RÉSUMÉ
The MYCN oncogene and histone deacetylases (HDACs) are key driver genes in the childhood cancer, neuroblastoma. We recently described a novel pyridobenzimidazole analogue, SE486-11, which enhanced the therapeutic effectiveness of HDAC inhibitors by increasing MYCN ubiquitination through effects on the deubiquitinase, ubiquitin-specific protease 5 (USP5). Here we describe the synthesis of a novel series of pyrimido[1,2-a]benzimidazole derivatives, and an evaluation of their cytopathic effects against non-malignant and human neuroblastoma cell lines. Among the tested analogues, 4-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (3a) was the most active compound against neuroblastoma cells (IC50 ≤ 2 µM), with low cytotoxicity (IC50 ≥ 15 µM) to normal cells. We show compound 3a bound to USP5 protein (Kd = 0.47 µM) in vitro and synergistically enhanced the efficacy of HDAC inhibitors against neuroblastoma cells. Moreover, knockdown of USP5 and MYCN in treated neuroblastoma cells showed that both USP5 and MYCN expression was necessary for the cytopathic activity of compound 3a, thus providing a clinically relevant rationale for further development of this of pyrimido[1,2-a]benzimidazole.
Sujet(s)
Inhibiteurs de désacétylase d'histone , Neuroblastome , Enfant , Humains , Benzimidazoles , Lignée cellulaire tumorale , Inhibiteurs de désacétylase d'histone/pharmacologie , Protéine du proto-oncogène N-Myc/génétique , Neuroblastome/traitement médicamenteux , Neuroblastome/génétique , Ubiquitin-specific proteasesRÉSUMÉ
MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R1 position. Replacing the trifluoromethyl group on the phenyl ring at the R2 position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.
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Momordica cochinchinensis is a herbal medicine used throughout Asia and this study investigated the antimelanoma potentials and molecular mechanisms of M. cochinchinensis seed with emphasis on extraction to optimise bioactivity. Overall, the aqueous extract was superior, with a wider diversity and higher concentration of proteins and peptides that was more cytotoxic to the melanoma cells than other extraction solvents. The IC50 of the aqueous extract on melanoma cells were similar to treatment with current anticancer drugs, vemurafenib and cisplatin. This cytotoxicity was cancer-specific with lower cytotoxic effects on HaCaT epidermal keratinocytes. Cytotoxicity correlated with MAPK signalling pathways leading to apoptosis and necrosis induced by triggering tumour necrosis factor receptor-1 (TNFR1), reducing the expression of nuclear factor kappa B (NF-kB), and suppression of BRAF/MEK. This efficacy of M. cochinchinensis seed extracts on melanoma cells provides a platform for future clinical trials as potent adjunctive therapy for metastatic melanoma.
RÉSUMÉ
MOTIVATION: Over 300 000 protein-protein interaction (PPI) pairs have been identified in the human proteome and targeting these is fast becoming the next frontier in drug design. Predicting PPI sites, however, is a challenging task that traditionally requires computationally expensive and time-consuming docking simulations. A major weakness of modern protein docking algorithms is the inability to account for protein flexibility, which ultimately leads to relatively poor results. RESULTS: Here, we propose DockNet, an efficient Siamese graph-based neural network method which predicts contact residues between two interacting proteins. Unlike other methods that only utilize a protein's surface or treat the protein structure as a rigid body, DockNet incorporates the entire protein structure and places no limits on protein flexibility during an interaction. Predictions are modeled at the residue level, based on a diverse set of input node features including residue type, surface accessibility, residue depth, secondary structure, pharmacophore and torsional angles. DockNet is comparable to current state-of-the-art methods, achieving an area under the curve (AUC) value of up to 0.84 on an independent test set (DB5), can be applied to a variety of different protein structures and can be utilized in situations where accurate unbound protein structures cannot be obtained. AVAILABILITY AND IMPLEMENTATION: DockNet is available at https://github.com/npwilliams09/docknet and an easy-to-use webserver at https://biosig.lab.uq.edu.au/docknet. All other data underlying this article are available in the article and in its online supplementary material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Sujet(s)
Algorithmes , , Humains , Protéome , Pharmacophore , Aire sous la courbe , Biologie informatiqueRÉSUMÉ
PURPOSE: Patients with an underlying telomere biology disorder (TBD) have variable clinical presentations, and they can be challenging to diagnose clinically. A genomic diagnosis for patients presenting with TBD is vital for optimal treatment. Unfortunately, many variants identified during diagnostic testing are variants of uncertain significance. This complicates management decisions, delays treatment, and risks nonuptake of potentially curative therapies. Improved application of functional genomic evidence may reduce variants of uncertain significance classifications. METHODS: We systematically searched the literature for published functional assays interrogating TBD gene variants. When possible, established likely benign/benign and likely pathogenic/pathogenic variants were used to estimate the assay sensitivity, specificity, positive predictive value, negative predictive value, and odds of pathogenicity. RESULTS: In total, 3131 articles were screened and 151 met inclusion criteria. Sufficient data to enable a PS3/BS3 recommendation were available for TERT variants only. We recommend that PS3 and BS3 can be applied at a moderate and supportive level, respectively. PS3/BS3 application was limited by a lack of assay standardization and limited inclusion of benign variants. CONCLUSION: Further assay standardization and assessment of benign variants are required for optimal use of the PS3/BS3 criterion for TBD gene variant classification.
