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Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.
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Descubrimiento de Drogas , Resonancia por Plasmón de Superficie , Resonancia por Plasmón de Superficie/métodos , Descubrimiento de Drogas/métodos , Proteínas , Cromatografía de Afinidad , Unión ProteicaRESUMEN
The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.
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Inmunoterapia Adoptiva , Anticuerpos de Cadena Única , Animales , Humanos , Inmunoterapia Adoptiva/métodos , Ligandos , Línea Celular Tumoral , Células Jurkat , Anticuerpos de Cadena Única/genética , Mamíferos/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismoRESUMEN
The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time- and dose-dependent downregulation of a proximal biomarker could be shown.
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Neoplasias , Factores de Transcripción , Animales , Ratones , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismoRESUMEN
Excess mortality not directly related to the virus has been shown to have increased during the COVID-19 pandemic. However, changes in heat-related mortality during the pandemic have not been addressed in detail. Here, we performed an observational study crossing daily mortality data collected in Portugal (SICO/DGS) with high-resolution temperature series (ERA5/ECMWF), characterizing their relation in the pre-pandemic, and how it aggravated during 2020. The combined result of COVID-19 and extreme temperatures caused the largest annual mortality burden in recent decades (~ 12 000 excess deaths [~ 11% above baseline]). COVID-19 caused the largest fraction of excess mortality during March to May (62%) and from October onwards (85%). During summer, its direct impact was residual, and deaths not reported as COVID-19 dominated excess mortality (553 versus 3 968). A prolonged hot spell led mortality to the upper tertile, reaching its peak in mid-July (+ 45% deaths/day). The lethality ratio (+ 14 deaths per cumulated ºC) was higher than that observed in recent heatwaves. We used a statistical model to estimate expected deaths due to cold/heat, indicating an amplification of at least 50% in heat-related deaths during 2020 compared to pre-pandemic years. Our findings suggest mortality during 2020 has been indirectly amplified by the COVID-19 pandemic, due to the disruption of healthcare systems and fear of population in attending healthcare facilities (expressed in emergency room admissions decreases). While lockdown measures and healthcare systems reorganization prevented deaths directly related to the virus, a significant burden due to other causes represents a strong secondary impact. This was particularly relevant during summer hot spells, when the lethality ratio reached magnitudes not experienced since the 2003 heatwaves. This severe amplification of heat-related mortality during 2020 stresses the need to resume normal healthcare services and public health awareness.
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COVID-19 , Pandemias , Control de Enfermedades Transmisibles , Calor , Humanos , Mortalidad , SARS-CoV-2RESUMEN
Humans have made such dramatic and permanent changes to Earth's landscapes that much of it is now substantially and irreversibly altered from its preanthropogenic state. Remote islands, until recently isolated from humans, offer insights into how these landscapes evolved in response to human-induced perturbations. However, little is known about when and how remote systems were colonized because archaeological data and historical records are scarce and incomplete. Here, we use a multiproxy approach to reconstruct the initial colonization and subsequent environmental impacts on the Azores Archipelago. Our reconstructions provide unambiguous evidence for widespread human disturbance of this archipelago starting between 700-60+50 and 850-60+60 Common Era (CE), ca. 700 y earlier than historical records suggest the onset of Portuguese settlement of the islands. Settlement proceeded in three phases, during which human pressure on the terrestrial and aquatic ecosystems grew steadily (i.e., through livestock introductions, logging, and fire), resulting in irreversible changes. Our climate models suggest that the initial colonization at the end of the early Middle Ages (500 to 900 CE) occurred in conjunction with anomalous northeasterly winds and warmer Northern Hemisphere temperatures. These climate conditions likely inhibited exploration from southern Europe and facilitated human settlers from the northeast Atlantic. These results are consistent with recent archaeological and genetic data suggesting that the Norse were most likely the earliest settlers on the islands.
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Ecosistema , Ambiente , Actividades Humanas , Migración Humana , Agricultura , Azores , Cambio Climático , Modelos Climáticos , Heces/química , HumanosRESUMEN
The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.
