Target Binding Sites Research Articles

Identification and characterization of circular RNAs in association with the feed efficiency in Hu lambs

BackgroundCircular RNA (circRNA), as a new members of noncoding RNA family, have vital functions in many biological processes by as microRNA sponges or competing endogenous RNAs (ceRNAs). However, little has been reported about the genetic mechanism of circRNAs regulation of feed efficiency in sheep.ResultsThis study aimed to explore the expression of circRNAs in the liver of Hu sheep with High-RFI (High residual feed intake) and Low-RFI (Low residual feed intake) using transcriptome sequencing. A total of 20,729 circRNAs were identified in two groups, in which 219 circRNAs were found as significantly differentially expressed. Several circRNAs were validated by using RT-PCR, sanger sequencing and RT-qPCR methods. These results demonstrated that the RNA-seq result and expression level of circRNAs identified are reliable. Subsequently, GO and KEGG enrichment analysis of the parental genes of the differentially expressed (DE) circRNAs were mainly involved in immunity response and metabolic process. Finally, the ceRNA regulatory networks analysis showed that the target binding sites for miRNA such as novel_41, novel_115, novel_171 and oar-miR-485-3p in the identified DE cirRNAs. Importantly, two metabolic (SHISA3 and PLEKHH2) and four (RTP4, CD274, OAS1, and RFC3) immune-related target mRNAs were identified from 4 miRNAs. Association analysis showed that the polymorphism (RTP4 c.399 A > G) in the target gene RTP4 were significantly associated with RFI (P < 0.05).ConclusionsAnalysis of sequencing data showed some candidate ceRNAs that may play key roles in the feed efficiency in sheep by regulating animal immune and metabolic. These results provide the basis data for further study of the biological functions of circRNAs in regulating sheep feed efficiency.

In silico identification of small molecule protein-protein interaction inhibitors: targeting hotspot regions at the interface of MXRA8 and CHIKV envelope protein

Chikungunya virus (CHIKV) is an arthritogenic arbovirus responsible for re-emerging epidemics of Chikungunya fever around the world for centuries. Chikungunya has become endemic in Africa, Southeast Asia, the Indian subcontinent, and subtropical regions of the Americas. The unavailability of antiviral therapy or vaccine against the CHIKV and its continuous re-emergence demands an urgent need to develop potential candidate therapeutics. CHIKV entry into the host cell is mediated by its envelope proteins engaging the cellular receptor MXRA8 to invade the susceptible cells. We report here two essential target binding sites at the CHIKV E1-E2 proteins by identifying hotspot regions at the E1-E2-MXRA8 binding interface. Further, we employed high throughput computational screening to identify potential small molecule protein-protein interaction (PPI) modulators which could effectively bind at the identified target sites. Molecular dynamics simulations and binding free energy calculations confirmed the stability of three compounds, viz., ZINC299817498, ZINC584908978, and LAS52155651, at both the predicted interface binding sites. The polar and charged residues at the interface were responsible for energetically holding the ligands at the binding sites. Altogether, our findings suggest that the predicted target binding sites at the E1-E2 dimer could be essential to block the receptor interaction as well as the fusion process of the CHIKV particles. Thus, we identified a few small molecule PPI inhibitors with great potential to block the E1-E2-MXRA8 interaction and act as promising templates to design anti-CHIKV drugs. Communicated by Ramaswamy H. Sarma

Evaluation of the binding performance of flavonoids to estrogen receptor alpha by Autodock, Autodock Vina and Surflex-Dock

