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By incorporating full flexibility and enabling the quantification of crucial parameters such as binding free energies and residence times, methods for investigating protein-ligand binding and unbinding via molecular dynamics provide details on the involved mechanisms at the molecular level. While these advancements hold promise for impacting drug discovery, a notable drawback persists: their relatively time-consuming nature limits throughput. Herein, we survey recent implementations which, employing a blend of enhanced sampling techniques, a clever choice of collective variables, and often machine learning, strive to enhance the efficiency of new and previously reported methods without compromising accuracy. Particularly noteworthy is the validation of these methods that was often performed on systems mirroring real-world drug discovery scenarios.
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Simulación de Dinámica Molecular , Unión Proteica , Proteínas , Ligandos , Proteínas/metabolismo , Proteínas/química , Descubrimiento de Drogas/métodos , Aprendizaje Automático , HumanosRESUMEN
The design and synthesis of a series of 2,7-diazaspiro[4.4]nonane derivatives as potent sigma receptor (SR) ligands, associated with analgesic activity, are the focus of this work. In this study, affinities at S1R and S2R were measured, and molecular modeling studies were performed to investigate the binding pose characteristics. The most promising compounds were subjected to in vitro toxicity testing and subsequently screened for in vivo analgesic properties. Compound 9d (AD258) exhibited negligible in vitro cellular toxicity and a high binding affinity to both SRs (KiS1R = 3.5 nM, KiS2R = 2.6 nM), but not for other pain-related targets, and exerted high potency in a model of capsaicin-induced allodynia, reaching the maximum antiallodynic effect at very low doses (0.6-1.25 mg/kg). Functional activity experiments showed that S1R antagonism is needed for the effects of 9d and that it did not induce motor impairment. In addition, 9d exhibited a favorable pharmacokinetic profile.
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Receptores sigma , Humanos , Ligandos , Receptores sigma/metabolismo , Unión Proteica , Dolor , Analgésicos/farmacología , Analgésicos/uso terapéuticoRESUMEN
How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.
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BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
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ADN Ligasa (ATP) , Reparación del ADN , Flavonoides , Mieloma Múltiple , Animales , Ratones , Anexina A5/genética , Anexina A5/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , ADN Ligasas/química , ADN Ligasas/genética , ADN Ligasas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Flavonoides/farmacología , Flavonoides/uso terapéutico , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Fenoles , Proteínas Recombinantes/metabolismoRESUMEN
Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.
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Cápsulas Bacterianas/enzimología , Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/enzimología , Cápsulas Bacterianas/genética , Proteínas Bacterianas/química , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/metabolismo , Metabolismo de los Hidratos de Carbono , Heptosas/biosíntesis , Polisacáridos/biosíntesis , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Fosfato de Piridoxal/metabolismoRESUMEN
ELAV-like (ELAVL) RNA-binding proteins play a pivotal role in post-transcriptional processes, and their dysregulation is involved in several pathologies. This work was focused on HuD (ELAVL4), which is specifically expressed in nervous tissues, and involved in differentiation and synaptic plasticity mechanisms. HuD represents a new, albeit unexplored, candidate target for the treatment of several relevant neurodegenerative diseases. The aim of this pioneering work was the identification of new molecules able to recognize and bind HuD, thus interfering with its activity. We combined virtual screening, molecular dynamics (MD), and STD-NMR techniques. Starting from around 51â¯000 compounds, four promising hits eventually provided experimental evidence of their ability to bind HuD. Among the selected best hits, folic acid was found to be the most interesting one, being able to well recognize the HuD binding site. Our results provide a basis for the identification of new HuD interfering compounds which may be useful against neurodegenerative syndromes.
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Proteína 4 Similar a ELAV/antagonistas & inhibidores , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Proteína 4 Similar a ELAV/metabolismo , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Relación Estructura-ActividadRESUMEN
l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.
