AimsTo characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon‐like peptide‐1 (GLP‐1) and glucagon receptors.Materials and methods MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP‐1 or glucagon receptors, to potentiate glucose‐stimulated insulin secretion (GSIS) in pancreatic β‐cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months.Results MEDI0382 potently activated rodent, cynomolgus and human GLP‐1 and glucagon receptors and exhibited a fivefold bias for activation of GLP‐1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP‐1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor‐mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP‐1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys.ConclusionsRepeated administration of MEDI0382 elicits profound weight loss in DIO mice and non‐human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP‐1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.
Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating transient receptor potential (melastatin) 2 (TRPM2) channels. Cell death has been attributed to the ability of these channels to raise cytosolic Ca 2 + . Recent studies however revealed that TRPM2 channels can also conduct Zn 2 + , but the physiological relevance of this property is enigmatic. Given that Zn 2 + is cytotoxic, we asked whether TRPM2 channels can permeate sufficient Zn 2 + to affect cell viability. To address this, we used the insulin secreting (INS1) β-cell line, human embryonic kidney (HEK)-293 cells transfected with TRPM2 and pancreatic islets. H 2 O 2 activation of TRPM2 channels increases the cytosolic levels of both Ca 2 + and Zn 2 + and causes apoptotic cell death. Interestingly, chelation of Zn 2 + alone was sufficient to prevent β-cell death. The source of the cytotoxic Zn 2 + is intracellular, found largely sequestered in lysosomes. Lysosomes express TRPM2 channels, providing a potential route for Zn 2 + release. Zn 2 + release is potentiated by extracellular Ca 2 + entry, indicating that Ca 2 + -induced Zn 2 + release leads to apoptosis. Knockout of TRPM2 channels protects mice from β-cell death and hyperglycaemia induced by multiple low-dose streptozotocin (STZ; MLDS) administration. These results argue that TRPM2-mediated, Ca 2 + -potentiated Zn 2 + release underlies ROS-induced β-cell death and Zn 2 + , rather than Ca 2 + , plays a primary role in apoptosis.
BACKGROUND AND PURPOSE The transient receptor potential melastatin‐3 (TRPM3) channel forms calcium‐permeable, non‐selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance. EXPERIMENTAL APPROACH We used TRPM3 channels over‐expressed in HEK 293 cells, with intracellular calcium measurement and whole‐cell patch‐clamp recording techniques. KEY RESULTS The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3‐dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (β) configuration of the side group, revealing previously unrecognized stereo‐selectivity and supporting existence of a specific binding site. A cis‐oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non‐steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects. CONCLUSIONS AND IMPLICATIONS Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.
Dual-acting glucagon-like peptide-1/glucagon receptor agonists such as G49 represent a novel therapeutic approach for patients with NASH and particularly those requiring PH. (Hepatology 2017;65:950-968).
Background and purpose: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. Experimental approach: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca 2 þ measurement. Key results: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca 2 þ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. Conclusions and implications:The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.
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