Molecular docking is a widely used method to predict the binding modes of small-molecule ligands to the target binding site. However, it remains a challenge to identify the correct binding conformation and the corresponding binding affinity for a series of structurally similar ligands, especially those with weak binding. An understanding of the various relative attributes of popular docking programs is required to ensure a successful docking outcome. In this study, we systematically compared the performance of three popular docking programs, Autodock, Autodock Vina, and Surflex-Dock for a series of structurally similar weekly binding flavonoids (22) binding to the estrogen receptor alpha (ERα). For these flavonoids-ERα interactions, Surflex-Dock showed higher accuracy than Autodock and Autodock Vina. The hydrogen bond overweighting by Autodock and Autodock Vina led to incorrect binding results, while Surflex-Dock effectively balanced both hydrogen bond and hydrophobic interactions. Moreover, the selection of initial receptor structure is critical as it influences the docking conformations of flavonoids-ERα complexes. The flexible docking method failed to further improve the docking accuracy of the semi-flexible docking method for such chemicals. In addition, binding interaction analysis revealed that 8 residues, including Ala350, Glu353, Leu387, Arg394, Phe404, Gly521, His524, and Leu525, are the key residues in ERα-flavonoids complexes. This work provides reference for assessing molecular interactions between ERα and flavonoid-like chemicals and provides instructive information for other environmental chemicals.

Predicting sequence and structural features of effective piRNA target binding sites.

Piwi-interacting RNA (piRNA) targets are usually identified through base pairing between the piRNA seed region and complementary bases on the target mRNAs, which often results in false predictions. Cross-linking immunoprecipitation (CLIP) study emerges as a promising method that enables accurate identification of PIWI-clade-based targets containing RNA-binding sites. In the present study, we have analyzed the piRNA-target CLIP-Seq datasets to uncover the additional characteristic features of piRNA targets. We studied important sequence and structural features using IP+ and IP- set targets that might enhance the accuracy of target site predictions. Analysis has revealed substantial enrichment of AU in target sites as well as in and around the 30 nts upstream and downstream of target sites in IP+ set relative to IP- set that might be contributing to lowering the minimal folding energy of target sites of IP+ set that might be easing the base pairing between piRNA and their targets. We have also found a lower MFE threshold (en) and higher miRanda score for piRNA targets. Interestingly, we have found that majority of the target sites are residing within 3'UTR, suggesting 3'UTR as a preferential target site like that of miRNA targets. Thus, we hypothesize that our findings on additional key features of piRNA target sites might be valuable in identifying the potential targets of piRNA accurately, which will aid in decrypting their functional importance.

MicroRNA MiR-490-5p suppresses pancreatic cancer through regulating epithelial-mesenchymal transition via targeting MAGI2 antisense RNA 3

ABSTRACT Pancreatic cancer with about 5% five-year overall survival rate remains a challenge. Invasion and migration of pancreatic cancer cells are the main factors leading to poor prognosis. MicroRNA-490-5p (miR-490-5p) has anti-cancer effects in a variety of tumors, but its role in pancreatic cancer has not been reported. The mRNA expressions of miR-490-5p, MAGI2 antisense RNA 3 (MAGI2-AS3), Matrix metalloproteinase (MMP)2, MMP9, N-cadherin, and E-cadherin were detected by quantitative real-time PCR, while the protein expressions of these genes except miR-490-5p were measured by Western blot analysis. The cell viability, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8), apoptosis and transwell assays. MiR-490-5p was abnormally low-expressed in pancreatic cancer, whose down-regulation generated enhanced effects on viability, migration and invasion in pancreatic cancer cells, as well as MAGI2-AS3 expression. MiR-490-5p mimic exerted the opposite effect on cells, which also down-regulated MMP2, MMP9, and N-cadherin protein expressions, while up-regulating E-cadherin protein expression. MAGI2-AS3, which was the targeted binding site of miR-490-5p, promoted viability, migration and invasion, and inhibited apoptosis of cancer cells. More importantly, miR-490-5p played an anti-cancer role in pancreatic cancer by targeting MAGI2-AS3 and regulating epithelial–mesenchymal transition (EMT), which was partially offset by MAGI2-AS3.

Sagacious epitope selection for vaccines, and both antibody-based therapeutics and diagnostics: tips from virology and oncology.