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We report the case of a 65-year-old female with an atypical presentation of renal lymphoma at computed tomography (CT), which was initially misinterpreted as a retroperitoneal hematoma. This case highlights the importance to keep a high level of suspicion in order to make a prompt diagnosis since treatment strategies differ significantly.
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Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.
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Neoplasias de la Mama , Proteínas de Unión al Calcio/inmunología , Técnicas de Visualización de Superficie Celular , Inmunoglobulina G/biosíntesis , Proteínas de la Membrana/inmunología , Femenino , Humanos , Ligandos , Células MCF-7RESUMEN
Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
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Fenilalanina Hidroxilasa/metabolismo , Quinolonas/química , Quinolonas/farmacología , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Fluorometría , Células HEK293 , Humanos , Enfermedades Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonurias/metabolismo , Resonancia por Plasmón de Superficie , TripsinaRESUMEN
This is the first attempt to apply an expert-based ecological vulnerability assessment of the effects of climate change on the main marine resources of Portugal. The vulnerability, exposure, sensitivity, adaptive capacity, and expected directional effects of 74 species of fish and invertebrates of commercial interest is estimated based on criteria related to their life-history and level of conservation or exploitation. This analysis is performed separately for three regions of Portugal and two scenarios of climate change (RCP 4.5 and RCP 8.5). To do that, the fourth assessment report IPCC framework for vulnerability assessments was coupled to the outputs of a physical-biogeochemical model allowing to weight the exposure of the species by the expected variability of the environmental variables in the future. The highest vulnerabilities were found for some migratory and elasmobranch species, although overall vulnerability scores were low probably due to the high adaptive capacity of species from temperate ecosystems. Among regions, the highest average vulnerability was estimated for the species in the Central region while higher vulnerabilities were identified under climate change scenario RCP 8.5 in the three regions, due to higher expected climatic variability. This work establishes the basis for the assessment of the vulnerability of the human activities relying on marine resources in the context of climate change.
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The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.
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ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/biosíntesis , Adenosina Trifosfato/metabolismo , Proteínas del Ojo/metabolismo , Técnicas de Inactivación de Genes , Genes Reporteros , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteómica/métodos , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.
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The genus Artemisia, often known collectively as "wormwood", has aroused great interest in the scientific community, pharmaceutical and food industries, generating many studies on the most varied aspects of these plants. In this review, the most recent evidence on health effects of edible Artemisia species and some of its constituents are presented and discussed, based on studies published until 2020, available in the Scopus, Web of Sciences and PubMed databases, related to food applications, nutritional and sesquiterpene lactones composition, and their therapeutic effects supported by in vivo and clinical studies. The analysis of more than 300 selected articles highlights the beneficial effect on health and the high clinical relevance of several Artemisia species besides some sesquiterpene lactones constituents and their derivatives. From an integrated perspective, as it includes therapeutic and nutritional properties, without ignoring some adverse effects described in the literature, this review shows the great potential of Artemisia plants and some of their constituents as dietary supplements, functional foods and as the source of new, more efficient, and safe medicines. Despite all the benefits demonstrated, some gaps need to be filled, mainly related to the use of raw Artemisia extracts, such as its standardization and clinical trials on adverse effects and its health care efficacy.
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Macroalgae are increasingly viewed as a source of secondary metabolites with great potential for the development of new drugs. In this development, in vitro studies are only the first step in a long process, while in vivo studies and clinical trials are the most revealing stages of the true potential and limitations that a given metabolite may have as a new drug. This literature review aims to give a critical overview of the secondary metabolites that reveal the most interesting results in these two steps. Phlorotannins show great pharmaceutical potential in in vivo models and, among the several examples, the anti-dyslipidemia activity of dieckol must be highlighted because it was more effective than lovastatin in an in vivo model. The IRLIIVLMPILMA tridecapeptide that exhibits an in vivo level of activity similar to the hypotensive clinical drug captopril should still be stressed, as well as griffithsin which showed such stunning results over a variety of animal models and which will probably move onto clinical trials soon. Regarding clinical trials, studies with pure algal metabolites are scarce, limited to those carried out with kahalalide F and fucoxanthin. The majority of clinical trials currently aim to ascertain the effect of algae consumption, as extracts or fractions, on obesity and diabetes.
