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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).

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

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.

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.