Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.
Ontology highlight
ABSTRACT:
SUBMITTER: Giubilaro J
PROVIDER: S-EPMC8333425 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.
| S-EPMC5722207 | biostudies-literature
Project description:The Rat Sarcoma (RAS) family (NRAS, HRAS, and KRAS) is endowed with GTPase activity to regulate various signaling pathways in ubiquitous animal cells. As proto-oncogenes, RAS mutations can maintain activation, leading to the growth and proliferation of abnormal cells and the development of a variety of human cancers. For the fight against tumors, the discovery of RAS-targeted drugs is of high significance. On the one hand, the structural properties of the RAS protein make it difficult to find inhibitors specifically targeted to it. On the other hand, targeting other molecules in the RAS signaling pathway often leads to severe tissue toxicities due to the lack of disease specificity. However, computer-aided drug design (CADD) can help solve the above problems. As an interdisciplinary approach that combines computational biology with medicinal chemistry, CADD has brought a variety of advances and numerous benefits to drug design, such as the rapid identification of new targets and discovery of new drugs. Based on an overview of RAS features and the history of inhibitor discovery, this review provides insight into the application of mainstream CADD methods to RAS drug design.
| S-EPMC9457765 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.
| S-EPMC3312097 | biostudies-other
Project description:The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-?-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.
| S-EPMC3877390 | biostudies-literature
Project description:The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5-PRMT7 axis as a target for therapy.
| S-EPMC4394339 | biostudies-literature
Project description:Clathrin-mediated endocytosis (CME) is a process vital to angiogenesis as well as general vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of CME have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for CME. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide a analysis of Arf6’s function in angiogenic endothelium, focusing on its role in lumenogenesis as well as its relation to actin and CME. We found that Arf6 localized to both filamentous actin and sites of CME in 2-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence. Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and CME.
| S-EPMC9980066 | biostudies-literature
Project description:SUMOylation is a reversible post-translational modification (PTM) involving covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. Dysregulation of SUMOylation and deSUMOylation results in cellular malfunction and is linked to various diseases, such as cancer. Sentrin-specific proteases (SENPs) were identified for the maturation of SUMOs and the deconjugation of SUMOs from their substrate proteins. Hence, this is a promising target tackling the dysregulation of the SUMOylation process. Herein, we report the discovery of a novel protein-protein interaction (PPI) inhibitor for SENP1-SUMO1 by virtual screening and subsequent medicinal chemistry optimization of the hit molecule. The optimized inhibitor ZHAWOC8697 showed IC50 values of 8.6 μM against SENP1 and 2.3 μM against SENP2. With a photo affinity probe the SENP target was validated. This novel SENP inhibitor represents a new valuable tool for the study of SUMOylation processes and the SENP-associated development of small molecule-based treatment options.
| S-EPMC9602571 | biostudies-literature
Project description:The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
| S-EPMC7251565 | biostudies-literature
Project description:G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, we wondered if the endosome-initiated signal encodes any discrete spatial information. Using the beta2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signalling specificity, based on endosome-mediated spatial encoding of intracellular second messenger production and 'location aware' downstream transcriptional control.
2014-11-01 | GSE57274 | GEO
Project description:This study shows that chemically and pharmacodynamically distinct agonists acting on the same GPCR can produce indistinguishable cellular responses and that this uniformity is conferred by endosomal signaling The ability of chemically distinct ligands to produce different effects on the same G protein-coupled receptor (GPCR) has interesting therapeutic implications but, if excessively propagated downstream, would introduce biological 'noise' compromising cognate ligand detection We asked if cells have the ability to limit the degree to which chemical diversity imposed at the ligand-GPCR interface is propagated to the downstream signal We carried out an unbiased analysis of the integrated cellular response elicited by two chemically and pharmacodynamically diverse β-adrenoceptor agonists, isoproterenol and salmeterol We show that both ligands generate an identical integrated response, and that this stereotyped output requires endocytosis We further demonstrate that the endosomal β2-AR signal confers uniformity on the downstream response because it is highly sensitive and saturable Based on these findings, we propose that GPCR signaling from endosomes functions as a biological noise filter to enhance reliability of cognate ligand detection
2016-11-30 | GSE87461 | GEO