A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

Discovery of a Novel Orally Active, Selective LPA Receptor Type 1 Antagonist, 4-(4-(2-Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin-1-yl)-4-oxobutanoic Acid, with a Distinct Molecular Scaffold.

Lysophosphatidic acid receptor 1 (LPAR1) antagonists show promise as potentially novel antifibrotic treatments. In a human LPAR1 ß-arrestin recruitment-based high-throughput screening campaign, we identified urea 19 as a hit with a LPAR1 IC50 value of 5.0 µM. Hit-to-lead activities revealed that one of the urea nitrogen atoms can be replaced by carbon and establish the corresponding phenylacetic amide as a lead structure for further optimization. Medicinal chemistry efforts led to the discovery of piperidine 18 as a potent and selective LPAR1 antagonist with oral activity in a mouse model of LPA-induced skin vascular leakage. The molecular scaffold of 18 shares no obvious structural similarity with any other LPAR1 antagonist disclosed so far.

Discovery of the Novel, Orally Active, and Selective LPA1 Receptor Antagonist ACT-1016-0707 as a Preclinical Candidate for the Treatment of Fibrotic Diseases.

Piperidine 3 is a potent and selective lysophosphatidic acid receptor subtype 1 receptor (LPAR1) antagonist that has shown efficacy in a skin vascular leakage target engagement model in mice. However, compound 3 has very high human plasma protein binding and high clearance in rats, which could significantly hamper its clinical development. Continued lead optimization led to the potent, less protein bound, metabolically stable, and orally active azetidine 17. Rat pharmacokinetics (PK) studies revealed that 17 accumulated in the liver. In vitro studies indicated that 17 is an organic anion co-transporting polypeptide 1B1 (OATP1B1) substrate. Although analogue 24 was no longer a substrate of OATP1B1, PK studies suggested that the compound undergoes enterohepatic recirculation. Replacing the carboxylic acidic side chain by a non-acidic sulfamide moiety and further fine-tuning of the scaffold yielded the potent, orally active LPAR1 antagonist 49, which was selected for preclinical development for the treatment of fibrotic diseases.

NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

Identifying early pulmonary arterial hypertension biomarkers in systemic sclerosis: machine learning on proteomics from the DETECT cohort.

Pulmonary arterial hypertension (PAH) is a devastating complication of systemic sclerosis (SSc). Screening for PAH in SSc has increased detection, allowed early treatment for PAH and improved patient outcomes. Blood-based biomarkers that reliably identify SSc patients at risk of PAH, or with early disease, would significantly improve screening, potentially leading to improved survival, and provide novel mechanistic insights into early disease. The main objective of this study was to identify a proteomic biomarker signature that could discriminate SSc patients with and without PAH using a machine learning approach and to validate the findings in an external cohort.Serum samples from patients with SSc and PAH (n=77) and SSc without pulmonary hypertension (non-PH) (n=80) were randomly selected from the clinical DETECT study and underwent proteomic screening using the Myriad RBM Discovery platform consisting of 313 proteins. Samples from an independent validation SSc cohort (PAH n=22 and non-PH n=22) were obtained from the University of Sheffield (Sheffield, UK).Random forest analysis identified a novel panel of eight proteins, comprising collagen IV, endostatin, insulin-like growth factor binding protein (IGFBP)-2, IGFBP-7, matrix metallopeptidase-2, neuropilin-1, N-terminal pro-brain natriuretic peptide and RAGE (receptor for advanced glycation end products), that discriminated PAH from non-PH in SSc patients in the DETECT Discovery Cohort (average area under the receiver operating characteristic curve 0.741, 65.1% sensitivity/69.0% specificity), which was reproduced in the Sheffield Confirmatory Cohort (81.1% accuracy, 77.3% sensitivity/86.5% specificity).This novel eight-protein biomarker panel has the potential to improve early detection of PAH in SSc patients and may provide novel insights into the pathogenesis of PAH in the context of SSc.

GPCR-induced YAP activation sensitizes fibroblasts to profibrotic activity of TGFß1.