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Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Asparaginasa/química , Asparaginasa/metabolismo , Asparaginasa/uso terapéutico , Asparagina/metabolismo , Niño , Preescolar , Glutaminasa/metabolismo , Humanos , Oncología Médica/métodos , Oncología Médica/normas , Oncología Médica/tendencias , Modelos Moleculares , Polietilenglicoles/química , Polietilenglicoles/uso terapéutico , Conformación Proteica , Mejoramiento de la Calidad , Adulto JovenRESUMEN
The mechanisms of inflammation and cancer are intertwined by complex networks of signaling pathways. Dysregulations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway underlie several pathogenic conditions related to chronic inflammatory states, autoimmune diseases and cancer. Historically, the potential application of JAK inhibition has been thoroughly explored, thus triggering an escalation of favorable results in this field. So far, five JAK inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of different diseases. Considering the complexity of JAK-depending processes and their involvement in multiple disorders, JAK inhibitors are the perfect candidates for drug repurposing and for the assessment of multitarget strategies. Herein we reviewed the recent progress concerning JAK inhibition, including the innovations provided by the release of JAKs crystal structures and the improvement of synthetic strategies aimed to simplify of the industrial scale-up.
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Antiinflamatorios/síntesis química , Antineoplásicos/síntesis química , Inhibidores de las Cinasas Janus/síntesis química , Quinasas Janus/antagonistas & inhibidores , Piperidinas/química , Pirazoles/química , Pirimidinas/química , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Aprobación de Drogas , Diseño de Fármacos , Humanos , Inflamación/tratamiento farmacológico , Inhibidores de las Cinasas Janus/farmacología , Neoplasias/tratamiento farmacológico , Nitrilos , Piperidinas/farmacología , Unión Proteica , Conformación Proteica , Pirazoles/farmacología , Pirimidinas/farmacología , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Inflammatory states are among the most common and most treated medical conditions. Inflammation comes along with swelling, pain and uneasiness in using the affected area. Inflammation is not always a simple symptom; more often is part of a defensive response of the body to an external threat or is a sign that the damaged tissue has not healed yet and needs to rest. The management of the pain associated with an inflammatory state could be a tricky task. In fact, most remedies simply quench the pain, leaving the inflammatory state unaltered. This review focuses on sesquiterpene lactones, a class of natural compounds, that represents a future promise in the treatment of inflammation. Sesquiterpene lactones are efficient inhibitors of multiple targets of the inflammatory process. Their natural sources are often ancient remedies with relevant traditional uses in folk medicines. This work also aims to elucidate how these compounds may represent the starting material for the development of new anti-inflammatory drugs.
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Inflamación , Antiinflamatorios , Edema , Humanos , Lactonas , FN-kappa B , Fitoquímicos , SesquiterpenosRESUMEN
[This corrects the article DOI: 10.1038/s42003-019-0587-z.].
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Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Carotenoides/metabolismo , Crocus/metabolismo , Proteínas de Plantas/metabolismo , Vacuolas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Vías Biosintéticas , Clonación Molecular , Crocus/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Cinética , Extractos Vegetales , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Distribución Tisular/fisiología , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.
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Artemisinin (ART) is an endoperoxide sesquiterpene lactone, commonly used in the treatment of malaria. Although it was isolated from Artemisia annuaL., a plant widely applied in Chinese Traditional Medicine, its mechanism of action remains uncertain and its clinical use is still limited due to its low solubility, its poor bioavailability and short in vivo half-life. Over time, several studies have been aimed towards the discovery of potent ART derivatives that could overcome clinical drawbacks. In this review, we focus on the multifaced aspects of ART and on the efforts spent to improve its pharmacological profile that so far culminated in the discovery of more effective drugs. Lastly, we outline the new perspectives in the ART-derivatives scenario.
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Antimaláricos/síntesis química , Artemisia/química , Artemisininas/síntesis química , Malaria/tratamiento farmacológico , Medicina Tradicional China , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/uso terapéutico , Artemisininas/química , Artemisininas/aislamiento & purificación , Artemisininas/uso terapéutico , Humanos , Estructura MolecularRESUMEN
Sesquiterpene compounds are widely known for their numerous pharmacological activities. Herein the focus of the authors was on α-Santonin, a sesquiterpene lactone from the Artemisia genus: the aim was to determine whether α-Santonin could be considered in the treatment of inflammation and pain. To this purpose, a small series of derivatives was designed and screened in silico against the enzyme COX-2 along with the parent compound. Drug-likeness parameters were also assessed. The compounds were eventually synthesized, and few were tested to determine their efficacy in the inhibition of COX-2 activity and expression. Overall, compound A2 was the only one with a detectable inhibitory potential of COX-2 activity whilst two of its ether derivatives demonstrated improved ability in the inhibition of COX-2 expression.