Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis.

Mounting evidence from the literature suggests that blocking S1P2 receptor (S1PR2) signaling could be effective for the treatment of idiopathic pulmonary fibrosis (IPF). However, only a few antagonists have been so far disclosed. A chemical enablement strategy led to the discovery of a pyridine series with good antagonist activity. A pyridazine series with improved lipophilic efficiency and with no CYP inhibition liability was identified by scaffold hopping. Further optimization led to the discovery of 40 (GLPG2938), a compound with exquisite potency on a phenotypic IL8 release assay, good pharmacokinetics, and good activity in a bleomycin-induced model of pulmonary fibrosis.

Discovery of 9-Cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (GLPG1205), a Unique GPR84 Negative Allosteric Modulator Undergoing Evaluation in a Phase II Clinical Trial.

GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5'-O-[γ-thio]triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein.

The Medium-Chain Fatty Acid Receptor GPR84 Mediates Myeloid Cell Infiltration Promoting Steatohepatitis and Fibrosis.

Medium-chain fatty acids (MCFAs) have been associated with anti-steatotic effects in hepatocytes. Expression of the MCFA receptor GPR84 (G protein-coupled receptor 84) is induced in immune cells under inflammatory conditions and can promote fibrogenesis. We aimed at deciphering the role of GPR84 in the pathogenesis of non-alcoholic steatohepatitis (NASH), exploring its potential as a therapeutic target. GPR84 expression is upregulated in liver from patients with non-alcoholic fatty liver disease (NAFLD), correlating with the histological degree of inflammation and fibrosis. In mouse and human, activated monocytes and neutrophils upregulate GPR84 expression. Chemotaxis of these myeloid cells by GPR84 stimulation is inhibited by two novel, small molecule GPR84 antagonists. Upon acute liver injury in mice, treatment with GPR84 antagonists significantly reduced the hepatic recruitment of neutrophils, monocytes, and monocyte-derived macrophages (MoMF). We, therefore, evaluated the therapeutic inhibition of GPR84 by these two novel antagonists in comparison to selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, in three NASH mouse models. Pharmacological inhibition of GPR84 significantly reduced macrophage accumulation and ameliorated inflammation and fibrosis, to an extent similar to selonsertib. In conclusion, our findings support that GPR84 mediates myeloid cell infiltration in liver injury and is a promising therapeutic target in steatohepatitis and fibrosis.

Antitumorigenic Effects of Inhibiting Ephrin Receptor Kinase Signaling by GLPG1790 against Colorectal Cancer Cell Lines In Vitro and In Vivo.

Erythropoietin-producing hepatocellular receptors (Eph) promote the onset and sustain the progression of cancers such as colorectal cancer (CRC), in which the A2 subtype of Eph receptor expression has been shown to correlate with a poor prognosis and has been identified as a promising therapeutic target. Herein, we investigated, in vitro and in vivo, the effects of treatment with GLPG1790, a potent pan-Eph inhibitor. The small molecule has selective activity against the EphA2 isoform in human HCT116 and HCT15 CRC cell lines expressing a constitutively active form of RAS concurrently with a wild-type or mutant form of p53, respectively. GLPG1790 reduced EPHA2 phosphorylation/activation and induced G1/S cell-cycle growth arrest by downregulating the expression of cyclin E and PCNA, while upregulating p21Waf1/Cip1 and p27Cip/Kip. The inhibition of ephrin signaling induced quiescence in HCT15 and senescence in HCT116 cells. While investigating the role of CRC-related, pro-oncogenic p53 and RAS pathways, we found that GLPG1790 upregulated p53 expression and that silencing p53 or inhibiting RAS (human rat sarcoma)/ERKs (extracellular signal-regulated kinase) signaling restrained the ability of GLPG1790 to induce senescence in HCT116 cells. On the other hand, HCT15 silencing of p53 predisposed cells to GLPG1790-induced senescence, whilst no effects of ERK inhibition were observed. Finally, GLPG1790 hindered the epithelial-mesenchymal transition, reduced the migratory capacities of CRC, and affected tumor formation in xenograft models in vivo more efficiently using HCT116 than HCT15 for xenografts. Taken together, our data suggest the therapeutic potential of GLPG1790 as a signal transduction-based therapeutic strategy in to treat CRC.