Tissue fibrosis is a pathological condition characterized by uncontrolled fibroblast activation that ultimately leads to organ failure. The TGFß1 pathway, one of the major players in establishment of the disease phenotype, is dependent on the transcriptional co-activators YAP/TAZ. We were interested whether fibroblasts can be sensitized to TGFß1 by activation of the GPCR/YAP/TAZ axis and whether this mechanism explains the profibrotic properties of diverse GPCR ligands. We found that LPA, S1P and thrombin cooperate in human dermal fibroblasts with TGFß1 to induce extracellular matrix synthesis, myofibroblast marker expression and cytokine secretion. Whole genome expression profiling identified a YAP/TAZ signature behind the synergistic profibrotic effects of LPA and TGFß1. LPA, S1P and thrombin stimulation led to activation of the Rho-YAP axis, an increase of nuclear YAP-Smad2 complexes and enhanced expression of profibrotic YAP/Smad2-target genes. More generally, dermal, cardiac and lung fibroblast responses to TGFß1 could be enhanced by increasing YAP nuclear levels (with GPCR ligands LPA, S1P, thrombin or Rho activator) and inhibited by decreasing nuclear YAP (with Rho inhibitor, forskolin, latrunculin B or 2-deoxy-glucose). Thus, we present here a conceptually interesting finding that fibroblast responses to TGFß1 can be predicted based on the nuclear levels of YAP and modulated by stimuli/treatments that change YAP nuclear levels. Our study contributes to better understanding of fibrosis as a complex interplay of signalling pathways and proposes YAP/TAZ as promising targets in the treatment of fibrosis.

The Antifibrotic Activity of Prostacyclin Receptor Agonism Is Mediated through Inhibition of YAP/TAZ.

Idiopathic pulmonary fibrosis is a life-threatening progressive disease characterized by loss of alveolar epithelial cells, inflammation, and aberrant fibroblast activation. The two currently approved therapies do not halt or reverse tissue remodeling, and therefore novel disease-modifying mechanisms are needed. Our results describe YAP/TAZ inhibition through prostacyclin (IP) receptor activation as a novel mechanism that suppresses profibrotic (myo)fibroblast activity. We investigated the antifibrotic properties of the selective IP receptor agonist ACT-333679 using primary human lung fibroblasts. ACT-333679 prevented transforming growth factor ß1-induced fibroblast-to-myofibroblast transition, proliferation, extracellular matrix synthesis, and IL-6 and PAI-1 secretion, and exerted relaxant effects in cell contraction assays. ACT-333679 treatment also reverted an established myofibroblast phenotype. Unbiased analysis of ACT-333679-induced whole-genome expression changes in transforming growth factor ß1-treated fibroblasts identified significant attenuation of genes regulated by YAP/TAZ, two transcriptional cofactors that are essential for fibrosis. We then demonstrated that ACT-333679, via elevation of cAMP, caused YAP/TAZ nuclear exclusion and subsequent suppression of YAP/TAZ-dependent profibrotic gene transcription. In summary, we offer a rationale for further exploring the potential of IP receptor agonists for the treatment of idiopathic pulmonary fibrosis.

Novel high-throughput myofibroblast assays identify agonists with therapeutic potential in pulmonary fibrosis that act via EP2 and EP4 receptors.

Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile α-smooth muscle actin (α-SMA)-expressing myofibroblasts that produce and secrete ECM are key effector cells of the disease and therefore represent a viable target for potential novel anti-fibrotic treatments. We used primary normal human lung fibroblasts (NHLF) in two novel high-throughput screening assays to discover molecules that inhibit or revert fibroblast-to-myofibroblast differentiation. A phenotypic high-content assay (HCA) quantified the degree of myofibroblast differentiation, whereas an impedance-based assay, multiplexed with MS / MS quantification of α-SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E1 (PGE1) alprostadil, which very effectively and potently attenuated and even reversed TGF-ß1-induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF-ß1-induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP2 and EP4, respectively). However, selective activation of the EP2 or the EP4 receptor was already sufficient to prevent or reverse TGF-ß1-induced NHLF myofibroblast transition. Our high-throughput assays identified chemical structures with potent anti-fibrotic properties acting through potentially novel mechanisms.

