification methods based on cell surface modification, named the biotin-avidin (BA) and cationic silica-polyanion (CSP) strategies were evaluated and compared with the traditional cell fractionation method to prepare highly enriched PM from freshly isolated C57 mouse hepatocytes. Employing different principles for PM modification, both methods were effective in the isolation of general and purified PM fraction. The CSP strategy showed better yield for the PM purification from freshly isolated hepatocytes. 189 non-redundant proteins were identified, including 49 from the BA method and 185 from CSP strategy. Many known and novel PM-associated proteins were also found. Our evaluation here should give implications for PM preparation from other freshly isolated tissue-derived cells. The hepatocyte PM proteins identified here should be taken as a references for the PM-related functional and diseases research.

Rh(2)(S-DOSP)(4)-catalyzed C-H activation to N,N-dimethylanilines is described. A double C-H activation was possible by using an excess of methyl aryldiazoacetate, and this provided a very direct approach to C(2)-symmetric anilines. [reaction--see text]

Rh(2)(S-DOSP)(4)-catalyzed C-H activation to N,N-dimethylanilines is described. A double C-H activation was possible by using an excess of methyl aryldiazoacetate, and this provided a very direct approach to C(2)-symmetric anilines. [reaction--see text]

An efficient C–H activation of primary benzylic positions by means of rhodium carbenoid induced C–H insertions is described. This key step was used in concise syntheses of (+)-imperanene and (−)-α-conidendrin.

Tetrakis[N-[4-dodecylphenyl)sulfonyl]-(S)-prolinate]dirhodium [Rh(2)(S-DOSP)(4)]-catalyzed decomposition of methyl aryldiazoacetates in the presence of substituted ethylbenzenes results in benzylic C-H activation by means of a rhodium-carbenoid-induced C-H insertion. A Hammet study showed that positive charge buildup occurred on the benzylic carbon in the transition state of the C-H activation step. C-H activation of toluene and isopropylbenzene is possible, but a competing double cyclopropanation occurs with these substrates. The C-H activation is highly regioselective and enantioselective, and in certain cases, moderate diastereoselectivity is also possible.

Page 1672. Eric C. Peters was mistakenly listed in the Acknowledgment of the published Communication rather than in the author list. Eric C. Peters should be included in the author list between Jonathan G. Swoboda and Charles Y. Cho, along with indication (‡) of his affiliation with The Genomics Institute of the Novartis Research Foundation. The first sentence of the Acknowledgment should now read as follows: The authors acknowledge the efforts of John Walker for experimental support.

The Rh2(S-DOSP)4-catalyzed reaction of vinyldiazoacetates with dihydronaphthalenes results in a highly enantioselective (91−99.6% ee) and diastereoselective (>98% de) C−H functionalization. The apparent intermolecular C−H insertion was demonstrated to be a combined C−H activation/Cope rearrangement followed by a retro-Cope rearrangement.