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Over 50% of patients with systemic LCH are not cured with front-line therapies, and data to guide salvage options are limited. We describe 58 patients with LCH who were treated with clofarabine. Clofarabine monotherapy was active against LCH in this cohort, including heavily pretreated patients with a systemic objective response rate of 92.6%, higher in children (93.8%) than adults (83.3%). BRAFV600E+ variant allele frequency in peripheral blood is correlated with clinical responses. Prospective multicentre trials are warranted to determine optimal dosing, long-term efficacy, late toxicities, relative cost and patient-reported outcomes of clofarabine compared to alternative LCH salvage therapy strategies.
Assuntos
Clofarabina , Histiocitose de Células de Langerhans , Humanos , Clofarabina/uso terapêutico , Clofarabina/administração & dosagem , Histiocitose de Células de Langerhans/tratamento farmacológico , Masculino , Feminino , Adulto , Adolescente , Criança , Pessoa de Meia-Idade , Pré-Escolar , Adulto Jovem , Idoso , Recidiva , Proteínas Proto-Oncogênicas B-raf/genética , Lactente , Resultado do Tratamento , Terapia de Salvação , Nucleotídeos de Adenina/uso terapêutico , Nucleotídeos de Adenina/administração & dosagem , Nucleotídeos de Adenina/efeitos adversos , Arabinonucleosídeos/uso terapêutico , Arabinonucleosídeos/administração & dosagem , Arabinonucleosídeos/efeitos adversosRESUMO
RAS mutations are frequently observed in childhood B-cell acute lymphoblastic leukemia (B-ALL) and previous studies have yielded conflicting results as to whether they are associated with a poor outcome. We and others have demonstrated that the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK) pathway can be activated through epigenetic mechanisms in the absence of RAS pathway mutations. Herein, we examined whether MAPK activation, as determined by measuring phosphorylated extracellular signal-regulated kinase (pERK) levels in 80 diagnostic patient samples using phosphoflow cytometry, could be used as a prognostic biomarker for pediatric B-ALL. The mean fluorescence intensity of pERK (MFI) was measured at baseline and after exogenous stimulation with or without pretreatment with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Activation levels (MFI stimulated/MFI baseline) ranged from 0.76 to 4.40 (median = 1.26), and inhibition indexes (MFI stimulated/MFI trametinib stimulated) ranged from 0.439 to 5.640 (median = 1.30), with no significant difference between patients with wildtype versus mutant RAS for either. Logistic regression demonstrated that neither MAPK activation levels nor RAS mutation status at diagnosis alone or in combination was prognostic of outcome. However, 35% of RAS wildtype samples showed MAPK inhibition indexes greater than the median, thus raising the possibility that therapeutic strategies to inhibit MAPK activation may not be restricted to patients whose blasts display Ras pathway defects.
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Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure-activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR-Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Modelos Moleculares , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When ß-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel ß-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.
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Amiloide/química , Espectroscopia de Ressonância Magnética/métodos , Zinco/química , Amiloide/metabolismo , Sítios de Ligação , Biologia Computacional , Histidina/química , Histidina/metabolismo , Metaloproteínas , Modelos Moleculares , Água/química , Zinco/metabolismoRESUMO
A computationally designed, allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non-enzymatic protein, is capable of efficient and site selective post-translational acylation of lysines in peptides with highly diverse sequences. Calmodulin's binding partners are involved in regulating a large number of cellular processes; this new chemical-biology tool will help to identify them and provide structural insight into their interactions with calmodulin.