The Small Molecule Ephrin Receptor Inhibitor, GLPG1790, Reduces Renewal Capabilities of Cancer Stem Cells, Showing Anti-Tumour Efficacy on Preclinical Glioblastoma Models.

Therapies against glioblastoma (GBM) show a high percentage of failure associated with the survival of glioma stem cells (GSCs) that repopulate treated tumours. Forced differentiation of GSCs is a promising new approach in cancer treatment. Erythropoietin-producing hepatocellular (Eph) receptors drive tumourigenicity and stemness in GBM. We tested GLPG1790, a first small molecule with inhibition activity versus inhibitor of various Eph receptor kinases, in preclinical GBM models using in vitro and in vivo assays. GLPG1790 rapidly and persistently inhibited Ephrin-A1-mediated phosphorylation of Tyr588 and Ser897, completely blocking EphA2 receptor signalling. Similarly, this compound blocks the ephrin B2-mediated EphA3 and EphB4 tyrosine phosphorylation. This resulted in anti-glioma effects. GLPG1790 down-modulated the expression of mesenchymal markers CD44, Sox2, nestin, octamer-binding transcription factor 3/4 (Oct3/4), Nanog, CD90, and CD105, and up-regulated that of glial fibrillary acidic protein (GFAP) and pro-neural/neuronal markers, ßIII tubulin, and neurofilaments. GLPG1790 reduced tumour growth in vivo. These effects were larger compared to radiation therapy (RT; U251 and T98G xenografts) and smaller than those of temozolomide (TMZ; U251 and U87MG cell models). By contrast, GLPG1790 showed effects that were higher than Radiotherapy (RT) and similar to Temozolomide (TMZ) in orthotopic U87MG and CSCs-5 models in terms of disease-free survival (DFS) and overall survival (OS). Further experiments were necessary to study possible interactions with radio- and chemotherapy. GLPG1790 demonstrated anti-tumor effects regulating both the differentiative status of Glioma Initiating Cells (GICs) and the quality of tumor microenvironment, translating into efficacy in aggressive GBM mouse models. Significant common molecular targets to radio and chemo therapy supported the combination use of GLPG1790 in ameliorative antiglioma therapy.

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells.

BACKGROUND: EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines. METHODS: EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts. RESULTS: Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts. CONCLUSIONS: Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

Reinvestigation of the Branimycin Stereochemistry at Position 17-C.

A conformational study of branimycin was performed using single-crystal X-ray crystallography to characterize the solid-state form, while a combination of NMR spectroscopy and molecular modeling was employed to gain information about the solution structure. Comparison of the crystal structure with its solution counterpart showed no significant differences in conformation, confirming the relative rigidity of the tricyclic system. However, these experiments revealed that the formerly proposed stereochemistry of branimycin at 17-C should be revised.

Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

Optimisation of 2-cyano-pyrimidine inhibitors of cathepsin K: improving selectivity over hERG.

Several structure-guided optimisation strategies were explored in order to improve the hERG selectivity profile of cathepsin K inhibitor 1, whilst maintaining its otherwise excellent in vitro and in vivo profile. Ultimately, attenuation of clogP and pK(a) properties proved a successful approach and led to the discovery of a potent analogue 23, which, in addition to the desired selectivity over hERG (>1000-fold), displayed a highly attractive overall profile.

2-Phenyl-9H-purine-6-carbonitrile derivatives as selective cathepsin S inhibitors.

Starting from previously disclosed equally potent cathepsin K and S inhibitor 4-propyl-6-(3-trifluoromethylphenyl)pyrimidine-2-carbonitrile 1, a novel 2-phenyl-9H-purine-6-carbonitrile scaffold was identified to provide potent and selective cathepsin S inhibitors.

Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors.

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC(50)=4nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.