Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit.

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA-mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

Discovery of PF-5190457, a Potent, Selective, and Orally Bioavailable Ghrelin Receptor Inverse Agonist Clinical Candidate

https://researchrepository.wvu.edu/wvu_herbwvfern/2465/thumbnail.jp

Evaluation and Synthesis of Polar Aryl- and Heteroaryl Spiroazetidine-Piperidine Acetamides as Ghrelin Inverse Agonists

Several polar heteroaromatic acetic acids and their piperidine amides were synthesized and evaluated as ghrelin or type 1a growth hormone secretagogue receptor (GHS-R1a) inverse agonists. Efforts to improve pharmacokinetic and safety profile was achieved by modulating physicochemical properties and, more specifically, emphasizing increased polarity of our chemical series. <i>ortho</i>-Carboxamide containing compounds provided optimal physicochemical, pharmacologic, and safety profile. pH-dependent chemical stability was also assessed with our series

Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H₂O₂ in living <i>Drosophila</i>

The role of hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in vivo. Here we describe a method for assessing changes in H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; within the mitochondrial matrix of living &lt;i&gt;Drosophila&lt;/i&gt;. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;. To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography–tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; production in mouse and cell culture studies

Discovery of PF-5190457, a Potent, Selective, and Orally Bioavailable Ghrelin Receptor Inverse Agonist Clinical Candidate

The identification of potent, highly selective orally bioavailable ghrelin receptor inverse agonists from a spiro-azetidino-piperidine series is described. Examples from this series have promising in vivo pharmacokinetics and increase glucose-stimulated insulin secretion in human whole and dispersed islets. A physicochemistry-based strategy to increase lipophilic efficiency for ghrelin receptor potency and retain low clearance and satisfactory permeability while reducing off-target pharmacology led to the discovery of <b>16h</b>. Compound <b>16h</b> has a superior balance of ghrelin receptor pharmacology and off-target selectivity. On the basis of its promising pharmacological and safety profile, <b>16h</b> was advanced to human clinical trials