Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

Giant supermagnonic Bloch point velocities in cylindrical ferromagnetic nanowires.

Achieving high velocities of magnetic domain walls is a crucial factor for their use as information carriers in modern nanoelectronic applications. In nanomagnetism and spintronics, these velocities are often limited either by internal domain wall instabilities, known as the Walker breakdown phenomenon, or by spin wave emission, known as the magnonic regime. In the rigid domain wall model, the maximum magnon velocity acts as an effective "speed of light", providing a relativistic analogy for the domain wall speed limitation. Cylindrical magnetic nanowires are an example of systems without the Walker breakdown phenomenon. Here we demonstrate that the magnonic limit could be outstandingly surpassed in cylindrical nanowires with high magnetization, such as iron. Our numerical modeling shows the Bloch point domain wall velocities as high as 14 km s-1, well above the magnonic limit estimated in the interval 1.7-2.0 km s-1. The key ingredient is the three-dimensional conical shape of the domain wall, which elongates and breaks during the dynamics, expelling backwards pairs of Bloch points. This leads to domain wall acceleration, the effect, which resembles the "jet propulsion". This effect will be very important for three-dimensional networks based on cylindrical magnetic nanowires.

Size-Dependence and High Temperature Stability of Radial Vortex Magnetic Textures Imprinted by Superconductor Stray Fields.

Swirling spin textures, including topologically nontrivial states, such as skyrmions, chiral domain walls, and magnetic vortices, have garnered significant attention within the scientific community due to their appeal from both fundamental and applied points of view. However, their creation, controlled manipulation, and stability are typically constrained to certain systems with specific crystallographic symmetries, bulk or interface interactions, and/or a precise stacking sequence of materials. Recently, a new approach has shown potential for the imprint of magnetic radial vortices in soft ferromagnetic compounds making use of the stray field of YBa2Cu3O7-δ superconducting microstructures in ferromagnet/superconductor (FM/SC) hybrids at temperatures below the superconducting transition temperature (TC). Here, we explore the lower size limit for the imprint of magnetic radial vortices in square and disc shaped structures as well as the persistence of these spin textures above TC, with magnetic domains retaining partial memory. Structures with circular geometry and with FM patterned to smaller radius than the superconductor island facilitate the imprinting of magnetic radial vortices and improve their stability above TC, in contrast to square structures where the presence of magnetic domains increases the dipolar energy. Micromagnetic modeling coupled with a SC field model reveals that the stabilization mechanism above TC is mediated by microstructural defects. Superconducting control of swirling spin textures, and their stabilization above the superconducting transition temperature by means of defect engineering holds promising prospects for shaping superconducting spintronics based on magnetic textures.

Three-dimensional magnetic nanotextures with high-order vorticity in soft magnetic wireframes.

Additive nanotechnology enable curvilinear and three-dimensional (3D) magnetic architectures with tunable topology and functionalities surpassing their planar counterparts. Here, we experimentally reveal that 3D soft magnetic wireframe structures resemble compact manifolds and accommodate magnetic textures of high order vorticity determined by the Euler characteristic, χ. We demonstrate that self-standing magnetic tetrapods (homeomorphic to a sphere; χ = + 2) support six surface topological solitons, namely four vortices and two antivortices, with a total vorticity of + 2 equal to its Euler characteristic. Alternatively, wireframe structures with one loop (homeomorphic to a torus; χ = 0) possess equal number of vortices and antivortices, which is relevant for spin-wave splitters and 3D magnonics. Subsequent introduction of n holes into the wireframe geometry (homeomorphic to an n-torus; χ < 0) enables the accommodation of a virtually unlimited number of antivortices, which suggests their usefulness for non-conventional (e.g., reservoir) computation. Furthermore, complex stray-field topologies around these objects are of interest for superconducting electronics, particle trapping and biomedical applications.

The Discovery of inhibitors of the SARS-CoV-2 S protein through computational drug repurposing.

SARS-CoV-2 must bind its principal receptor, ACE2, on the target cell to initiate infection. This interaction is largely driven by the receptor binding domain (RBD) of the viral Spike (S) protein. Accordingly, antiviral compounds that can block RBD/ACE2 interactions can constitute promising antiviral agents. To identify such molecules, we performed a virtual screening of the Selleck FDA approved drugs and the Selleck database of Natural Products using a multistep computational procedure. An initial set of candidates was identified from an ensemble docking process using representative structures determined from the analysis of four 3 µ s molecular dynamics trajectories of the RBD/ACE2 complex. Two procedures were used to construct an initial set of candidates including a standard and a pharmacophore guided docking procedure. The initial set was subsequently subjected to a multistep sieving process to reduce the number of candidates to be tested experimentally, using increasingly demanding computational procedures, including the calculation of the binding free energy computed using the MMPBSA and MMGBSA methods. After the sieving process, a final list of 10 candidates was proposed, compounds which were subsequently purchased and tested ex-vivo. The results identified estradiol cypionate and telmisartan as inhibitors of SARS-CoV-2 entry into cells. Our findings demonstrate that the methodology presented here enables the discovery of inhibitors targeting viruses for which high-resolution structures are available.