Assessment of Peripheral Serotonin Synthesis Using Stable Isotope-Labeled Tryptophan.

Serotonin (5-HT) is synthesized from dietary tryptophan (Trp) and plays an important role in numerous diseases of the central nervous system and periphery. Stable isotope tracers enable safe monitoring of metabolic rates. Here we demonstrate measurement of peripheral 5-HT synthesis in healthy subjects by monitoring the produced [13 C10 ]-5-HT (h-5-HT) in EDTA-whole blood from three doses of orally administered [13 C11 ]-Trp (h-Trp) tracer. h-Trp was rapidly absorbed and distributed in a multiphasic manner, followed by a slower terminal elimination phase. The h-5-HT synthesis rate was dependent on h-Trp dose, appeared linear up to 12 hours postdose, and could be reliably assessed for the two highest doses. The human data was compared to similar studies in rats and dogs, finding larger interspecies differences in the h-5-HT synthesis rate than in 5-HT levels. In future studies, the h-5-HT synthesis rate can be used to assess disease-dysregulated 5-HT synthesis or quantify the pharmacodynamics of 5-HT synthesis inhibitors.

A fully automated image analysis method to quantify lung fibrosis in the bleomycin-induced rat model.

Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone.

S1P1 Modulator-Induced G αi Signaling and ß-Arrestin Recruitment Are Both Necessary to Induce Rapid and Efficient Reduction of Blood Lymphocyte Count In Vivo.

S1P1 (sphingosine-1-phosphate receptor 1) agonists prevent lymphocyte egress from secondary lymphoid organs and cause a reduction in the number of circulating blood lymphocytes. We hypothesized that S1P1 receptor modulators with pathway-selective signaling properties could help to further elucidate the molecular mechanisms involved in lymphocyte trapping. A proprietary S1P1 receptor modulator library was screened for compounds with clear potency differences in ß-arrestin recruitment and G protein alpha i subunit (G αi) protein-mediated signaling. We describe here the structure-activity relationships of highly potent S1P1 modulators with apparent pathway selectivity for ß-arrestin recruitment. The most differentiated compound, D3-2, displayed a 180-fold higher potency in the ß-arrestin recruitment assay (EC50 0.9 nM) compared with the G αi-activation assay (167 nM), whereas ponesimod, a S1P1 modulator that is currently in advanced clinical development in multiple sclerosis, was equipotent in both assays (EC50 1.5 and 1.1 nM, respectively). Using these novel compounds as pharmacological tools, we showed that although a high potency in ß-arrestin recruitment is required to fully internalize S1P1 receptors, the potency in inducing G αi signaling determines the rate of receptor internalization in vitro. In contrast to ponesimod, the compound D3-2 did not reduce the number or circulating lymphocytes in rats despite high plasma exposures. Thus, for rapid and maximal S1P1 receptor internalization a high potency in both G αi signaling and ß-arrestin recruitment is mandatory and this translates into efficient reduction of the number of circulating lymphocytes in vivo.

Cenerimod, a novel selective S1P1 receptor modulator with unique signaling properties.

Sphingosine-1-phosphate receptor 1 (S1P1 ) modulators sequester circulating lymphocytes within lymph nodes, thereby preventing potentially pathogenic autoimmune cells from exiting into the blood stream and reaching inflamed tissues. S1P1 receptor modulation may thus offer potential to treat various autoimmune diseases. The first nonselective S1P1-5 receptor modulator FTY720/fingolimod/Gilenya® has successfully demonstrated clinical efficacy in relapsing forms of multiple sclerosis. However, cardiovascular, hepatic, and respiratory side-effects were reported and there is a need for novel S1P1 receptor modulators with better safety profiles. Here, we describe the discovery of cenerimod, a novel, potent and selective S1P1 receptor modulator with unique S1P1 receptor signaling properties and absence of broncho- and vasoconstrictor effects ex vivo and in vivo. Cenerimod dose-dependently lowered circulating lymphocyte counts in rats and mice after oral administration and effectively attenuated disease parameters in a mouse experimental autoimmune encephalitis (EAE) model. Cenerimod has potential as novel therapy with improved safety profile for autoimmune diseases with high unmet medical need.