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Dépistage génétique , Variation génétique , Humains , Variation génétique/génétique , Génome humain , Génomique , Télomère/génétiqueRÉSUMÉ
Introduction: COVID-19 pandemic has been threatening public health and economic development worldwide for over two years. Compared with the original SARS-CoV-2 strain reported in 2019, the Omicron variant (B.1.1.529.1) is more transmissible. This variant has 34 mutations in its Spike protein, 15 of which are present in the Receptor Binding Domain (RBD), facilitating viral internalization via binding to the angiotensin-converting enzyme 2 (ACE2) receptor on endothelial cells as well as promoting increased immune evasion capacity. Methods: Herein we compared SARS-CoV-2 proteins (including ORF3a, ORF7, ORF8, Nucleoprotein (N), membrane protein (M) and Spike (S) proteins) from multiple ancestral strains. We included the currently designated original Variant of Concern (VOC) Omicron, its subsequent emerged variants BA.1, BA2, BA3, BA.4, BA.5, the two currently emerging variants BQ.1 and BBX.1, and compared these with the previously circulating VOCs Alpha, Beta, Gamma, and Delta, to better understand the nature and potential impact of Omicron specific mutations. Results: Only in Omicron and its subvariants, a bias toward an Asparagine to Lysine (N to K) mutation was evident within the Spike protein, including regions outside the RBD domain, while none of the regions outside the Spike protein domain were characterized by this mutational bias. Computational structural analysis revealed that three of these specific mutations located in the central core region, contribute to a preference for the alteration of conformations of the Spike protein. Several mutations in the RBD which have circulated across most Omicron subvariants were also analysed, and these showed more potential for immune escape. Conclusion: This study emphasizes the importance of understanding how specific N to K mutations outside of the RBD region affect SARS-CoV-2 conformational changes and the need for neutralizing antibodies for Omicron to target a subset of conformationally dependent B cell epitopes.
Sujet(s)
COVID-19 , Lysine , Humains , Lysine/génétique , Asparagine , SARS-CoV-2/génétique , Cellules endothéliales , Pandémies , Glycoprotéine de spicule des coronavirus/génétique , COVID-19/génétique , MutationRÉSUMÉ
Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.
Sujet(s)
Dynamines , Quinazolinones , Humains , Dynamines/antagonistes et inhibiteurs , dGTPases/métabolisme , Dynamique mitochondriale , Quinazolinones/pharmacologieRÉSUMÉ
Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Šresolution. VCP-6 binds deeper in the binding channel than T4 with the 3',5'-dichlorophenyl ring binding in the 'forward' mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.