Assuntos
Substituição de Aminoácidos , Calmodulina/genética , Calmodulina/metabolismo , Lisina/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Acilação , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/química , Humanos , Lisina/análise , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Engenharia de ProteínasRESUMO
To evaluate the role of COX-2 and 5-LOX as dual inhibitors in controlling the cancer cell proliferation, a set of two series having 42 compounds of 1, 2, 3-Tethered Indole-3-glyoxamide derivatives were synthesized by employing click chemistry approach and were also evaluated for their in vitro cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) inhibitory activities with in vivo anti-inflammatory and in vitro anti-proliferative potencies. Among the compounds tested, compounds 11q and 13s displayed excellent inhibition of COX-2 (IC50 0.12⯵M) with good COX-2 selectivity index (COX-2/COX-1) of 0.058 and 0.046 respectively. Compounds 11q and 13s also demonstrated comparable 5-LOX inhibitory activity with IC50 7.73 and 7.43⯵M respectively to that of standard Norhihydroguaiaretic acid (NDGA: IC50 7.31⯵M). Among all the selected cell lines, prostate cancer cell line DU145 was found to be susceptible to this class of compounds. Among all the tested compounds, compounds 11g, 11i, 11k, 11q, 13r, 13s and 13u demonstrated excellent to moderate anti-proliferative activity with IC50s ranging between 6.29 and 18.53⯵M. Compounds 11q and 11g demonstrated better anti-proliferative activities against DU145 cancer cell line with IC50 values 8.17 and 8.69⯵M respectively when compared to the standard drug etoposide (VP16; IC50 9.80⯵M). Compounds 11g, 11k, 11q, 13s and 13u showed good dual COX-2/5-LOX inhibitory potentials with excellent anti-proliferative activity. Results from carrageenan-induced hind paw edema demonstrated that compounds 11b, 11l, 11q and 13q exhibited significant anti-inflammatory activity with 69-77% inhibition at 3â¯h, 75-82% inhibition at 5â¯h when compared to the standard drug indomethacin (66.6% at 3â¯h and 77.94% at 5â¯h). Ulcerogenic study revealed that compounds 11q and 13q did not cause any gastric ulceration. In vitro tubulin assay resuted that compound 11q interfered with microtubulin dynamic and act as tubulin polymerization inhibitor. In silico molecular docking studies demonstrated that compounds 11q and 13s are occupying the colchicines binding site of tubulin polymer and 11q illustrated very good binding affinities towards COX-2 and 5-LOX.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Compostos de Sulfonilureia/farmacologia , Triazóis/farmacologia , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Araquidonato 5-Lipoxigenase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/síntese química , Indóis/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Compostos de Sulfonilureia/síntese química , Compostos de Sulfonilureia/química , Triazóis/química , Tubulina (Proteína)/metabolismoRESUMO
INTRODUCTION AND HYPOTHESIS: Hormonal contraceptive use is common practice worldwide. Although the effects of hormone treatments in the pelvic region are well established, there is no clear evidence regarding their effects on incontinence, bladder, bowel, vaginal and sexual symptoms in premenopausal women. We hypothesized that hormonal contraceptives affect pelvic floor function. We therefore performed a comprehensive systematic review of published studies to determine the influence of hormonal contraception on pelvic floor functions. METHODS: Electronic literature databases were searched from database inception to March 2015. Keywords and medical subject headings searched for included terms and word variations for 'contraception', and 'bowel', 'vaginal', 'sexual' and 'urinary' symptoms. Studies were eligible if they looked at these symptoms in women taking hormonal contraception. Two reviewers independently screened studies for inclusion, and extracted data on study characteristics, quality and results. Data were combined where possible. RESULTS: Of the 429 citations identified, 13 studies were included in the review. Data were meta-analysed where possible and presented as prevalence. The results indicate statistically significant links between interstitial cystitis and oral contraceptive use at any point (ever) (OR 2.31, 95 % CI 1.03 - 5.16; p = 0.04) and vulvar vestibulitis and current oral contraceptive use (OR 2.10, 95 % CI 1.26 - 3.49; p = 0.004). The evidence is unclear in other areas. CONCLUSIONS: Our results indicate that oral contraceptives may have an effect on pelvic floor function. They could increase the risk of painful bladder and vulvar vestibulitis, but their effect on dyspareunia is inconsistent. However, robustly collected prospective data to establish causal associations are needed.
