38 research outputs found
Molecular Profile of Barrett's Esophagus and Gastroesophageal Reflux Disease in the Development of Translational Physiological and Pharmacological Studies
Barrett's esophagus (BE) is a premalignant condition caused by gastroesophageal reflux disease (GERD), where physiological squamous epithelium is replaced by columnar epithelium. Several in vivo and in vitro BE models were developed with questionable translational relevance when implemented separately. Therefore, we aimed to screen Gene Expression Omnibus 2R (GEO2R) databases to establish whether clinical BE molecular profile was comparable with animal and optimized human esophageal squamous cell lines-based in vitro models. The GEO2R tool and selected databases were used to establish human BE molecular profile. BE-specific mRNAs in human esophageal cell lines (Het-1A and EPC2) were determined after one, three and/or six-day treatment with acidified medium (pH 5.0) and/or 50 and 100 ”M bile mixture (BM). Wistar rats underwent microsurgical procedures to generate esophagogastroduodenal anastomosis (EGDA) leading to BE. BE-specific genes (keratin (KRT)1, KRT4, KRT5, KRT6A, KRT13, KRT14, KRT15, KRT16, KRT23, KRT24, KRT7, KRT8, KRT18, KRT20, trefoil factor (TFF)1, TFF2, TFF3, villin (VIL)1, mucin (MUC)2, MUC3A/B, MUC5B, MUC6 and MUC13) mRNA expression was assessed by real-time PCR. Pro/anti-inflammatory factors (interleukin (IL)-1ÎČ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, tumor necrosis factor α, interferon Îł, granulocyte-macrophage colony-stimulating factor) serum concentration was assessed by a Luminex assay. Expression profile in vivo reflected about 45% of clinical BE with accompanied inflammatory response. Six-day treatment with 100 ”M BM (pH 5.0) altered gene expression in vitro reflecting in 73% human BE profile and making this the most reliable in vitro tool taking into account two tested cell lines. Our optimized and established combined in vitro and in vivo BE models can improve further physiological and pharmacological studies testing pathomechanisms and novel therapeutic targets of this disorder
Discovery of inhibitory fragments that selectively target Spire2âFMN2 interaction
Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2âFMN2 but not the Spire1âFMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1Hâ15N HSQC nuclear magnetic resonance. Extensive structureâactivity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2âFMN2 interaction
Exploring the Surface of the Ectodomain of the PD-L1 Immune Checkpoint with Small-Molecule Fragments
Development of small molecules targeting the PD-L1/PD-1 interface is advancing both in industry and academia, but only a few have reached early-stage clinical trials. Here, we take a closer look at the general druggability of PD-L1 using in silico hot spot mapping and nuclear magnetic resonance (NMR)-based characterization. We found that the conformational elasticity of the PD-L1 surface strongly influences the formation of hot spots. We deconstructed several generations of known inhibitors into fragments and examined their binding properties using differential scanning fluorimetry (DSF) and protein-based nuclear magnetic resonance (NMR). These biophysical analyses showed that not all fragments bind to the PD-L1 ectodomain despite having the biphenyl scaffold. Although most of the binding fragments induced PD-L1 oligomerization, two compounds, TAH35 and TAH36, retain the monomeric state of proteins upon binding. Additionally, the presence of the entire ectodomain did not affect the binding of the hit compounds and dimerization of PD-L1. The data demonstrated here provide important information on the PD-L1 druggability and the structure-activity relationship of the biphenyl core moiety and therefore may aid in the design of novel inhibitors and focused fragment libraries for PD-L1.This research has been supported by Grants Maestro 2017/26/A/ST5/00572 (to T.A.H.) , Sonata UMO-2020/39/D/ST4/01344 (to E.S.) , Preludium UMO-2021/41/N/ST4/03485 (to M.Z.) , and Preludium UMO-2020/37/N/ST4/02691 (to D.M.) from the National Science Centre, Poland. X.d.C. thanks the Basque Country Government for the predoctoral and EGONLABUR grants
Design of indole- and MCR-based macrocycles as p53-MDM2 antagonists
Macrocycles were designed to antagonize the protein-protein interaction p53-MDM2 based on the three-finger pharmacophore F19W23L25. The synthesis was accomplished by a rapid, one-pot synthesis of indole-based macrocycles based on Ugi macrocyclization. The reaction of 12 different α,Ï-amino acids and different indole-3-carboxaldehyde derivatives afforded a unique library of macrocycles otherwise difficult to access. Screening of the library for p53-MDM2 inhibition by fluorescence polarization and 1H,15N HSQC NMR measurements confirm MDM2 binding
Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments
Delayed neutrophil apoptosis in granulomatosis with polyangiitis: dysregulation of neutrophil gene signature and circulating apoptosis-related proteins
Endothelial cells response to neutrophilâderived extracellular vesicles miRNAs in antiâPR3 positive vasculitis
In vasculitis disorders, inflammation affects blood vessels. Granulomatosis with polyangiitis (GPA) is a chronic systemic vasculitis distinguished by the presence of antiâproteinaseâ3 autoantibodies (antiâPR3). In this study we analyzed the molecular signature of human umbilical endothelial cells (HUVECs) in response to neutrophilâderived extracellular vesicles (EVs). EVs were obtained from antiâPR3âactivated neutrophils, purified and characterized by flow cytometry, nanoparticle tracking and miRNA screening. HUVECs were stimulated with EVs and miRNA/mRNA expression was measured. Cell culture media proteins were identified by antibody microarrays and selected cytokines were measured. Comparison of differentially expressed miRNAs/mRNAs between nonâstimulated and EVâstimulated HUVECs revealed two regulatory patterns. Significant upâregulation of 14 mRNA transcripts (including CXCL8, DKK1, IL1RL1, ANGPTâ2, THBS1 and VCAMâ1) was accompanied by 11 miRNAs silencing (including miRâ661, miRâ664aâ3p, miRâ377â3p, miRâ30dâ5p). Significant downâregulation was observed for nine mRNA transcripts (including FASLG, CASP8, STAT3, GATA3, IRAK1 and IL6) and accompanied by upâregulation of 10 miRNAs (including miRâ223â3p, miRâ142â3p, miRâ211â5p). Stimulated HUVECs released ILâ8, Dickkopfârelated protein 1 (DKKâ1), soluble interleukin (IL)â1 like receptorâ1 (ST2), growth differentiation factor 15 (GDFâ15), angiopoietinâ2, endoglin, thrombospondinâ1 and vascular adhesion moleculeâ1 (VCAMâ1). Moreover, transfection of HUVECs with mimics of highly expressed in EVs miRâ223â3p or miRâ142â3p, stimulated production of ILâ8, ST2 and endoglin. Cytokines released by HUVECs were also elevated in blood of patients with GPA. The most increased were ILâ8, DKKâ1, ST2, angiopoietinâ2 and ILâ33. Inâvitro stimulation of HUVECs by neutrophilâderived EVs recapitulates contribution of endothelium in autoimmune vasculitis. Proinflammatory phenotype of released cytokines corresponds with the regulatory network of miRNAs/mRNAs comprising both EVs miRNA and endothelial cell transcripts