Sujet(s)
Amyloïdose , Préalbumine , Humains , Préalbumine/métabolisme , Conformation des protéines , Modèles moléculaires , Sites de fixation , Cristallographie aux rayons X , Amyloïde , Protéines amyloïdogènes , HalogènesRÉSUMÉ
PURPOSE OF REVIEW: This study is aimed at reviewing the recent progress in Drp1 inhibition as a novel approach for reducing doxorubicin-induced cardiotoxicity and for improving cancer treatment. RECENT FINDINGS: Anthracyclines (e.g. doxorubicin) are one of the most common and effective chemotherapeutic agents to treat a variety of cancers. However, the clinical usage of doxorubicin has been hampered by its severe cardiotoxic side effects leading to heart failure. Mitochondrial dysfunction is one of the major aetiologies of doxorubicin-induced cardiotoxicity. The morphology of mitochondria is highly dynamic, governed by two opposing processes known as fusion and fission, collectively known as mitochondrial dynamics. An imbalance in mitochondrial dynamics is often reported in tumourigenesis which can lead to adaptive and acquired resistance to chemotherapy. Drp1 is a key mitochondrial fission regulator, and emerging evidence has demonstrated that Drp1-mediated mitochondrial fission is upregulated in both cancer cells to their survival advantage and injured heart tissue in the setting of doxorubicin-induced cardiotoxicity. Effective treatment to prevent and mitigate doxorubicin-induced cardiotoxicity is currently not available. Recent advances in cardio-oncology have highlighted that Drp1 inhibition holds great potential as a targeted mitochondrial therapy for doxorubicin-induced cardiotoxicity.
Sujet(s)
Protéines mitochondriales , Tumeurs , Humains , Protéines mitochondriales/génétique , Protéines mitochondriales/métabolisme , Protéines mitochondriales/pharmacologie , Cardiotoxicité/prévention et contrôle , Dynamines/métabolisme , Dynamines/pharmacologie , Mitochondries/métabolisme , Doxorubicine/effets indésirables , Tumeurs/traitement médicamenteux , Tumeurs/métabolismeRÉSUMÉ
Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.
Sujet(s)
Telomerase , Biologie , Humains , Mutation , ARN/génétique , Telomerase/génétique , Telomerase/métabolisme , Télomère/génétique , Télomère/métabolismeRÉSUMÉ
The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.
Sujet(s)
Syndrome de Bloom/génétique , ADN cruciforme/génétique , Instabilité du génome/génétique , Allèles , Protéines de transport/génétique , Lignée cellulaire , ADN topoisomérases de type I/génétique , Humains , Mutation/génétique , Liaison aux protéines/génétique , RecQ helicases/génétique , Recombinaison génétique/génétique , SolubilitéRÉSUMÉ
Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 µg/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 ± 0.22, in non-replicating stage). Using "click" chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study. GroEL-2 identified a potent and specific target protein for RRA 2 compound in whole cell extract of Mtb H37Ra.
Sujet(s)
Protéines bactériennes/analyse , Mycobacterium tuberculosis , Alcynes , Antibactériens , Antituberculeux/composition chimique , Antituberculeux/pharmacologie , Vaccin BCG , Biotine , Extrait cellulaire , Tests de sensibilité microbienne , Protéines , Thiols , TriazolesRÉSUMÉ
AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.
Sujet(s)
Dynamines/antagonistes et inhibiteurs , Antienzymes/pharmacologie , Hydralazine/pharmacologie , Mitochondries du myocarde/effets des médicaments et des substances chimiques , Dynamique mitochondriale/effets des médicaments et des substances chimiques , Infarctus du myocarde/prévention et contrôle , Lésion de reperfusion myocardique/prévention et contrôle , Myocytes cardiaques/effets des médicaments et des substances chimiques , Animaux , Antioxydants/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Modèles animaux de maladie humaine , Dynamines/métabolisme , Femelle , Cellules HeLa , Humains , Préparation de coeur isolé , Mâle , Souris de lignée C57BL , Souris transgéniques , Mitochondries du myocarde/métabolisme , Mitochondries du myocarde/anatomopathologie , Infarctus du myocarde/enzymologie , Infarctus du myocarde/anatomopathologie , Lésion de reperfusion myocardique/enzymologie , Lésion de reperfusion myocardique/anatomopathologie , Myocytes cardiaques/enzymologie , Myocytes cardiaques/anatomopathologie , Stress oxydatif/effets des médicaments et des substances chimiques , Transduction du signalRÉSUMÉ
Dihydrodipicolinate synthase (DHDPS), responsible for the first committed step of the diaminopimelate pathway for lysine biosynthesis, has become an attractive target for the development of new antibacterial and herbicidal agents. Herein, we report the discovery and exploration of the first inhibitors of E. coli DHDPS which have been identified from screening lead and are not based on substrates from the lysine biosynthesis pathway. Over 50 thiazolidinediones and related analogues have been prepared in order to thoroughly evaluate the structure-activity relationships against this enzyme of significant interest.
