Severity of polycystic kidney disease revealed by multiparametric MRI.

PURPOSE: We aimed to compare multiple MRI parameters, including relaxation rates ( R 1 $$ {R}_1 $$ , R 2 $$ {R}_2 $$ , and R 1 ρ $$ {R}_{1\rho } $$ ), ADC from diffusion weighted imaging, pool size ratio (PSR) from quantitative magnetization transfer, and measures of exchange from spin-lock imaging ( S ρ $$ {S}_{\rho } $$ ), for assessing and predicting the severity of polycystic kidney disease (PKD) over time. METHODS: Pcy/Pcy mice with CD1 strain, a mouse model of autosomal dominant PKD, were imaged at 5, 9, and 26 wk of age using a 7T MRI system. Twelve-week normal CD1 mice were used as controls. Post-mortem paraffin tissue sections were stained using hematoxylin and eosin and picrosirius red to identify histological changes. RESULTS: Histology detected segmental cyst formation in the early stage (week 5) and progression of PKD over time in Pcy kidneys. In T 2 $$ {T}_2 $$ -weighted images, small cysts appeared locally in cystic kidneys in week 5 and gradually extended to the whole cortex and outer stripe of outer medulla region from week 5 to week 26. Regional PSR, R 1 $$ {R}_1 $$ , R 2 $$ {R}_2 $$ , and R 1 ρ $$ {R}_{1\rho } $$ decreased consistently over time compared to normal kidneys, with significant changes detected in week 5. Among all the MRI measures, R 2 $$ {R}_2 $$ and R 1 ρ $$ {R}_{1\rho } $$ allow highest detectability to PKD, while PSR and R 1 $$ {R}_1 $$ have highest correlation with pathological indices of PKD. Using optimum MRI parameters as regressors, multiple linear regression provides reliable prediction of PKD progression. CONCLUSION: R 2 $$ {R}_2 $$ , R 1 $$ {R}_1 $$ , and PSR are sensitive indicators of the presence of PKD. Multiparametric MRI allows a comprehensive analysis of renal changes caused by cyst formation and expansion.

Treatment effects of soluble guanylate cyclase modulation on diabetic kidney disease at single-cell resolution.

Diabetic kidney disease (DKD) is the most common cause of renal failure. Therapeutics development is hampered by our incomplete understanding of animal models on a cellular level. We show that ZSF1 rats recapitulate human DKD on a phenotypic and transcriptomic level. Tensor decomposition prioritizes proximal tubule (PT) and stroma as phenotype-relevant cell types exhibiting a continuous lineage relationship. As DKD features endothelial dysfunction, oxidative stress, and nitric oxide depletion, soluble guanylate cyclase (sGC) is a promising DKD drug target. sGC expression is specifically enriched in PT and stroma. In ZSF1 rats, pharmacological sGC activation confers considerable benefits over stimulation and is mechanistically related to improved oxidative stress regulation, resulting in enhanced downstream cGMP effects. Finally, we define sGC gene co-expression modules, which allow stratification of human kidney samples by DKD prevalence and disease-relevant measures such as kidney function, proteinuria, and fibrosis, underscoring the relevance of the sGC pathway to patients.

The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling.

BACKGROUND: CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied. AIM: We aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling. METHODS: CD148 forms, wild-type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin-1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme-linked immunosorbent assay, and Western blotting. RESULTS: Both CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF-induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)-expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells. CONCLUSION: Our data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1-CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.

Direct Blood Pressure-Independent Anti-Fibrotic Effects by the Selective Nonsteroidal Mineralocorticoid Receptor Antagonist Finerenone in Progressive Models of Kidney Fibrosis.