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and vaccine development. This importance is focused on the target binding site—epitope, where epitope selection as a part of design thinking beyond traditional antigen selection using whole cell or whole protein immunization can positively impact success. With purified recombinant protein production and peptide synthesis to display limited/selected epitopes, intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for. Many of these factors stem from the location of the epitope that can impact accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even noncompetitive inhibition sites. By incorporating novel computational methods for predicting antigen changes to model-informed drug discovery and development, superior vaccines and antibody-based therapeutics or diagnostics can be easily designed to mitigate failures. With detailed examples, this review highlights the new opportunities, factors, and methods of predicting antigenic changes for consideration in sagacious epitope selection.

MiR-29c-3p May Promote the Progression of Alzheimer's Disease through BACE1.

The aim of this study was to explore the specific role of miR-29c-3p in Alzheimer's disease (AD). Animal models of AD were established by injecting streptozotocin (STZ) into mice through the lateral ventricle, while cell models of AD were induced by 10 μM β-amyloid (Aβ). We detected miR-29c-3p and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) contents and measured AD cell proliferation and apoptosis. A low miR-29c-3p level and a high BACE1 level were detected in the brain tissue of AD animal models and AD cell models. Aβ-processed cells had markedly lower proliferation activity, higher apoptosis, increased phosphorylation of tau protein was over phosphorylated, but the overexpression of miR-29c-3p or the silencing of BACE1 significantly enhanced the cell proliferation activity and reduced cell apoptosis by regulating the contents of related proteins. Inhibition of miR-29c-3p or overexpression of BACE1 aggravated Aβ-induced side effects. We used Targetscan7.2 to predict the downstream target genes of miR-29c-3p. Then, we detected that there were target binding sites between miR-29c-3p and BACE1. The rescue experiment identified BACE1 as a functional target for miR-29c-3p. AD leads to decreased miR-29c-3p level and increased BACE1 level. MiR-29c-3p has specific binding sites with the 3′-untranslated region (3′-UTR) of BACE1 and thus negatively regulates the BACE1 level, thereby affecting the progression of AD.

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm.

ABSTRACTDevelopment of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.

Site-specific protein modification by 3-n-butylphthalide in primary hepatocytes: Covalent protein adducts diminished by glutathione and N-acetylcysteine

Aims3-n-Butylphthalide (NBP) is widely used for the treatment of cerebral ischaemic stroke but can causeliver injury in clinical practice. This study aims to elucidate the underlying mechanisms and propose potential preventive strategies. Main methodsNBP and its four major metabolites, 3-hydroxy-NBP (3-OH-NBP), 10-hydroxy-NBP, 10-keto-NBP and NBP-11-oic acid, were synthesized and evaluated in primary human or rat hepatocytes (PHHs, PRHs). NBP-related substances or amino acid adducts were identified and semi-quantitated by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The target proteins and binding sites were identified by shotgun proteomics based on peptide mass fingerprinting coupled with tandem mass spectrometry and verified by molecular docking. Key findingsThe toxicity of NBP and its four major metabolites were compared in both PHHs and PRHs, and 3-OH-NBP was found to be the most toxic metabolite. 3-OH-NBP induced remarkable cell death and oxidative stresses in hepatocytes, which correlated well with the levels of glutathione and N-acetylcysteine adducts (3-GSH-NBP and 3-NAC-NBP) in cell supernatants. Additionally, 3-OH-NBP covalently conjugated with intracellular Cys, Lys and Ser, with preferable binding to Cys sites at Myh9 Cys1380, Prdx4 Cys53, Vdac2 Cys48 and Vdac3 Cys36. Furthermore, we found that CYP3A4 induction by rifampicin augmented NBP-induced cell toxicity and supplementing with GSH or NAC alleviated the oxidative stresses and reactive metabolites caused by 3-OH-NBP. SignificanceOur work suggests that glutathione depletion, mitochondrial injury and covalent protein modification are the main causes of NBP-induced hepatotoxicity, which may be prevented by exogenous GSH or NAC supplementation and avoiding concomitant use of CYP3A4 inducers.

Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression

Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets.We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, ‘R‘). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R.At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation.Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog.Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs.Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. DisclosuresSilveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

Lysine Covalent Antagonists of Melanoma Inhibitors of Apoptosis Protein.

We have recently reported on Lys-covalent agents that, based on aryl-sulfonyl fluorides, were designed to target binding site Lys 311 in the X-linked inhibitor of apoptosis protein (XIAP). Similar to XIAP, melanoma-IAP (ML-IAP), a less well-characterized IAP family protein, also presents a lysine residue (Lys 135), which is in a position equivalent to that of Lys 311 of XIAP. On the contrary, two other members of the IAP family, namely, cellular-IAPs (cIAP1 and cIAP2), present a glutamic acid residue in that position. Hence, in the present work, we describe the derivation and characterization of the very first potent ML-IAP Lys-covalent inhibitor with cellular activity. The agent can be used as a pharmacological tool to further validate ML-IAP as a drug target and eventually for the development of ML-IAP-targeted therapeutics.

Neural stem/precursor cells dynamically change their epigenetic landscape to differentially respond to BMP signaling for fate switching during brain development.

During neocortical development, tight regulation of neurogenesis-to-astrogenesis switching of neural precursor cells (NPCs) is critical to generate a balanced number of each neural cell type for proper brain functions. Accumulating evidence indicates that a complex array of epigenetic modifications and the availability of extracellular factors control the timing of neuronal and astrocytic differentiation. However, our understanding of NPC fate regulation is still far from complete. Bone morphogenetic proteins (BMPs) are renowned as cytokines that induce astrogenesis of gliogenic late-gestational NPCs. They also promote neurogenesis of mid-gestational NPCs, although the underlying mechanisms remain elusive. By performing multiple genome-wide analyses, we demonstrate that Smads, transcription factors that act downstream from BMP signaling, target dramatically different genomic regions in neurogenic and gliogenic NPCs. We found that histone H3K27 trimethylation and DNA methylation around Smad-binding sites change rapidly as gestation proceeds, strongly associated with the alteration of accessibility of Smads to their target binding sites. Furthermore, we identified two lineage-specific Smad-interacting partners-Sox11 for neurogenic and Sox8 for astrocytic differentiation-that further ensure Smad-regulated fate-specific gene induction. Our findings illuminate an exquisite regulation of NPC property change mediated by the interplay between cell-extrinsic cues and -intrinsic epigenetic programs during cortical development.

MiR-671-5p negatively regulates SMAD3 to inhibit migration and invasion of osteosarcoma cells

To explore the role of miR-671-5p in regulating the migration and invasion of osteosarcoma and the underlying mechanisms. The differentially expressed microRNAs (miRNAs) in osteosarcoma were screened in the NCBI online database, and the target proteins of these miRNAs were predicted and their functions were analyzed. Osteosarcoma cells were transfected with a plasmid overexpressing miR-671-5p, and the transfection efficiency was assessed using quantitative real-time PCR (qRT-PCR). The changes in the migration and invasion of the transfected cells were examined with Transwell assay, and the expressions of proteins related with epithelial-mesenchymal transition (EMT) were detected using Western blotting. Dual-luciferase reporter assay was performed to determine whether the 3'UTR of SMAD3 contained a targeted binding site of miR-671-5p. MiR-671-5p was significantly down-regulated in both osteosarcoma tissues and osteosarcoma cells (P < 0.05). The osteosarcoma cells overexpressing miR-671-5p showed significantly reduced migration and invasion abilities (P < 0.05) with obviously lowered expressions of EMT-related proteins (P < 0.05). SMAD3 was highly expressed in osteosarcoma cells (P < 0.05), and dual-luciferase reporter assay confirmed the presence of a targeted binding site between miR-671-5p and the 3'UTR of SMAD3 (P < 0.05). In osteosarcoma cells transfected with a SMAD3-overexpressing plasmid (P < 0.05), the high expression of SMAD3 significantly inhibited by miR-671-5p overexpression (P < 0.05). Transwell assay demonstrated that SMAD3 overexpression significantly promoted the migration and invasion of osteosarcoma cells (P < 0.05), and while miR-671-5p overexpression obviously reversed this effect (P < 0.05). MiR-671-5p can inhibit the invasion and migration of osteosarcoma cells by negatively regulating SMAD3.