Sujet(s)
Antienzymes/pharmacologie , Composés hétérocycliques/pharmacologie , Hydro-lyases/antagonistes et inhibiteurs , Thiazolidinediones/pharmacologie , Relation dose-effet des médicaments , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Escherichia coli/enzymologie , Composés hétérocycliques/synthèse chimique , Composés hétérocycliques/composition chimique , Hydro-lyases/métabolisme , Structure moléculaire , Relation structure-activité , Thiazolidinediones/synthèse chimique , Thiazolidinediones/composition chimiqueRÉSUMÉ
Despite the important roles played by protein-protein interactions (PPIs) in disease, they have been long considered as 'undruggable'. However, recent advances have suggested that PPIs are druggable but may not follow conventional rules of 'drug ability'. Here we explore which physicochemical parameters are essential for a PPI modulator to be a clinical drug by analysing the physicochemical properties of small-molecule PPI modulators in the market, in clinical trials, and published. Our analysis reveals that those compounds currently on the market have a larger range of values for most of the physicochemical parameters, whereas those in clinical trials fit much more stringently to standard drug-like parameters. This observation was particularly true for molecular weight, clog P and topological polar surface area, where aside from a few outliers, most of the compounds in clinical trials fit within standard drug-like parameters. This implies that the newer PPI modulators are more drug-like than those currently on the market, suggesting that designing new PPI-specific screening libraries should remain within standard drug-like parameters in order to obtain a clinical candidate. Taken together, our analysis has important implications for designing future drug discovery campaigns aimed at targeting PPIs.
RÉSUMÉ
The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.
Sujet(s)
Anti-inflammatoires/pharmacologie , Protéines conçues par répétition de motifs ankyrine/pharmacologie , Antigène macrophage 1/métabolisme , Monocytes/effets des médicaments et des substances chimiques , Infarctus du myocarde/traitement médicamenteux , Myocardite/traitement médicamenteux , Myocarde/métabolisme , Sepsie/traitement médicamenteux , Animaux , Techniques d'exposition à la surface cellulaire , Cellules cultivées , Protéines conçues par répétition de motifs ankyrine/génétique , Modèles animaux de maladie humaine , Épitopes , Oxygénation extracorporelle sur oxygénateur à membrane , Humains , Antigène macrophage 1/génétique , Macrophages/effets des médicaments et des substances chimiques , Macrophages/immunologie , Macrophages/métabolisme , Simulation de docking moléculaire , Monocytes/immunologie , Monocytes/métabolisme , Infarctus du myocarde/immunologie , Infarctus du myocarde/métabolisme , Infarctus du myocarde/physiopathologie , Myocardite/immunologie , Myocardite/métabolisme , Myocardite/physiopathologie , Myocarde/immunologie , Myocarde/anatomopathologie , Étude de validation de principe , Liaison aux protéines , Infarctus du myocarde avec sus-décalage du segment ST/immunologie , Infarctus du myocarde avec sus-décalage du segment ST/métabolisme , Sepsie/immunologie , Sepsie/métabolisme , Sepsie/physiopathologie , Fonction ventriculaire gauche/effets des médicaments et des substances chimiquesRÉSUMÉ
Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.
Sujet(s)
Carcinogenèse/effets des médicaments et des substances chimiques , Inhibiteurs de désacétylase d'histone/pharmacologie , Protéine du proto-oncogène N-Myc/génétique , Neuroblastome/traitement médicamenteux , Ubiquitin-specific proteases/génétique , Protéines de poisson-zèbre/génétique , Animaux , Animal génétiquement modifié/génétique , Apoptose/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Évaluation préclinique de médicament , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Hétérogreffes , Humains , Souris , Neuroblastome/génétique , Neuroblastome/anatomopathologie , Bibliothèques de petites molécules/pharmacologie , Vorinostat/pharmacologie , Danio zébré/génétiqueRÉSUMÉ
Insecticides remain valuable tools for the control of insect pests that significantly impact human health and agriculture. A deeper understanding of insecticide targets is important in maintaining this control over pests. Our study systematically investigates the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, we were able to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dß1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dα6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in ß1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dß1 examined here provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dß1 as a key component of nAChRs targeted by both these insecticide classes. A comparison of resistance phenotypes found in this study to resistance reported in insect pests reveals a strong conservation of subunit targets across many different insect species and that mutations have been identified in most of the receptor subunits that our findings would predict to have the potential to confer resistance.
