Development of G-quadruplex Stabilizers as Anticancer Drug Therapy and Selective Agonists of the Human Oxytocin Receptor as a Therapeutic Tool for Neuropsychiatric Disorders

The principal aim of pharmacology in drug discovery is the characterization of drug activity by system-independent scales that allow prediction of drug activity in all cellular systems. This thesis concentrated in two drug targets: telomeres and GPCRs. Telomere biology is a validated anticancer drug target. G-protein-coupled receptors (GPCRs) recognize external ligands and transmit signals to cellular G-proteins (guanine-nucleotide-binding proteins) to elicit a response. These receptors are tractable for drug discovery because they are on the cell surface therefore drugs do not need to penetrate the cell to produce an effect [1]. In 2000, nearly half of all prescription drugs in the US were targeted towards GPCRs [2]. The projects in this dissertation will describe two different approaches to the successful identification of small-molecules that interact with their validated targets. The first project focused on the identification of new G-quadruplex stabilizers and their ability to inhibit human telomerase as an anticancer drug modality. The second project focused on the efforts made towards the identification of novel small molecules that selectively activate the human oxytocin receptor to regulate complex behaviors in animal models with therapeutic implications in various neuropsychiatric disorders

Light, medium-weight or heavy? The nature of the first supermassive black hole seeds

Observations of hyper-luminous quasars at $z>6$ reveal the rapid growth of supermassive black holes (SMBHs $>10^9 \rm M_{\odot}$) whose origin is still difficult to explain. Their progenitors may have formed as remnants of massive, metal free stars (light seeds), via stellar collisions (medium-weight seeds) and/or massive gas clouds direct collapse (heavy seeds). In this work we investigate for the first time the relative role of these three seed populations in the formation of $z>6$ SMBHs within an Eddington-limited gas accretion scenario. To this aim, we implement in our semi-analytical data-constrained model a statistical description of the spatial fluctuations of Lyman-Werner (LW) photo-dissociating radiation and of metal/dust enrichment. This allows us to set the physical conditions for BH seeds formation, exploring their relative birth rate in a highly biased region of the Universe at $z>6$. We find that the inclusion of medium-weight seeds does not qualitatively change the growth history of the first SMBHs: although less massive seeds ($<10^3 \rm M_\odot$) form at a higher rate, the mass growth of a $\sim 10^9 \rm M_\odot$ SMBH at $z<15$ is driven by efficient gas accretion (at a sub-Eddington rate) onto its heavy progenitors ($10^5 \rm M_\odot$). This conclusion holds independently of the critical level of LW radiation and even when medium-weight seeds are allowed to form in higher metallicity galaxies, via the so-called super-competitive accretion scenario. Our study suggests that the genealogy of $z \sim 6$ SMBHs is characterized by a rich variety of BH progenitors, which represent only a small fraction ($< 10 - 20\%$) of all the BHs that seed galaxies at $z > 15$.Comment: (21 pages, 18 figures, 2 tables. Accepted for publication in MNRAS

HTS navigator: freely accessible cheminformatics software for analyzing high-throughput screening data

Summary: We report on the development of the high-throughput screening (HTS) Navigator software to analyze and visualize the results of HTS of chemical libraries. The HTS Navigator processes output files from different plate readers' formats, computes the overall HTS matrix, automatically detects hits and has different types of baseline navigation and correction features. The software incorporates advanced cheminformatics capabilities such as chemical structure storage and visualization, fast similarity search and chemical neighborhood analysis for retrieved hits. The software is freely available for academic laboratories

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs

Role of extracellular microvesicles in celiac disease as potential pathogenetic agents and biomarkers of intestinal inflammation

BACKGROUND AND AIM: Celiac Disease (CD) is a chronic intestinal disease caused by the ingestion of gluten. Microvesicles (MVs) belong to a heterogeneous population, released by cells both in homeostasis and pathological conditions. MVs can be considered mediators of inflammation and potential biomarkers. The aim of this study is: 1) to evaluate the possible role of MVs in the propagation of inflammation in CD, using MVs purified by supernatant of duodenal biopsies from CD patients; 2) to identify potential biomarkers by proteomic analysis of pasma-derived MVs from CD patients. MATERIAL AND METHODS: MVs were isolated by molecular exclusion chromatography and ultracentrifugation respectively from plasma and culture supernatant of duodenal biopsies of 10 active CD, 5 remission CD and 6 controls. Proteomic analysis of plasma-derived MVs was performed by mass spectrometry. The possible effects of duodenal-derived MVs on confluent Caco-2 cells were evaluated by measuring Transepithelial Electrical Resistance (TEER) and analyzing the expression of actin, tissue transglutaminase (TG2) and Zonula Occludens-1 (ZO-1). The dosage of IL-8 in the Caco-2 culture supernatant was carried out by ELISA test. The statistical analysis of the data obtained was performed using the Student's t-test. RESULTS: The proteomic analysis of circulating MVs showed 8 proteins from desmosome and cytoskeleton (desmoglein-1 and gamma-enteric actin) associated with the active phase of the disease. Caco-2 cells, treated with the MVs purified from the duodenal biopsies of active CD patients showed: 1) rearrangement of actin filaments; 2) increased expression of TG2; 3) decreased expression of the ZO-1 protein, although an alteration of intestinal permeability was not observed. The analysis of Caco-2 cell supernatants showed a statistically significant increase in IL-8 (p &lt;0.05), in the presence of MVs isolated from biopsies of active CD patients, compared to remission CD patients and controls. CONCLUSIONS: MVs isolated from plasma of active CD patients could represent potential diagnostic and prognostic biomarkers. Although they don’t induce changes in intestinal permeability, MVs could contribute to inflammatory cascade increasing IL-8 production

Colloidal Aggregation Causes Inhibition of G Protein-Coupled Receptors

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field

Loss of Primary Cilia Potentiates BRAF/MAPK Pathway Activation in Rhabdoid Colorectal Carcinoma: A Series of 21 Cases Showing Ciliary Rootlet CoiledCoil (CROCC) Alterations

A rhabdoid colorectal tumor (RCT) is a rare cancer with aggressive clinical behavior. Recently, it has been recognized as a distinct disease entity, characterized by genetic alterations in the SMARCB1 and Ciliary Rootlet Coiled-Coil (CROCC). We here investigate the genetic and immunophenotypic profiling of 21 RCTs using immunohistochemistry and next-generation sequencing. Mismatch repair-deficient phenotypes were identified in 60% of RCTs. Similarly, a large proportion of cancers exhibited the combined marker phenotype (CK7-/CK20-/CDX2-) not common to classical adenocarcinoma variants. More than 70% of cases displayed aberrant activation of the mitogen-activated protein kinase (MAPK) pathway with mutations prevalently in BRAF V600E. SMARCB1/INI1 expression was normal in a large majority of lesions. In contrast, ciliogenic markers including CROCC and γ-tubulin were globally altered in tumors. Notably, CROCC and γ-tubulin were observed to colocalize in large cilia found on cancer tissues but not in normal controls. Taken together, our findings indicate that primary ciliogenesis and MAPK pathway activation contribute to the aggressiveness of RCTs and, therefore, may constitute a novel therapeutic target

Citokininek, a növényi in vitro fejlődés kulcsregulátorai

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