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Anticoncepcionais Femininos/uso terapêutico , Cistite Intersticial/epidemiologia , Dispositivos Intrauterinos Medicados , Levanogestrel/uso terapêutico , Sintomas do Trato Urinário Inferior/epidemiologia , Anticoncepcionais Orais Hormonais/uso terapêutico , Preparações de Ação Retardada/uso terapêutico , Dispareunia/epidemiologia , Feminino , Humanos , Acetato de Medroxiprogesterona/uso terapêutico , Diafragma da Pelve/fisiopatologia , Pré-Menopausa , Vulvodinia/epidemiologiaRESUMO
INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome is a difficult condition to treat. The purpose of this systematic review is to assess the effectiveness of various complementary therapies available for treatment. METHODS: This review was conducted in adherence with Preferred Reporting Items for Systematic Reviews. Citations were retrieved using a comprehensive database search (from inception to July 2014: CINAHL, Cochrane, EMBASE, Medline and SIGEL and grey literature). Studies that fulfilled the inclusion criteria were selected. Eligibility consisted of women with bladder pain syndrome, an intervention of alternative/complementary therapies and an outcome of improvement of symptoms. Information regarding study characteristics and primary outcomes was collated. The Cochrane risk of bias scale was used to evaluate the quality of the studies included. RESULTS: A total of 1,454 citations were identified, 11 studies fulfilled the inclusion criteria (4 randomised control trials [RCTs] and 7 prospective studies). The key interventions studied were acupuncture, relaxation therapy, physical therapy, hydrogen-rich therapy, diet and nitric oxide synthetase. CONCLUSION: Therapies with the potential for benefit in patients with bladder pain syndrome are dietary management, acupuncture and physical therapy. These findings were obtained from small studies and hence caution is advised. Robustly designed multicentre RCTs on these complementary therapies are needed to guide patients and clinicians.
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Terapias Complementares/métodos , Cistite Intersticial/terapia , Terapia por Acupuntura/métodos , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Modalidades de Fisioterapia , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Terapia de Relaxamento/métodos , Resultado do TratamentoRESUMO
We have shown that deâ novo designed peptides self-assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self-assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion-like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.
Assuntos
Cobre/química , Oxigênio/química , Peptídeos/química , Sítios de Ligação , Catálise , Peptídeos/síntese químicaRESUMO
Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.
Assuntos
Enzimas/química , Mutação , Proteínas/química , Catálise , Dicroísmo Circular , Cinética , Ressonância Magnética Nuclear Biomolecular , Proteínas/genética , Proteólise , Espectrofotometria UltravioletaRESUMO
Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, ß-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with ß-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with ß-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the ß-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing ß-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the ß-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.
Assuntos
Fluorescência , Bicamadas Lipídicas/química , Meliteno , Triptofano , Meliteno/análogos & derivados , Meliteno/química , Estrutura Secundária de Proteína , Triptofano/análogos & derivados , Triptofano/químicaRESUMO
tRNA- Encoded Peptides (tREPs) have recently been discovered as new functional peptides and hold promise as therapeutics for anti-parasitic applications. In this study, in silico investigations were conducted to design tRNA-encoded peptides with the potential to target over-expressed receptors in breast cancer cells. tRNA genes were translated into corresponding peptides (tREPs) using computational tools. The tREPs, which were predicted as anticancer peptides, were then screened for various ADMET properties. Molecular docking studies were conducted for three cancer target receptors, the Estrogen Receptor (ER), Peroxisome Proliferator-Activated Receptor (PPAR) and the Epidermal Growth Factor Receptor (EGFR). Based on the docking results, specific tREPs were screened and molecular dynamics simulations were performed, and the binding energies were further explored using MMPBSA calculations. The peptide Pep1 (DWIAWRHHNDIVSWLTCGPRFKSWS) and Pep2 (GFIAWWSRHLELAQTRFKSWWS) exhibited a good binding affinity against the Estrogen Receptor (ER) and the Peroxisome Proliferator-Activated Receptor Alpha (PPAR) cancer target. The Pep1-ER and Pep1-PPAR complex maintained an average of two hydrogen bonds throughout the simulation and demonstrated a higher negative binding free energy of -72.27 kcal/mol and -65.16 kcal/mol respectively, as calculated by MMPBSA. Therefore, the tREPs designed as anticancer peptides in this study provide novel approaches for potential anticancer therapeutic modalities.Communicated by Ramaswamy H. Sarma.
