NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors

Lévay, Magdolna; Krobert, Kurt A.; Wittig, Karola; Voigt, Niels; Bermudez, Marcel; Wolber, Gerhard; Dobrev, Dobromir; Levy, Finn Olav; Wieland, Thomas

doi:10.1124/jpet.113.207266

Cited by 85 publications

(77 citation statements)

References 54 publications

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“…Before starting phagocytosis assays with latex beads, as described above, MC were incubated with Y27632 at a concentration of 10µM for 15 minutes at 37°C, as described in [42]. NSC23766 was used at two different concentrations (10 and 50µM) for incubation of MC for 30 minutes at 37°C, as described in [43]. …”

Section: Methodsmentioning

confidence: 99%

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Marek¹,

Becker²,

Fahlbusch³

et al. 2018

Cell Physiol Biochem

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Background/Aims: Healing of mesangioproliferative glomerulonephritis involves degradation of excess extracellular matrix, resolution of hypercellularity by apoptosis and phagocytosis of apoptotic cells. Integrin receptors participate in the regulation of phagocytosis. In mice deficient for alpha8 integrin (Itga8-/-) healing of glomerulonephritis is delayed. As Itga8 is abundant in mesangial cells (MC) which are non-professional phagocytes, we hypothesized that Itga8 facilitates phagocytosis of apoptotic cells and matrix components by MC. Methods: MC were isolated from wild type (WT) and Itga8-/- mice. Latex beads were coated with matrix components. Apoptosis was induced by cisplatin in macrophages and in DiI-stained MC. After coincubation of latex beads or apoptotic cells with MC, the phagocytosis rate was detected in WT and Itga8-/- MC via fluorescence microscopy and FACS analysis. Results: Itga8-/- MC showed reduced phagocytosis of matrix-coated beads and apoptotic cells compared to WT MC. Reduction of stress fibers was observed in Itga8-/- compared to WT MC. Inhibition of cytoskeletal reorganization by inhibition of Rac1 or ROCK during phagocytosis significantly decreased the rate of phagocytosis by WT MC but not by Itga8-/- MC. Conclusion: The expression of Itga8 facilitates phagocytosis in MC, likely mediated by Itga8-cytoskeleton interactions. An impairment of MC phagocytosis might thus contribute to a delayed glomerular regeneration in Itga8-/- mice.

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Section: Methodsmentioning

confidence: 99%

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Marek¹,

Becker²,

Fahlbusch³

et al. 2018

Cell Physiol Biochem

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“…3). Within the subset of five ligands selected based on both active and inactive receptor conformations, only NSC-322661 and -13316 showed some chemical identity, both quinolonebased ligands, which is a scaffold that has been shown to bind and antagonize the muscarinic receptors, such as NSC-23766 (45). Additionally, NSC-143491 is an anthraquinone-based ligand.…”

Section: Subtype Selectivity Of Modulators On Agonist-mediated Responmentioning

confidence: 99%

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Miao

Goldfeld

Moo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M 2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [ 3 H]Nmethylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M 2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M 1 and M 3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.GPCR | allosteric modulators | ensemble docking | affinity | cooperativity

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“…To rule out the potential off‐target effects of NSC23766 observed when used at high concentrations,27 we used another structurally different Rac1 inhibitor, called EHT1864. As shown in Figure S4, also with EHT1864, a higher dose of inhibitor was necessary to significantly reduce platelet aggregation in diabetic conditions compared with control subjects.…”

Section: Resultsmentioning

confidence: 99%

Rac1 Modulates Endothelial Function and Platelet Aggregation in Diabetes Mellitus

Schiattarella

Carrizzo

Ilardi

et al. 2018

JAHA

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BackgroundVascular complications and abnormal platelet function contribute to morbidity and mortality in diabetes mellitus. We hypothesized that the Rho‐related GTPase protein, Rac1, can influence both endothelial and platelet function and might represent a potential novel therapeutic target in diabetes mellitus.Methods and ResultsWe used both in vitro and ex vivo approaches to test the effects of pharmacological inhibition of Rac1 during hyperglycemic condition. We evaluated the effect of NSC23766, a pharmacological inhibitor of Rac1, on vascular function in diabetic mice and platelet aggregation in diabetic subjects. We demonstrated that the administration of NSC23766 protects from hyperglycemia‐induced endothelial dysfunction, restoring NO levels, and reduces oxidative stress generated by nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, we identified Rho‐associated coiled‐coil serine/threonine kinase‐1 as a downstream target of Rac1. Moreover, we reported that during hyperglycemic conditions, human platelets showed hyperactivation of Rac1 and impaired NO release, which were both partially restored after NSC23766 treatment. Finally, we characterized the antiplatelet effect of NSC23766 during hyperglycemic conditions, demonstrating the additional role of Rac1 inhibition in reducing platelet aggregation in diabetic patients treated with common antiplatelet drugs.ConclusionsOur data suggest that the pharmacological inhibition of Rac1 could represent a novel therapeutic strategy to reduce endothelial dysfunction and platelet hyperaggregation in diabetes mellitus.

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NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors

Cited by 85 publications

References 54 publications

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Rac1 Modulates Endothelial Function and Platelet Aggregation in Diabetes Mellitus

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