INTRODUCTION: The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated clinical benefits in chronic kidney disease patients with type 2 diabetes. Precise molecular mechanisms responsible for these benefits are incompletely understood. Here, we investigated potential direct anti-fibrotic effects and mechanisms of nonsteroidal MR antagonism by finerenone or SGLT2 inhibition by empagliflozin in 2 relevant mouse kidney fibrosis models: unilateral ureter obstruction and sub-chronic ischemia reperfusion injury. METHODS: Kidney fibrosis was induced in mice via unilateral ureteral obstruction or ischemia. In a series of experiments, mice were treated orally with the MR antagonist finerenone (3 or 10 mg/kg), the SGLT2 inhibitor empagliflozin (10 or 30 mg/kg), or in a direct comparison of both drugs. Interstitial myofibroblast accumulation was quantified via alpha-smooth muscle actin and interstitial collagen deposition via Sirius Red/Fast Green staining in both models. Secondary analyses included the assessment of inflammatory cells, kidney mRNA expression of fibrotic markers as well as functional parameters (serum creatinine and albuminuria) in the ischemic model. Blood pressure was measured via telemetry in healthy conscious compound-treated animals. RESULTS: Finerenone dose-dependently decreased pathological myofibroblast accumulation and collagen deposition with no effects on systemic blood pressure and inflammatory markers in the tested dose range. Reduced kidney fibrosis was paralleled by reduced kidney plasminogen activator inhibitor-1 (PAI-1) and naked cuticle 2 (NKD2) expression in finerenone-treated mice. In contrast, treatment with empagliflozin strongly increased urinary glucose excretion in both models and reduced ischemia-induced albuminuria but had no effects on kidney myofibroblasts or collagen deposition. DISCUSSION/CONCLUSION: Finerenone has direct anti-fibrotic properties resulting in reduced myofibroblast and collagen deposition accompanied by a reduction in renal PAI-1 and NKD2 expression in mouse models of progressive kidney fibrosis at blood pressure-independent dosages.

Establishment of a novel, cell-based autotaxin assay.

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin ß1, integrin ß3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes.

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators.

The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery.

Allosteric targeting of the FFA2 receptor (GPR43) restores responsiveness of desensitized human neutrophils.

The G protein-coupled free fatty acid receptor 2 (FFA2R) is highly expressed on neutrophils and was previously described to regulate neutrophil activation. Allosteric targeting of G protein-coupled receptors (GPCRs) is increasingly explored to create distinct pharmacology compared to endogenous, orthosteric ligands. The consequence of allosteric versus orthosteric FFA2R activation for neutrophil response, however, is currently largely elusive. Here, different FFA2R desensitization profiles in human neutrophils following allosteric or orthosteric activation are reported. Using a set of neutrophil functional assays to measure calcium flux, pERK1/2, chemotaxis, cellular degranulation, and oxidative burst together with holistic and pathway-unbiased whole cell sensing based on dynamic mass redistribution, it is found that the synthetic positive allosteric modulator agonist 4-CMTB potently activates neutrophils and simultaneously alters FFA2R responsiveness toward the endogenous, orthosteric agonist propionic acid (C3) after homologous and heterologous receptor desensitization. Stimulation with C3 or the hierarchically superior chemokine receptor activator IL-8 led to strong FFA2R desensitization and rendered neutrophils unresponsive toward repeated stimulation with C3. In contrast, stimulation with allosteric 4-CMTB engaged a distinct composition of signaling pathways as compared to orthosteric receptor activation and was able to activate neutrophils that underwent homologous and heterologous desensitization with C3 and IL-8, respectively. Moreover, allosteric FFA2R activation could re-sensitize FFA2 toward the endogenous agonist C3 after homologous and heterologous desensitization. Given the fact that receptor desensitization is critical in neutrophils to sense and adapt to their current environment, these findings are expected to be useful for the discovery of novel pharmacological mechanisms to modulate neutrophil responsiveness therapeutically.

Pharmacological inhibition of Vanin-1 is not protective in models of acute and chronic kidney disease.

Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3-/- (Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.

Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels.

Dynamic mass redistribution (DMR) and cellular dielectric spectroscopy (CDS) are label-free biosensor technologies that capture real-time integrated cellular responses upon exposure to extra- and intracellular stimuli. They register signaling routes that are accompanied by cell shape changes and/or molecular movement of cells proximal to the biosensor to which they are attached. Here, we report the unexpected observation that robust DMR and CDS signatures are also elicited upon direct stimulation of G protein-activated inwardly rectifying potassium (GIRK) channels, which are involved in the regulation of excitability in the heart and brain. Using ML297, a small-molecule GIRK activator, along with channel blockers and cytoskeletal network inhibitors, we found that GIRK activation exerts its effects on cell shape by a mechanism which depends on actin but not the microtubule network. Because label-free real-time biosensing (i) quantitatively determines concentration dependency of GIRK activators, (ii) accurately assesses the impact of GIRK channel blockers, (iii) is high throughput-compatible, and (iv) visualizes previously unknown cellular consequences downstream of direct GIRK activation, we do not only provide a novel experimental strategy for identification of GIRK ligands but also an entirely new angle to probe GIRK (ligand) biology. We envision that DMR and CDS may add to the repertoire of technologies for systematic exploitation of ion channel function and, in turn, to the identification of novel GIRK ligands in order to treat cardiovascular and neurological disorders.