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tRNA-Encoded Peptides (tREPs), encoded by small open reading frames (smORFs) within tRNA genes, have recently emerged as a new class of functional peptides exhibiting antiparasitic activity. The discovery of tREPs has led to a re-evaluation of the role of tRNAs in biology and has expanded our understanding of the genetic code. This presents an immense, unexplored potential in the realm of tRNA-peptide interactions, paving the way for groundbreaking discoveries and innovative applications in various biological functions. This study explores the antimicrobial potential of tREPs against protein targets by employing a computational method that uses verified data sources and highly recognized predictive algorithms to provide a sorted list of likely antimicrobial peptides, which were then filtered for toxicity, cell permeability, allergenicity and half-life. These peptides were then docked with screened protein targets and computationally validated using molecular dynamics (MD) simulations for 150 ns and the binding free energy was estimated. The peptides Pep2 (VVLWRKPRVRKTG) and Pep6 (HRLRLRRRKPWW) exhibited good binding affinities of -110.5 +/- 2.5 and -129.0 +/- 3.9, respectively, with RMSD values of 0.4 and 0.25 nm against the fucose-binding lectin (7NEF) and the 30S ribosome of Mycobacterium smegmatis (5O5J) protein targets. The 7NEF-Pep2 and 5O5J-Pep6 complexes indicated higher negative binding free energies of -52.55 kcal/mol and -55.52 kcal/mol respectively, as calculated by Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Thus, the tREPs derived peptides designed as a part of this study, provide novel approaches for potential anti-bacterial therapeutic modalities.Communicated by Ramaswamy H. Sarma.
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Transfer RNAs (tRNA) are non-coding RNAs. Encouraged by biological applications discovered for peptides derived from other non-coding genomic regions, we explore the possibility of deriving epitope-based vaccines from tRNA encoded peptides (tREP) in this study. Epitope-based vaccines have been identified as an effective strategy to mitigate safety and specificity concerns observed in vaccine development. In this study, we explore the potential of tREP as a source for epitope-based vaccines for virus pathogens. We present a computational workflow that uses verified data sources and community-validated predictive tools to produce a ranked list of plausible epitope-based vaccines starting from tRNA sequences. The top epitope, bound to the predicted HLA molecule, for the virus pathogen is computationally validated through 200 ns molecular dynamics (MD) simulations followed by binding free energy calculations. The simulation results indicate that two tRNA encoded epitope-based vaccines, RRHIDIVV and IMVRFSAE for Mamastrovirus 3 and Norovirus GII, respectively, are likely candidates. Peptides originating from tRNAs provide unexplored opportunities for vaccine design. Encouraged by our previous experimental study, which established the inhibitory properties of tREPs against infectious parasites, we have proposed a computationally validated set of peptides derived from tREPs as vaccines for viral pathogens.
Assuntos
Biologia Computacional , Simulação de Dinâmica Molecular , Peptídeos , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/química , Biologia Computacional/métodos , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Humanos , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/química , Epitopos/química , Epitopos/imunologia , Epitopos/genética , Norovirus/genética , Norovirus/imunologia , Norovirus/químicaRESUMO
Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.
RESUMO
UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.
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The placement of orthodontic bonded brackets may be accomplished by either a direct or indirect technique. Most orthodontists will agree that brackets can be positioned more accurately on study casts than directly on teeth in the mouth. And, also direct bonding is more demanding to the orthodontist. Yet, very few orthodontists routinely use an indirect bonding technique. The reasons commonly given for not using the indirect method are difficulty in achieving consistent and predictable adhesion to the teeth, excess of composite around the bracket margins, failure to get all the brackets to adhere to the teeth e the expense of the materials. These disadvantages can be overcome by a new simplified method of bonding outlined in this article; additionally it has advantages of direct bonding also.
Assuntos
Colagem Dentária/métodos , Braquetes Ortodônticos , Resinas Compostas/química , Colagem Dentária/instrumentação , Materiais para Moldagem Odontológica/química , Humanos , Tinta , Modelos Dentários , Desenho de Aparelho Ortodôntico , Radiografia Panorâmica , Cimentos de Resina/química , Propriedades de Superfície , Fatores de Tempo , Coroa do Dente/anatomia & histologia , Coroa do Dente/diagnóstico por imagemRESUMO
This study developed a method to simultaneously determine 73 multi-class pesticides in okra fruit using LC-MS/MS and GC-MS/MS. The sample was extracted with acetonitrile and subsequent clean-up through dispersive-SPE method. The quantification level of the technique was 0.01 µg g-1 and compliance to the MRLs fixed by the regulatory bodies like EU and FSSAI. The recovery at 10, 50, and 100 µg kg-1 spiked levels; intra and inter-day precision at 50 µg kg-1 were found within 70-120% with RSD less than 15% with LC-MS/MS and GC-MS/MS. Measurement uncertainty was in the range of 1.81 to 12.91 µg kg-1 estimated at 50 µg kg-1. The matrix effects were slightly higher for LC than GC-compatible pesticides. Risk assessment for pesticides detected in the field and market samples found no hazardous to the consumers except profenofos. The proposed method is highly sensitive, reproducible for the complex matrix like okra, and meets the regulatory standards.
