Norbert Redemann

The Dissociation of Cohesin from Chromosomes in Prophase Is Regulated by Polo-like Kinase

The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.

Structure of a Cbs-Domain Pair from the Regulatory Gamma1 Subunit of Human Ampk in Complex with AMP and Zmp.

AMP-activated kinase (AMPK) is central to sensing energy status in eukaryotic cells via binding of AMP and ATP to CBS (cystathionine beta-synthase) domains in the regulatory gamma subunit. The structure of a CBS-domain pair from human AMPK gamma1 in complex with the physiological activator AMP and the pharmacological activator ZMP (AICAR) is presented.

Normalization of Prandial Blood Glucose and Improvement of Glucose Tolerance by Liver-Specific Inhibition of SH2 Domain–Containing Inositol Phosphatase 2 (SHIP2) in Diabetic KKAy Mice: SHIP2 Inhibition Causes Insulin-Mimetic Effects on Glycogen Metabolism, Gluconeogenesis, and Glycolysis

Type 2 diabetes is characterized by a progressive resistance of peripheral tissues to insulin. Recent data have established the lipid phosphatase SH2 domain–containing inositol phosphatase 2 (SHIP2) as a critical negative regulator of insulin signal transduction. Mutations in the SHIP2 gene are associated with type 2 diabetes. Here, we used hyperglycemic and hyperinsulinemic KKAy mice to gain insight into the signaling events and metabolic changes triggered by SHIP2 inhibition in vivo. Liver-specific expression of a dominant-negative SHIP2 mutant in KKAy mice increased basal and insulin-stimulated Akt phosphorylation. Protein levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were significantly reduced, and consequently the liver produced less glucose through gluconeogenesis. Furthermore, SHIP2 inhibition improved hepatic glycogen metabolism by modulating the phosphorylation states of glycogen phosphorylase and glycogen synthase, which ultimately increased hepatic glycogen content. Enhanced glucokinase and reduced pyruvate dehydrogenase kinase 4 expression, together with increased plasma triglycerides, indicate improved glycolysis. As a consequence of the insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis, the liver-specific inhibition of SHIP2 improved glucose tolerance and markedly reduced prandial blood glucose levels in KKAy mice. These results support the attractiveness of a specific inhibition of SHIP2 for the prevention and/or treatment of type 2 diabetes.

An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

Inhibition of SH2‐domain containing inositol phosphatase 2 (SHIP2) in insulin producing INS1E cells improves insulin signal transduction and induces proliferation

Inhibition of the lipid phosphatase SH2‐domain containing inositol phosphatase 2 (SHIP2) in L6‐C10 muscle cells, in 3T3‐L1 adipocytes and in the liver of db/db mice has been shown to ameliorate insulin signal transduction and established SHIP2 as a negative regulator of insulin action. Here we show that SHIP2 inhibition in INS1E insulinoma cells increased Akt, glycogen synthase kinase 3 and extracellular signal‐regulated kinases 1 and 2 phosphorylation. SHIP2 inhibition did not prevent palmitate‐induced apoptosis, but increased cell proliferation. Our data raise the interesting possibility that SHIP2 inhibition exerts proliferative effects in β‐cells and further support the attractiveness of a specific inhibition of SHIP2 for the treatment of type 2 diabetes.

A high-throughput, image-based screen to identify kinases involved in brown adipocyte development

Multiple kinases are potential targets to enhance the development and function of brown fat. Finding the kinases that power brown fat Obesity and its associated health risks are a critical and growing problem. Unlike white adipocytes, which store energy, brown adipocytes dissipate energy in the form of heat. Expansion or activation of brown fat has been proposed as a strategy to combat obesity. Using high-throughput analysis that combined RNA interference with pharmacological inhibitors, Perdikari et al. identified kinases that affected brown adipocyte proliferation, differentiation, or formation (when these cells acquire the ability to dissipate energy as heat). Of the 190 brown fat–regulating kinases, they investigated in detail the role of AMPK in promoting the formation of brown adipocytes. Their results highlight kinases that could be therapeutically targeted to enhance the development and function of brown fat. Brown adipose tissue (BAT) is responsible for thermogenesis that is not associated with shivering through the process of converting chemical energy into heat through uncoupling protein 1 (UCP1) in the mitochondria. Thus, expanding or activating BAT could be a potential tool against obesity. To analyze the effect of kinase signaling on brown adipocyte formation, a process that describes the acquisition of the ability to dissipate energy as heat, we performed lentiviral-mediated short hairpin knockdown or used pharmacological inhibitors in a high-content and high-throughput in vitro image-based screen. We identified 190 kinases that either stimulated or inhibited brown adipocyte proliferation, differentiation, or formation. Among these kinases, we found that 5′ AMP–activated protein kinase (AMPK) promoted the formation of brown adipocytes abundant inUCP1. Together, our results provide insight into the kinases, particularly AMPK, that regulate brown adipocyte formation.

Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction:

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

Discovery of BI 99179, a potent and selective inhibitor of type I fatty acid synthase with central exposure.

Based on a high-throughput screen, cyclopentanecarboxanilides were identified as a new chemotype of non-covalent inhibitors of type I fatty acid synthase (FAS). Starting from initial hits we aimed at generating a tool compound suitable for the in vivo validation of FAS as a therapeutic target. Optimisation yielded BI 99179 which is characterised by high potency, remarkably high selectivity and significant exposure (both peripheral and central) upon oral administration in rats.

Low concentrations of UD-CG 212 enhance myocyte contractility by an increase in calcium responsiveness in the presence of inorganic phosphate

Recent data show that UD-CG 212 in nanomolar concentrations increases myofibrillar Ca++ responsiveness of chemically skinned cardiac preparations in the presence of elevated inorganic phosphate. We studied the effects of UD-CG 212 on cell shortening of intact myocytes and in addition measured the intracellular calcium transients with the aid of INDO-1 fluorescence in the presence of 5 mM inorganic phosphate.The validity of our experimental system was first tested with the calcium channel opener Bay k 8644. Bay k 8644 at 10−8 M did not significantly influence myocyte shortening ( + 13.9 ± 4.6%, n = 9) but at 10−7 M and 10−6 M significantly increased contraction by 40.1 +- 13.6%and52.5 ± 17.0% respectively. Bay k 8644 at 10−8 M increased the INDO-1 fluorescence ratio by 17.3 ± 4.7% (P < 0.01; n = 9), and at 10−7 M by 21.5 + 4.3% (P < 0.01; n = 9), whereas 10−6 M Bay k 8644 had no significant effect on peak INDO-1 ratio. However, 10−7and 10−6 M Bay k 8644 accelerated and broadened the calcium transients.Cell shortening of guinea pig ventricular myocytes electrically stimulated at 1 Hz was significantly increased by UD-CG 212 (10−9-10−7 M) and isoprena line(3 × 10−8 M). An increase of 37.0 ± 14.0% (P < 0.05; n = 9) was observed at 10−9 M UD-CG 212, 90.5±18.2% (P<0.05; n=9) at 10−8 M UD-CG 212, 164.0 ± 34.9% (P < 0.05; n = 9) at 10−7 M UD-CG 212, and 258.2 ± 67.4% (P < 0.05; n = 9) at 3 × 10-8 M isoprenaline. Peak INDO-1 fluorescence ratios were not significantly (P > 0.05) influenced after addition of 10−9 M and 10−8 M UD-CG 212, but significantly increased by 19.4 ± 4.9%(P < 0.05; n = 9) at 10−7 MUD-CG 212 and by 81.1 ± 11.1% (P < 0.05; n = 9) at 3 x 10−8 M isoprenaline.In conclusion, UD-CG 212 (10−9 - 10−7 M) Concentration-dependently increased myocyte shortening in the presence of 5 mM inorganic phosphate. Low concentrations of 10−9 and 10−8 M UD-CG 212 increased myocyte contractility without altering the peak INDO-1 fluorescence ratio whereas 10−7 M UD-CG 212 and 3 × 10−8 M isoprenaline increased cell shortening as well as peak INDO-1 fluorescence ratio. These data suggest that low concentrations of UD-CG 212 increase myocyte contractility by enhancing myofibrillar calcium responsiveness whereas higher concentrations elevate intracellular calcium probably via increased intracellular CAMP brought about by phosphodiesterase inhibition.