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Péptidos/farmacología , Fenoles/farmacología , Algas Marinas/química , Animales , Fármacos Antiobesidad , Antihipertensivos , Antioxidantes , Benzofuranos , Humanos , Péptidos/uso terapéutico , Fenoles/uso terapéutico , Algas Marinas/metabolismo , Estigmasterol/análogos & derivados , Estigmasterol/farmacología , Estigmasterol/uso terapéutico , Xantófilas/farmacología , Xantófilas/uso terapéuticoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.
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Fenilalanina Hidroxilasa/metabolismo , Fenilalanina Hidroxilasa/ultraestructura , Dominio Catalítico , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fenilalanina/metabolismo , Fenilcetonurias/genética , Fenilcetonurias/fisiopatología , Unión Proteica , Conformación Proteica , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Rayos XRESUMEN
Biosynthesis of hydrogen sulfide (H2S), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.
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Cistationina betasintasa/antagonistas & inhibidores , Cistationina gamma-Liasa/antagonistas & inhibidores , Sulfuro de Hidrógeno/metabolismo , Piridinas/farmacología , Sulfurtransferasas/antagonistas & inhibidores , Dicroismo Circular , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fluorometría/métodos , Humanos , Azul de Metileno , Piridinas/química , Sulfurtransferasas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Global temperatures have increased considerably over the last decades, directly impacting the number, intensity and duration of extreme events such as heat waves. Climate model projections accounting for anthropogenic factors indicate that deadly mega-heat waves are likely to become more frequent in the future. Although the atmospheric features and social-economic related impacts of heat waves have already been documented in various regions around the world, for other highly populated regions, such as the Metropolitan Region of Rio de Janeiro (MRRJ), a similar objective assessment is still needed. Heat waves directly impact the public health sector and particularly the less wealthy and elderly population groups. During February 2010, an elevated mortality peak occurred during a 8-day period (from 2 to 9 Feb 2010) characterized as a heat wave episode in MRRJ. A total excess of 737 deaths was recorded with the elderly group registering the highest mortality incidence. During this heat wave period, a quasi-stationary anticyclonic anomaly forced in altitude by a Rossby wave train was established over the south Brazilian coast. At the surface, the meteorological scenario from January 2010 to the heat wave period was marked by clear sky conditions, large precipitation deficits, and enhanced diabatic heating. During the heat wave period, warm and dry air masses were advected from interior regions towards the MRRJ, exacerbating temperature conditions by pronounced subsidence and adiabatic heating mechanisms. All these conditions contributed to pronounced positive temperature anomalies, reinforced by land-atmosphere feedbacks.
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Atmósfera/química , Clima , Exposición a Riesgos Ambientales/estadística & datos numéricos , Calor , Mortalidad/tendencias , Anciano , Brasil , Cambio Climático , Humanos , Incidencia , Salud PúblicaRESUMEN
The respiratory chain of some prokaryotes was shown to be organized in supercomplexes. This association has been proposed to improve enzyme stability and the overall efficiency of the oxidative phosphorylation process. Here, we have revisited recent data on the supercomplexes of Bacillus subtilis respiratory chain, by means of 1D and 2D-BN-PAGE, sucrose gradient fractionation of solubilized membranes, and mass spectrometry analysis of BN-PAGE bands detected in gel for succinate and cytochrome c oxidoreductase activities. The cytochrome bc:caa3 oxygen oxidoreductase supercomplex was observed in different stoichiometries, (bc)4:(caa3)2, (bc)2:(caa3)4 and 2[(bc)2:(caa3)4], suggesting for the first time the string association model of supercomplexes in a Gram positive bacterium. In addition, the presence of a succinate:quinone oxidoreductase:nitrate reductase supercomplex was confirmed by the co-localized succinate:nitroblue tetrazolium and methylviologen:nitrate oxidoreductase activities detected in gel and corroborated by LC-MS/MS analysis.