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Chlorantraniliprole belongsto theanthranilic diamide group is widely used against broad range of lepidopteron pests in a variety of vegetable and rice pests includingyellow rice stem borer and leaf folder. Supervised field trials were conducted duringRabi (2018-2019) and Kharif (2019) to evaluate the dissipation pattern and risk assessment of chlorantraniliprole 18.5% SC in paddy ecosystem following foliar application at 30 and 60 g a.i. ha-1 in two different cropping seasons.Modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique was used for the extraction of CAP residues with acetonitrile and determined by LC-MS/MS (ESI +).The limit of quantification (LOQ) was 0.01 µg g-1 for paddy leaf, straw, husk, and brown rice, respectively and 0.005 µg g-1 for soil. The average recoveries obtained were 84.30-88.92% from paddy leaf, 94.25-97.81% from straw, 90.21-93.38% from husk, 93.57-96.40% from brown rice and 89.93-91.14% from soil. The residues in paddy leaf dissipated within 35-40 days with a half-life of 4.33-5.07 days in Rabi and 3.92-4.86 days in Kharif at 30 and 60 g a.i. ha-1, respectively. The residues in soil dissipated within 15-21 days with a half-life of 14.44-15.75 days in Rabi and 13.33-14.44 days in Kharif at respective doses. At harvest chlorantraniliprole residues were not detected in straw, husk, and brown rice. The dietary risk of paddy leaf (green fodder) for cattle was found safe for consumption as the hazard index is less than one. Soil ecological risk assessment was found to be less than one (RQ < 0.1) for earthworms (Eisenia foetida) and arthropods (Aphidiusrhopalosiphi). The presentmethod could be useful inthe analysis ofchlorantraniliproleresidues in different cereals and vegetable crop ecosystems and application at recommended dose is safe for the final produce at harvest.
Assuntos
Oryza , Resíduos de Praguicidas , Poluentes do Solo , Animais , Bovinos , Cromatografia Líquida , Ecossistema , Espectrometria de Massas em Tandem , Resíduos de Praguicidas/análise , Meia-Vida , Solo/química , Oryza/química , Poluentes do Solo/análiseRESUMO
Norovirus (NoV) belongs to the Calciviridae family that causes diarrhoea, vomiting, and stomach pain in people who have acute gastroenteritis (AGE). Identifying multi-epitope dependent vaccines for single stranded positive sense viruses such as NoV has been a long due. Although efforts have been in place to look into the candidate epitopes, understanding molecular mimicry and finding new epitopes for inducing immune responses against the T/B-cells which play an important role for the cell-mediated and humoral immunity was not dealt with in great detail. The current study focuses on identifying new epitopes from various databases that were filtered for antigenicity, allergenicity, and toxicity. The adjuvant ß-defensin along with different linkers were used for vaccine construction. Further, the binding relationship between the vaccine construct and toll-like immune receptor (TLR3) complex was determined using a molecular docking analysis, followed by molecular dynamics simulation of 100 ns. The vaccine candidate developed expresses good solubility with a score of 0.530, Z-score of -4.39 and molecular docking score of -140.4 ± 12.1. The MD trajectories reveal that there is a stability between TLR3 and the developed vaccine candidate with an average of 0.91 nm RMSD value and also the system highest occupancy H-bond formed between GLU127 of TLR3 and TYR10 of vaccine candidate (61.55%). Four more H-bonds exist with an occupancy of more than 32% between TLR3 and the vaccine candidates which makes it stable. Thus, the multi-epitope based vaccine developed in the present study forms the basis for further experimental investigations to develop a potentially good vaccine against NoV.Communicated by Ramaswamy H. Sarma.