Dissecting the Pathogenesis of Diabetic Retinopathy Based on the Biological ceRNA Network and Genome Variation Disturbance.

Background Diabetic retinopathy (DR) is the most important manifestation of diabetic microangiopathy. It is essential to explore the gene regulatory relationship and genomic variation disturbance of biological networks in DR progression. Methods In this study, we constructed a comprehensive lncRNA-mRNA ceRNA network of DR procession (CLMN) and explored its topological characteristics. Results Modular and functional analysis indicated that the organization of CLMN performed fundamental and specific functions in diabetes and DR pathology. The differential expression of hub ceRNA nodes and positive correlation reveals the highly connected ceRNA regulation and important roles in the regulating of DR pathology. A large proportion of SNPs in the TFBS, DHS, and enhancer regions of lncRNAs will affect lncRNA transcription and further cause expression variation. Some SNPs were found to disrupt the lncRNA functional elements such as miRNA target binding sites. These results indicate the complex nature of genotypic effects in the disturbing of CLMN and further contribute to gene expression variation and different disease phenotypes. Conclusion The identification of individual genomic variations and analysis of biological network disturbance by these genomic variations will help provide more personalized treatment plans and promote the development of precision medicine for DR.

CircBAGE2 (hsa_circ_0061259) regulates CCND1 and PDCD10 expression by functioning as an miR-103a-3p ‘sponge’ to alter the proliferation and apoptosis of prostate cancer cells*

Abstract Objective The aim of the article is to explore the function of circBAGE2 (hsa_circ_0061259) in prostate cancer (PCa) cells. Methods Sequencing results of circBAGE2 were verified by quantitative RT PCR (qRT-PCR) and Sanger sequencing. Agarose gel electrophoresis was used to detect the resistance of GAPDH, BAGE2, and circBAGE2 to RNase R and their expression as cDNA and gDNAin 22RV1 cells. The biological functions of circBAGE2 were investigated by CCK8 assay and flow cytometry in 22RV1 cells transfected with siRNAs. Multiple databases were used to predict the target binding sites between circRNAs, miRNAs, and mRNAs. Western blotting was used to detect the expression of CCND1 and PDCD10. Results CircBAGE2 was significantly upregulated in PCa samples and PCa cells compared to that in matched normal tissues and normal cells, and CircBAGE2 knockdown inhibits cell proliferation and promotes apoptosis. Downregulation of circBAGE2 compromised the expression of CCND1 and PDCD10. The 3′ UTRs of CCND1 and PDCD10 were matched by miR-103a-3p, which shared binding sites with circBAGE2. Conclusion CircBAGE2 contributes to PCa progression by upregulating CCND1 and PDCD10 expression through its role as a ‘sponge’ of miR-103a-3p. CircBAGE2 may be a potential therapeutic target for PCa.

A Bridge to Alkaloid Synthesis.

The construction of a structurally rigid architecture with chiral complexity, necessary to enhance the interaction with binding sites of drug targets, has been adapted as an intriguing approach in drug development. In the past few years, we have been interested in the synthesis of biologically significant and bridged alkaloids via novel synthetic methods and strategies based on recognition of the privileged pattern. Therefore, nitroso-ene and aza-Wacker cyclizations were elevated for the first time to construct bridged alkaloids, such as hosieine A, kopsone, melinonine-E and strychnoxanthine. Mechanistic investigations, including computational calculations for nitroso-ene reaction and deuterated experiments for aza-Wacker reaction, enable us to gain more insights into the chemical reactivity and selectivity of specific functional groups in developing viable synthetic methods.

Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes.

G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.

MicroRNA-206 relieves irradiation-induced neuroinflammation by regulating connexin 43.

Radiation therapy has been widely used for the treatment of various types of cancer; however, it may cause neuroinflammation during the pathological process of the disease. Astrocytes, the most abundant cell type in the central nervous system, have been confirmed to play vital roles in various diseases. Connexin (Cx)43, the main Cx type in astrocytes, which has been identified as a direct target gene of microRNA (miR)-206, was found to be involved in diseases pathologies in regions with astrocytes. The aim of the present study was to investigate the mechanism through which γ-radiation may cause astrocyte neuroinflammation and determine the specific mechanism underlying the effects of miR-206 in irradiation-induced HA-1800 cells. A dual-luciferase reporter system was used to predict and verify the target binding site between Cx43 and miR-206. HA-1800 cell viability and apoptosis were determined using a MTT assay and flow cytometry, respectively. In addition, the HA-1800 cells were induced by γ-radiation, then the protein and mRNA expression levels of Cx43, miR-206 and cleaved-caspase-3 were determined using western blot and reverse transcription-quantitative PCR analyses, respectively. ELISA was also performed to evaluate the concentrations of different inflammatory cytokines (TNF-α, IL-β, IL-6 and IFN-γ). The dual-luciferase reporter system indicated that Cx43 was a direct target of miR-206. miR-206 mimics increased the expression level of miR-206 in the astrocytes. Irradiation suppressed cell proliferation, increased apoptotic cells and enhanced cleaved-caspase-3 expression and inflammatory cytokines secretion in astrocytes. Furthermore, miR-206 was found to be downregulated and its expression was inversely associated with that of Cx43 in γ-radiation-induced astrocytes. Overexpression of miR-206 enhanced miR-206 and suppressed Cx43 expression, while Cx43 was upregulated in HA-1800 cells transfected with miR-206 mimic + Cx43-plasmid. However, the expression level of miR-206 was not significantly different in the Cx43-plasmid transfected group. In addition, it was found that miR-206 mimics relieved irradiation-induced neuroinflammation, which was confirmed by increased cell viability, and reduced cell apoptosis and cleaved caspase-3 protein expression, as well as decreased inflammatory cytokine secretion. Furthermore, all the effects of miR-206 mimics on γ-radiation-induced astrocytes were reversed by Cx43-plasmid. In summary, the results of the present study indicated that miR-206 may relieve irradiation-induced neural damage by regulating Cx43, which may provide a novel research direction and a potential therapeutic target for the clinical treatment of inflammation-associated neuronal injury following irradiation.

MiRNA target-binding sites as regulators of genes involved in the lipid metabolism might explain the hypercholesterolaemia in FH patients

Background and Aims: Familial hypercholesterolemia (FH) is due to mutations in LDLR, APOB and PCSK9, however, about 50% of clinical FH patients do not have an identifiable genetic cause. MicroRNAs (small non-coding RNAs) are negative regulators of gene expression and creation of new biding sites can be the cause of hypercholesterolaemia in mutation-negative families. The present work aims to analyse miRNAs targets as regulators of genes involved in lipid metabolism in FH patients.

Rational Multitargeted Drug Design Strategy from the Perspective of a Medicinal Chemist.

The development of multitarget-directed ligands (MTDLs) has become a widely focused research topic, but rational design remains as an enormous challenge. This paper reviews and discusses the design strategy of incorporating the second activity into an existing single-active ligand. If the binding sites of both targets share similar endogenous substrates, MTDLs can be designed by merging two lead compounds with similar functional groups. If the binding sites are large or adjacent to the solution, two key pharmacophores can be fused directly. If the binding regions are small and deep inside the proteins, the linked-pharmacophore strategy might be the only way. The added pharmacophores of second targets should not affect the binding mode of the original ones. Moreover, the inhibitory activities of the two targets need to be adjusted to achieve an optimal ratio.