As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC50 of approximately 4 μM and exhibited a substantially slower rate of onset than the typical pore blocker La3+, together with almost no current recovery upon wash-out over 4 min. For the less Ca2+-selective Orai1 E106D pore mutant, ICRAC inhibition was significantly reduced. FRET experiments indicated that neither STIM1–STIM1 oligomerization nor STIM1–Orai1 coupling was affected by these compounds.These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.Measurements and Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.
Modification
of proteins with polyubiquitin chains is a key regulatory
mechanism to control cellular behavior and alterations in the ubiquitin
system are linked to many diseases. Linear (M1-linked) polyubiquitin
chains play pivotal roles in several cellular signaling pathways mediating
immune and inflammatory responses and apoptotic cell death. These
chains are formed by the linear ubiquitin chain assembly complex (LUBAC),
a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L,
and SHARPIN. Herein, we describe the discovery of inhibitors targeting
the active site cysteine of the catalytic subunit HOIP using fragment-based
covalent ligand screening. We report the synthesis of a diverse library
of electrophilic fragments and demonstrate an integrated use of protein
LC–MS, biochemical ubiquitination assays, chemical synthesis,
and protein crystallography to enable the first structure-based development
of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based
assays and chemoproteomics, we demonstrate that these compounds effectively
penetrate mammalian cells to label and inhibit HOIP and NF-κB
activation, making them suitable hits for the development of selective
probes to study LUBAC biology. Our results illustrate the power of
fragment-based covalent ligand screening to discover lead compounds
for challenging targets, which holds promise to be a general approach
for the development of cell-permeable inhibitors of thioester-forming
E3 ubiquitin ligases.
α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analogue
N
-oxalylglycine (NOG) and its cell-permeable pro-drug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolysed to yield methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived TCA-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase (GDH). These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration, and have important implications for the use of (D)MOG in studying αKG-dependent signalling and metabolism.
The tetrahydronaphthalene-benzoxazine glucocorticoid receptor (GR) partial agonist 4b was optimized to produce potent full agonists of GR. Aromatic ring substitution of the tetrahydronaphthalene leads to weak GR antagonists. Discovery of an "agonist trigger" substituent on the saturated ring of the tetrahydronaphthalene leads to increased potency and efficacious GR agonism. These compounds are efficacy selective in an NFkB GR agonist assay (representing transrepression effects) over an MMTV GR agonist assay (representing transactivation effects). 52 and 60 have NFkB pIC(50) = 8.92 (105%) and 8.69 (92%) and MMTV pEC(50) = 8.20 (47%) and 7.75 (39%), respectively. The impact of the trigger substituent on agonism is modeled within GR and discussed. 36, 52, and 60 have anti-inflammatory activity in a mouse model of inflammation after topical dosing with 52 and 60, having an effect similar to that of dexamethasone. The original lead was discovered by a manual agreement docking method, and automation of this method is also described.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.