Pro-Angiogenic Effects of Latent Heparanase and Thrombin Receptor-Mediated Pathways-Do They Share a Common Ground in Melanoma Cells?

Heparanase (HPSE) is an endo-ß-D-glucuronidase that cleaves heparan sulphate (HS) chains of proteoglycans (HSPGs). Besides a remodelling of the extracellular matrix, HPSE increases the bioavailability of pro-angiogenic mediators, such as HS-associated vascular endothelial growth factor (VEGF), thereby contributing to metastatic niche formation. Notably, HPSE also induces release of VEGF from tumour cells independent of its enzymatic activity, but the underlying molecular mechanisms remain unresolved. We found that exogenous addition of latent HPSE stimulates VEGF release from human MV3 melanoma cells. The same effect was noted upon direct stimulation of thrombin receptor (protease-activated receptor 1 [PAR-1]) by Thrombin Receptor Activator Peptide 6 (TRAP-6). The matricellular ligand cysteine-rich 61 protein (Cyr61) was identified as pathway component since Cyr61 knockdown in MV3 cells abolished the VEGF release by TRAP-6 and HPSE. Since both TRAP-6 and HPSE mediated an up-regulation of phosphorylated focal adhesion kinase, which could be blocked by antagonizing PAR-1, we postulated a crosstalk between latent HPSE and PAR-1 in promoting pro-angiogenic pathways. To test this hypothesis at a molecular level, we applied dynamic mass redistribution (DMR) technique measuring intracellular mass relocation as consequence of direct receptor activation. Indeed, latent HPSE evoked a concentration-dependent DMR signal in MV3 cells as TRAP-6 did. Both could be modulated by targeting G-protein receptor signalling in general or by the PAR-1 inhibitor RWJ 56110. Using cells devoid of cell surface HS synthesis, we could confirm HPSE effects on PAR-1, independent of HSPG involvement. These data indicate, for the first time, a crosstalk between latent HPSE, thrombin receptor activation and G-protein signalling in general.

Lack of beta-arrestin signaling in the absence of active G proteins.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

Temporal Bias: Time-Encoded Dynamic GPCR Signaling.

Evidence suggests that cells can time-encode signals for secure transport and perception of information, and it appears that this dynamic signaling is a common principle of nature to code information in time. G-protein-coupled receptor (GPCR) signaling networks are no exception as their composition and signal transduction appear temporally flexible. In this review, we discuss the potential mechanisms by which GPCRs code biological information in time to create 'temporal bias.' We highlight dynamic signaling patterns from the second messenger to the receptor-ligand level and shed light on the dynamics of G-protein cycles, the kinetics of ligand-receptor interaction, and the occurrence of distinct signaling waves within the cell. A dynamic feature such as temporal bias adds to the complexity of GPCR signaling bias and gives rise to the question whether this trait could be exploited to gain control over time-encoded cell physiology.

Label-Free Dynamic Mass Redistribution and Bio-Impedance Methods for Drug Discovery.

Label-free biosensors are increasingly employed in drug discovery. Cell-based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the detection of dynamic mass redistribution (DMR) and impedance biosensors using cellular dielectric spectroscopy (CDS) capture changes of the cytoskeleton of living cells in real time. Because signal transduction correlates with changes in cell morphology, DMR and CDS biosensors are exquisitely suited for recording integrated cell responses in an unbiased, yet pathway-specific manner without the use of labels that may interfere with cell function. Described in this unit are several experimental approaches utilizing optical label-free system capturing dynamic mass redistribution (DMR) in living cells (Epic System) and an impedance-based CDS technology (CellKey). In addition, potential pitfalls associated with these assays and alternative approaches for overcoming such technical challenges are discussed. © 2017 by John Wiley & Sons, Inc.