Selexipag Active Metabolite ACT-333679 Displays Strong Anticontractile and Antiremodeling Effects but Low ß-Arrestin Recruitment and Desensitization Potential.

Prostacyclin (PGI2) receptor (IP receptor) agonists, which are indicated for the treatment of pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation, and extracellular matrix synthesis. Selexipag (Uptravi, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide) is the first nonprostanoid IP receptor agonist, it is available orally and was recently approved for the treatment of PAH. In this study we show that the active metabolite of selexipag and the main contributor to clinical efficacy ACT-333679 (previously known as MRE-269) behaved as a full agonist in multiple PAH-relevant receptor-distal-or downstream-cellular assays with a maximal efficacy (Emax) comparable to that of the prototypic PGI2 analog iloprost. In PASMC, ACT-333679 potently induced cellular relaxation (EC50 4.3 nM) and inhibited cell proliferation (IC50 4.0 nM) as well as extracellular matrix synthesis (IC50 8.3 nM). In contrast, ACT-333679 displayed partial agonism in receptor-proximal-or upstream-cAMP accumulation assays (Emax 56%) when compared with iloprost and the PGI2 analogs beraprost and treprostinil (Emax ∼100%). Partial agonism of ACT-333679 also resulted in limited ß-arrestin recruitment (Emax 40%) and lack of sustained IP receptor internalization, whereas all tested PGI2 analogs behaved as full agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related receptor-proximal readouts.

MMP-7 is a predictive biomarker of disease progression in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with poor prognosis, which is characterised by destruction of normal lung architecture and excessive deposition of lung extracellular matrix. The heterogeneity of disease progression in patients with IPF poses significant obstacles to patient care and prevents efficient development of novel therapeutic interventions. Blood biomarkers, reflecting pathobiological processes in the lung, could provide objective evidence of the underlying disease. Longitudinally collected serum samples from the Bosentan Use in Interstitial Lung Disease (BUILD)-3 trial were used to measure four biomarkers (metalloproteinase-7 (MMP-7), Fas death receptor ligand, osteopontin and procollagen type I C-peptide), to assess their potential prognostic capabilities and to follow changes during disease progression in patients with IPF. In baseline BUILD-3 samples, only MMP-7 showed clearly elevated protein levels compared with samples from healthy controls, and further investigations demonstrated that MMP-7 levels also increased over time. Baseline levels of MMP-7 were able to predict patients who had higher risk of worsening and, notably, baseline levels of MMP-7 could predict changes in FVC as early as month 4. MMP-7 shows potential to be a reliable predictor of lung function decline and disease progression.

Serotonin biosynthesis as a predictive marker of serotonin pharmacodynamics and disease-induced dysregulation.

The biogenic amine serotonin (5-HT) is a multi-faceted hormone that is synthesized from dietary tryptophan with the rate limiting step being catalyzed by the enzyme tryptophan hydroxylase (TPH). The therapeutic potential of peripheral 5-HT synthesis inhibitors has been demonstrated in a number of clinical and pre-clinical studies in diseases including carcinoid syndrome, lung fibrosis, ulcerative colitis and obesity. Due to the long half-life of 5-HT in blood and lung, changes in steady-state levels are slow to manifest themselves. Here, the administration of stable isotope labeled tryptophan (heavy "h-Trp") and resultant in vivo conversion to h-5-HT is used to monitor 5-HT synthesis in rats. Dose responses for the blockade of h-5-HT appearance in blood with the TPH inhibitors L-para-chlorophenylalanine (30 and 100 mg/kg) and telotristat etiprate (6, 20 and 60 mg/kg), demonstrated that the method enables robust quantification of pharmacodynamic effects on a short time-scale, opening the possibility for rapid screening of TPH1 inhibitors in vivo. In the bleomycin-induced lung fibrosis rat model, the mechanism of lung 5-HT increase was investigated using a combination of synthesis and steady state 5-HT measurement. Elevated 5-HT synthesis measured in the injured lungs was an early predictor of disease induced increases in total 5-HT.

