Design, synthesis and preliminary biological evaluation of 3-cyclopropyl-4-phenoxy-1H-pyrazole derivatives as small molecular ligands of RAGE

Receptor for advanced glycation end products (RAGE) is a multiligand receptor belonging to the immunoglobulin superfamily and plays a crucial role in the development of many human diseases such as neurodegenerative diseases, diabetes, cardiovascular diseases and cancer.1 RAGE is involved in a number of cell processes such as neuroinflammation, apoptosis, proliferation and autophagy, and therefore it is of considerable interest as a promising drug target for innovative therapeutic approaches. It consists of an extracellular region, a short hydrophobic transmembrane spanning region, and a highly charged amino acid cytoplasmatic tail. The extracellular region contains a signal peptide, followed by one N-terminal V-type immunoglobulin domain and two C-type (C1 and C2) immunoglobulin domains.2 RAGE is able to interact with a large number of pro-inflammatory and regulatory molecules, such as advanced glycation end-products (AGEs), quinolinic acid, beta amyloid (A\u3b2), high mobility group box 1 (HMGB1), S100/calgranulin family proteins.3,4 However, due to the structural heterogeneity of these endogenous ligands, little is known about the key pharmacophore elements for ligand-RAGE interaction and the specific mode of binding. On these grounds, we aimed at designing new small molecules able to bind the VC1 extracellular domains of RAGE, in order to clarify the structural features that account for RAGE affinity and activation, and to identify new drug-like compounds. Following a process of structural simplification of known pyrazole-5-carboxamide RAGE ligands,1 we planned a set of novel derivatives characterized by a variously functionalized 3-cyclopropyl-4-phenoxy-1H-pyrazole scaffold (Figure 1). The design and synthesis of the new putative RAGE ligands will be presented and discussed, together with the results of their in vitro screening by means of a surface plasmon resonance (SPR)-based assay to estimate their binding ability to the RAGE extracellular domain. References 1. Bongarzone S., Savickas V., Luzi F., Gee A. D. J. Med. Chem. 2017, 60, 7213-7232. 2. Hudson B. I., Carter A. M., Harja E., Kalea A. Z., Arriero M., Yang H., Grant P. J., Schmidt A. M. FASEB J. 2008, 22, 1572-1580. 3. Xue J., Rai V., Singer D., Chabierski S., Xie J., Reverdatto S., Burz D. S., Schmidt A. M., Hoffmann R., Shekhtman A. Structure 2011, 19, 722\u2013732. 4. Koch M., Chitayat S., Dattilo B. M., Schiefner A., Diez J., Chazin W. J., Fritz, G. Structure 2010, 18, 1342-1352

Targeting SMYD3 to sensitize homologous recombination-proficient tumors to PARP-mediated synthetic lethality

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors

Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication

BACKGROUND: Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication. METHODOLOGY/PRINCIPAL FINDINGS: We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro. CONCLUSIONS/SIGNIFICANCE: We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins

Evolutionary Dynamics of Vibrio cholerae O1 following a Single-Source Introduction to Haiti

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins

Small-molecule inhibitors of lysine methyltransferases SMYD2 and SMYD3: Current trends

Lysine methyltransferases SMYD2 and SMYD3 are involved in the epigenetic regulation of cell differentiation and functioning. Overexpression and deregulation of these enzymes have been correlated to the insurgence and progression of different tumors, making them promising molecular targets in cancer therapy even if their role in tumors is not yet fully understood. In this light, selective small-molecule inhibitors are required to fully understand and validate these enzymes, as this is a prerequisite for the development of successful targeted therapeutic strategies. The present review gives a systematic overview of the chemical probes developed to selectively target SMYD2 and SMYD3, with particular focus on the structural features important for high inhibitory activity, on the mode of inhibition and on the efficacy in cell-based and in in vivo models

Gas product distribution from polyethylene pyrolysis

The disposal of plastic waste is extensively studied nowadays. Of the treatments available, thermal degradation is attracting great interest because it offers the possibility of recovering energy and useful chemicals. PE pyrolysis has already been discussed in a previous paper [1] where the rate of decomposition of a sample was modeled using a detailed kinetic scheme. Its predictions were validated by thermogravimetric experimental results across wide operative pressure and heating rate ranges. The gas product distribution was derived from the random scission hypothesis. Thus, the ratio of alkane, alkene and dialkene species was 1:2:1 and, within the same class of products, the distribution was equimolecular, except for those species which are more favored by allyl resonance. This paper aims to continue the previous work in attempting to model gas products correctly. The principal improvements made here are the introduction of backbiting reactions into the kinetic scheme to predict the larger amounts of selected products and the consideration of the physical aspects of the degradation process. These phenomena are responsible for the production rate and the distribution of volatiles. A simplified model of the in-depth mass transfer through bubble formation is presented which describes the degradation progress for a small-sized polyethylene sample. The model predictions are compared with both the experimental results of polyethylene pyrolysis performed under various temperature conditions and with thermogravimetric analysis. (C) 1999 Elsevier Science B.V. All rights reserved