Holistic Methods for the Analysis of cNMP Effects.

Cyclic nucleotide monophosphates (cNMPs) typify the archetype second messenger in living cells and serve as molecular switches with broad functionality. cAMP and cGMP are the best-described cNMPs; however, there is a growing body of evidence indicating that also cCMP and cUMP play a substantial role in signal transduction. Despite research efforts, to date, relatively little is known about the biology of these noncanonical cNMPs, which is due, at least in part, to methodological issues in the past entailing setbacks of the entire field. Only recently, with the use of state-of-the-art techniques, it was possible to revive noncanonical cNMP research. While high-sensitive detection methods disclosed relevant levels of cCMP and cUMP in mammalian cells, knowledge about the biological effectors and their physiological interplay is still incomplete. Holistic biophysical readouts capture cell responses label-free and in an unbiased fashion with the advantage to detect concealed aspects of cell signaling that are arduous to access via traditional biochemical assay approaches. In this chapter, we introduce the dynamic mass redistribution (DMR) technology to explore cell signaling beyond established receptor-controlled mechanisms. Both common and distinctive features in the signaling structure of cCMP and cUMP were identified. Moreover, the integrated response of whole live cells revealed a hitherto undisclosed additional effector of the noncanonical cNMPs. Future studies will show how holistic methods will become integrated into the methodological arsenal of contemporary cNMP research.

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13.

Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD6 assembles with Gαi1/Gαq (but not with Gαs, Gαo and Ga12/13), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD4, a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD4 assembles-in a DVL-independent manner-with Gα12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Gαi1, Gαo, Gαs, and Gαq The FZD4-G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD4 green fluorescent protein-transfected cells depend on Gα12/13 Furthermore, expression of FZD4 and Gα12 or Gα13 in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Gα12/13 signaling, underlining the functionality of an FZD4-Gα12/13-RHO signaling axis. In summary, Gα12/13-mediated WNT/FZD4 signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD4 signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.

A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor.

Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as the first sequential activator and corroborate its two-step activation in living cells by tracking integrated responses with innovative label-free biosensors that visualize multiple signaling inputs in real time. We validate this unique pharmacology with traditional cellular readouts, including mutational and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information.

The molecular mechanism by which saturated lysophosphatidylcholine attenuates the metastatic capacity of melanoma cells.

Lysophophatidylcholine (LysoPC) is an abundant constituent in human plasma. Patients with malignant cancer diseases have attenuated LysoPC plasma levels, and thus LysoPC has been examined as a metabolic biomarker for cancer prediction. Preclinical studies have shown that solid tumor cells drastically degrade LysoPCs by incorporating their free fatty acids into cell membrane phospholipids. In this way, LysoPC C18:0 reduced the metastatic spread of murine melanoma B16.F10 cells in mice. Although membrane rigidification may have a key role in the attenuation of metastasis, evidence for this has yet to be shown. Therefore, the present study aimed to determine how LysoPC reduces the metastatic capacity of B16.F10 cells. Following cellular preincubation with LysoPC C18:0 at increasing concentrations and lengths of time, cell migration was most significantly attenuated with 450 µm LysoPC C18:0 at 72 h. Biosensor measurements suggest that, despite their abundance in B16.F10 cells, LysoPC-sensitive G protein-coupled receptors do not appear to contribute to this effect. Instead, the attenuated migration appears to result from changes in cell membrane properties and their effect on underlying signaling pathways, most likely the formation of focal adhesion complexes. Treatment with 450 µm LysoPC C18:0 activates protein kinase C (PKC)δ to phosphorylate syndecan-4, accompanied by deactivation of PKCα. Subsequently, focal adhesion complex formation was attenuated, as confirmed by the reduced activity of focal adhesion kinase (FAK). Interestingly, 450 µm LysoPC C18:1 did not affect FAK activity, explaining its lower propensity to affect migration and metastasis. Therefore, membrane rigidification by LysoPC C18:0 appears to prevent the formation of focal adhesion complexes, thus affecting integrin activity as a key for metastatic melanoma spread.

N-Benzylbenzamides: A Novel Merged Scaffold for Orally Available Dual Soluble Epoxide Hydrolase/Peroxisome Proliferator-Activated Receptor γ Modulators.

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 µM/PPARγ EC50 = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αßγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.