Novel S1P1 receptor agonists - Part 4: Alkylaminomethyl substituted aryl head groups.

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.

Novel S1P1 receptor agonists - Part 5: From amino-to alkoxy-pyridines.

In a previous communication we reported on the discovery of aminopyridine 1 as a potent, selective and orally active S1P1 receptor agonist. More detailed studies revealed that this compound is phototoxic in vitro. As a result of efforts aiming at eliminating this undesired property, a series of alkoxy substituted pyridine derivatives was discovered. The photo irritancy factor (PIF) of these alkoxy pyridines was significantly lower than the one of aminopyridine 1 and most compounds were not phototoxic. Focused SAR studies showed, that 2-, 3-, and 4-pyridine derivatives delivered highly potent S1P1 receptor agonists. While the 2-pyridines were clearly more selective against S1PR3, the corresponding 3- or 4-pyridine analogues showed significantly longer oral half-lives and as a consequence longer pharmacological duration of action after oral administration. One of the best compounds, cyclopentoxy-pyridine 45b lacked phototoxicity, showed EC50 values of 0.7 and 140 nM on S1PR1 and S1PR3, respectively, and maximally reduced the blood lymphocyte count for at least 24 h after oral administration of 10 mg/kg to Wistar rats.

Comparison of Macitentan and Bosentan on Right Ventricular Remodeling in a Rat Model of Non-vasoreactive Pulmonary Hypertension.

AIMS: We compared the efficacy of macitentan, a novel dual endothelin A/endothelin B receptor antagonist, with that of another dual endothelin receptor antagonist, bosentan, in a rat model of non-vasoreactive pulmonary hypertension (PH) with particular emphasis on right ventricular (RV) remodeling. METHODS AND RESULTS: Unlike monocrotaline or hypoxic/sugen rats, bleomycin-treated rats presented a non-vasoreactive PH characterized by the absence of pulmonary dilatation to adenosine. We therefore chose the bleomycin rat model to compare the effects of the maximally effective doses of macitentan and bosentan on pulmonary vascular and RV remodeling. Macitentan (100 mg·kg(-1)·d(-1)), but not bosentan (300 mg·kg(-1)·d(-1)), significantly prevented pulmonary vascular remodeling, RV hypertrophy, and cardiomyocyte diameter increase. Cardiac protection by macitentan was associated with a significant attenuation of genes related to cell hypertrophy and extracellular matrix remodeling. Microautoradiography and high performance liquid chromatography analysis showed greater distribution of macitentan than bosentan in the RV and pulmonary tissue. CONCLUSIONS: Macitentan was more efficacious than bosentan in preventing the development of pulmonary and RV hypertrophies in a model of non-vasoreactive PH. Greater ability to distribute into the tissue could contribute to the greater structural improvement by macitentan compared with bosentan.

FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction.

FTY720 phosphate (FTY720-P; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, monodihydrogen phosphate ester) is a nonselective sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P2 receptor subtype. However, we have recently shown that FTY720-P displays significant S1P2 receptor agonist activity in recombinant cells and fibroblasts expressing endogenous S1P2 receptors. To elucidate the S1P2-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and small interfering RNA-based pathway inhibition in recombinant Chinese hamster ovary (CHO)-S1P2 cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P2 cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα12/13 knockdown and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα12/13/Rho/ROCK signaling. S1P similarly activated Gα12/13/Rho/ROCK signaling via S1P2 receptors, whereas the two selective S1P1 receptor agonists (Z,Z)-5-(3-chloro-4-[(2R)-2,3-dihydroxy-propoxy]-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (ponesimond) and 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871) were inactive. In lung myofibroblasts, which mainly expressed the S1P2 receptor subtype, we showed that FTY720-P selectively activated the Gα12/13/Rho/ROCK pathway via the S1P2 receptor. Moreover, the activation of the Gα12/13/Rho/ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or phenotypic assays in native expression systems can uncover activation of additional pathways, such as Gα12/13/Rho/ROCK signaling.