A bottleneck in drug discovery is the identification of the molecular targets of a compound (mode of action, MoA) and of its off-target effects. Previous approaches to elucidate drug MoA include analysis of chemical structures, transcriptional responses following treatment, and text mining. Methods based on transcriptional responses require the least amount of information and can be quickly applied to new compounds. Available methods are inefficient and are not able to support network pharmacology. We developed an automatic and robust approach that exploits similarity in gene expression profiles following drug treatment, across multiple cell lines and dosages, to predict similarities in drug effect and MoA. We constructed a "drug network" of 1,302 nodes (drugs) and 41,047 edges (indicating similarities between pair of drugs). We applied network theory, partitioning drugs into groups of densely interconnected nodes (i.e., communities). These communities are significantly enriched for compounds with similar MoA, or acting on the same pathway, and can be used to identify the compoundtargeted biological pathways. New compounds can be integrated into the network to predict their therapeutic and off-target effects. Using this network, we correctly predicted the MoA for nine anticancer compounds, and we were able to discover an unreported effect for a well-known drug. We verified an unexpected similarity between cyclin-dependent kinase 2 inhibitors and Topoisomerase inhibitors. We discovered that Fasudil (a Rho-kinase inhibitor) might be "repositioned" as an enhancer of cellular autophagy, potentially applicable to several neurodegenerative disorders. Our approach was implemented in a tool (Mode of Action by NeTwoRk Analysis, MANTRA, http://mantra.tigem.it).computational drug discovery | drug repurposing | systems biology | chemotherapy
The thyroid-stimulating hormone͞thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSH͞TSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit dw ͞pit dw ) or of a functional TSHR (tshr hyt ͞tshr hyt and TSHRknockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium͞iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH͞TSHR pathway is in controlling genes involved in iodide metabolism such as sodium͞iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid. T he mouse thyroid gland begins to develop at embryonic day (E)8.5 as an endodermal thickening in the floor of the primitive pharynx. After loosing all connections with the pharynx, the thyroid bud migrates caudally, reaching its final position in front of the trachea ϷE13 (1). Only after completion of migration do thyroid follicular cells begin their differentiative program and express thyroid-specific genes such as thyroglobulin (Tg), thyroid-stimulating hormone͞thyrotropin (TSH) receptor (TSHR), thyroperoxidase (TPO), and the sodium͞iodide symporter (NIS) (2). Finally, primitive follicles appear, and the gland displays its final morphological organization. Since E8.5, thyroid precursor cells express a combination of transcription factors such as TTF-1 (encoded by the titf1͞nkx2.1 gene) (3), TTF-2 (encoded by the titf2͞foxe1 gene) (4), and Pax8 (5). Gene-targeting experiments demonstrated that all these factors are required for the early stages of thyroid development (6-8). However, it still is unclear what the mechanisms are that lead to the initiation of functional differentiation that only occurs at E14.TSH is known as the main regulator of the adult thyroid gland. Indeed, after binding to its receptor, TSH stimulates the thyroid cells in almost every aspect of their metabolism including synthesis and secretion of thyroid hormones (9). Several groups have demonstrated clearly that TSH regulates mRNA levels of several thyroid-specific genes such as Tg (10-13), TPO (13-15), and NIS (16,17).TSH also stimulates the aggregation of porcine thyroid cells in follicles (18), and its presence is necessary to maintain the follicular architecture (19). In the rat, there is a temporal correlation between the increased expression of TSHR and the formation of follicles. Indeed, TSHR mRNA is expressed by E15 (3, 20), and its expression increases on E17-E18. At this stage, thyroid-speci...
Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.
We collected a massive and heterogeneous dataset of 20 255 gene expression profiles (GEPs) from a variety of human samples and experimental conditions, as well as 8895 GEPs from mouse samples. We developed a mutual information (MI) reverse-engineering approach to quantify the extent to which the mRNA levels of two genes are related to each other across the dataset. The resulting networks consist of 4 817 629 connections among 20 255 transcripts in human and 14 461 095 connections among 45 101 transcripts in mouse, with a inter-species conservation of 12%. The inferred connections were compared against known interactions to assess their biological significance. We experimentally validated a subset of not previously described protein–protein interactions. We discovered co-expressed modules within the networks, consisting of genes strongly connected to each other, which carry out specific biological functions, and tend to be in physical proximity at the chromatin level in the nucleus. We show that the network can be used to predict the biological function and subcellular localization of a protein, and to elucidate the function of a disease gene. We experimentally verified that granulin precursor (GRN) gene, whose mutations cause frontotemporal lobar degeneration, is involved in lysosome function. We have developed an online tool to explore the human and mouse gene networks.
The development and the function of central nervous system depend on thyroid hormones. In humans, the lack of thyroid hormones causes cretinism, a syndrome of severe mental deficiency. It is assumed that thyroid hormones affect the normal development and function of the brain by activating or suppressing target gene expression because several genes expressed in the brain have been shown to be under thyroid hormone control. Among these, the Rhes gene, encoding a small GTP-binding protein, is predominantly expressed in the striatal region of the brain. To clarify the role of Rhes in vivo, we disrupted the Rhes gene by homologous recombination in embryonic stem cells and generated mice homozygous for the Rhes null mutation (Rhes ؊/؊ ). Rhes ؊/؊ mice were viable but weighed less than wild-type mice. Furthermore, they showed behavioral abnormalities, displaying a gender-dependent increase in anxiety levels and a clear motor coordination deficit but no learning or memory impairment. These results suggest that Rhes disruption affects selected behavioral competencies.The thyroid hormones thyroxine (T 4 ) and triiodothyronine (T 3 ) have many physiological effects. They exert their actions in all tissues examined and affect many metabolic pathways. Some of the most prominent effects of thyroid hormones occur during fetal development and in early childhood. In humans, the lack of adequate levels of thyroid hormones in the first trimester of life, such as in iodine deficiency (endemic cretinism) (8, 9), or in developmental abnormalities of the thyroid gland (congenital hypothyroidism) (22, 28, 55) results in cretinism, a syndrome of severe mental deficiency, which may be accompanied by retarded growth and/or neurological deficits, such as spastic diplegia. Many of these developmental effects are not reversed by later treatment with hormone, indicating that thyroid hormone acts in a specific developmental window. Therefore, adequate levels of thyroid hormone are required for normal central nervous system development.To date, several specific central nervous system genes whose expression is controlled by thyroid hormone have been identified. The expression of these genes may be decreased (2, 5) or increased (1, 18) in hypothyroidism. Furthermore, the total or partial absence of thyroid hormones may also affect either mRNA stability (43, 54) or the mRNA translational process (43,57,60). The identification of thyroid hormone target genes in the central nervous system and the understanding of their function in central nervous system development are important to understanding the pathogenesis of neurological cretinism at the molecular level.In order to understand the molecular basis of neurological cretinism, we studied the Rhes (Ras homolog enriched in striatum) gene (24). Rhes is predominantly expressed in the striatum, and its expression is controlled by thyroid hormones (59). Interestingly, several lines of evidence indicate that in neurological cretinism, there is damage of striatum, which determines a striatopallidal syndr...
Helper dependent adenoviral (HDAd) vectors can mediate long-term, high level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor A (SR-A) and with scavenger receptor expressed on endothelial cells I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs).
The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (alpha-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8-10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.
Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor b (TGF-b) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-b signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-b antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.
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