Sujet(s)
Drosophila melanogaster , Résistance aux insecticides/génétique , Insecticides/pharmacologie , Récepteurs nicotiniques , Animaux , Protéines de Drosophila/métabolisme , Drosophila melanogaster/génétique , Drosophila melanogaster/métabolisme , Association médicamenteuse , Macrolides/pharmacologie , Mutation , Néonicotinoïdes/pharmacologie , Pyridines/pharmacologie , Récepteurs nicotiniques/effets des médicaments et des substances chimiques , Récepteurs nicotiniques/génétique , Récepteurs nicotiniques/métabolisme , Composés du soufre/pharmacologieRÉSUMÉ
COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), whilst commonly characterised as a respiratory disease, is reported to have extrapulmonary manifestations in multiple organs. Extrapulmonary involvement in COVID-19 includes autoimmune-like diseases such as Guillain-Barré syndrome and Kawasaki disease, as well as the presence of various autoantibodies including those associated with autoimmune diseases such a systemic lupus erythematosus (e.g. ANA, anti-La). Multiple strains of SARS-CoV-2 have emerged globally, some of which are found to be associated with increased transmissibility and severe disease. We performed an unbiased comprehensive mapping of the potential for cross-reactivity with self-antigens across multiple SARS-CoV-2 proteins and compared identified immunogenic regions across multiples strains. Using the Immune Epitope Database (IEDB) B cell epitope prediction tool, regions predicted as antibody epitopes with high prediction scores were selected. Epitope sequences were then blasted to eight other global strains to identify mutations within these regions. Of the 15 sequences compared, eight had a mutation in at least one other global strain. Predicted epitopes were then compared to human proteins using the NCBI blast tool. In contrast to studies focusing on short sequences of peptide identity, we have taken an immunological approach to selection criteria for further analysis and have identified 136 alignments of 6-23 amino acids (aa) in 129 human proteins that are immunologically likely to be cross-reactive with SARS-CoV-2. Additionally, to identify regions with significant potential to interfere with host cell function-or promote immunopathology, we identified epitope regions more likely to be accessible to pathogenic autoantibodies in the host, selected using a novel combination of sequence similarity, and modelling protein and alignment localization with a focus on extracellular regions. Our analysis identified 11 new predicted B-cell epitopes in host proteins, potentially capable of explaining key aspects of COVID-19 extrapulmonary pathology, and which were missed in other in silico studies which used direct identity rather than immunologically related functional criteria.
RÉSUMÉ
The role of proliferation-associated protein 2G4 (PA2G4), alternatively known as ErbB3-binding protein 1 (EBP1), in cancer has become apparent over the past 20 years. PA2G4 expression levels are correlated with prognosis in a range of human cancers, including neuroblastoma, cervical, brain, breast, prostate, pancreatic, hepatocellular, and other tumors. There are two PA2G4 isoforms, PA2G4-p42 and PA2G4-p48, and although both isoforms of PA2G4 regulate cellular growth and differentiation, these isoforms often have opposing roles depending on the context. Therefore, PA2G4 can function either as a contextual tumor suppressor or as an oncogene, depending on the tissue being studied. However, it is unclear how distinct structural features of the two PA2G4 isoforms translate into different functional outcomes. In this review, we examine published structures to identify important structural and functional components of PA2G4 and consider how they may explain its crucial role in the malignant phenotype. We will highlight the lysine-rich regions, protein-protein interaction sites, and post-translational modifications of the two PA2G4 isoforms and relate these to the functional cellular role of PA2G4. These data will enable a better understanding of the function and structure relationship of the two PA2G4 isoforms and highlight the care that will need to be undertaken for those who wish to conduct isoform-specific structure-based drug design campaigns.