[Quality in medicine as a deontological requirement]. / La calidad en medicina como obligación deontológica.

Medical practice has two main dimensions, one scientific and the other human, and in both, the commitment of the doctor is essential. Medical deontology, as a branch of ethics that deals with the duties and obligations of doctors, embodies in the codes of deontology the ethical principles and standards of mandatory compliance. From this point, medical practice should guarantee that patients receive an quality medical care, and to be treated with dignity and respect. In December 2022, the General Council of Official Medical Associations of Spain published a new version of the Spanish Code of Medical Ethics that aims, among other purposes, to standardize medical care of scientific and human quality. Medical practice supported by scientifically-based medicine, lex artis medica, patient safety, professional responsibility, honesty and integrity are some of the aspects that in this updated document of medical ethics frame and define the work of the doctor for a performance of medicine with quality.

Investigation of Zebrafish Embryo Membranes at Epiboly Stage through Electrorotation Technique.

A preliminary exploration of the physiology and morphology of the zebrafish embryo (ZFE) during the late-blastula and early-gastrula stages through its electrical properties was performed, applying the electrorotation (ROT) technique. This method, based on induced polarizability at the interfaces, was combined with an analytical spherical shell model to obtain the best fit of empirical data and the desired information, providing a means of understanding the role of different membranes. Suspended in two solutions of low conductivity, the major compartments of the ZFE were electrically characterized, considering morphological data from both observed records and data from the literature. Membrane integrity was also analyzed for dead embryos. The low permeability and relatively high permittivity obtained for the chorion probably reflected both its structural characteristics and external conditions. Reasonable values were derived for perivitelline fluid according to the influx of water that occurs after the fertilization of the oocyte. The so-called yolk membrane, which comprises three different and contiguous layers at the epiboly stage, showed atypical electrical values of the membrane, as did the yolk core with a relatively low permittivity. The internal morphological complexity of the embryo itself could be addressed in future studies by developing an accurate geometric model.

Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

La calidad en medicina como obligación deontológica / Quality in medicine as a deontological requirement

La práctica médica tiene dos dimensiones fundamentales una la científica, y otra la humana, y en ambas es esencial el compromiso del médico. La deontología médica, como rama de la ética que se ocupa de los deberes y obligaciones de los médicos, plasma en los códigos de deontología los principios y están dares éticos de obligado cumplimiento, a fin de que la práctica médica pueda garantizar que los pacientes reciban una atención médica de calidad, y que se les trate con dignidad y respeto. En diciembre del año 2022 el Consejo General de Colegios Oficiales de Médicos de España publicó una nueva versión del Código de Deontología Médica español que pretende, entre otros fines, normativizar una asistencia médica de calidad científica y humana. Práctica médica sustentada en medicina con base científica, lex artis médica, seguridad del paciente, responsabilidad profesional, honestidad e integridad son algunos de los aspectos que en este documento actualizado de deontología médica enmarcan y define el quehacer del médico para un desempeño de medicina con calidad (AU)

Medical practice has two main dimensions, one scientific and the other human, and in both, the com mitment of the doctor is essential. Medical deontology, as a branch of ethics that deals with the duties and obligations of doctors, embodies in the codes of deontology the ethical principles and standards of mandatory compliance. From this point, medical practice should guarantee that patients receive an quality medical care, and to be treated with dignity and respect. In December 2022, the General Council of Official Medical Associations of Spain published a new version of the Spanish Code of Medical Ethics that aims, among other purposes, to standardize medical care of scientific and human quality. Medical practice sup ported by scientifically-based medicine, lex artis médica, patient safety, professional responsibility, honesty and integrity are some of the aspects that in this updated document of medical ethics frame and define the work of the doctor for a performance of medicine with quality (AU)

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

Discovery and Rational Mutagenesis of Methionine Sulfoxide Reductase Biocatalysts To Expand the Substrate Scope of the Kinetic Resolution of Chiral Sulfoxides.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.

Autochthonous Peruvian Natural Plants as Potential SARS-CoV-2 Mpro Main Protease Inhibitors.

Over 750 million cases of COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), have been reported since the onset of the global outbreak. The need for effective treatments has spurred intensive research for therapeutic agents based on pharmaceutical repositioning or natural products. In light of prior studies asserting the bioactivity of natural compounds of the autochthonous Peruvian flora, the present study focuses on the identification SARS-CoV-2 Mpro main protease dimer inhibitors. To this end, a target-based virtual screening was performed over a representative set of Peruvian flora-derived natural compounds. The best poses obtained from the ensemble molecular docking process were selected. These structures were subjected to extensive molecular dynamics steps for the computation of binding free energies along the trajectory and evaluation of the stability of the complexes. The compounds exhibiting the best free energy behaviors were selected for in vitro testing, confirming the inhibitory activity of Hyperoside against Mpro, with a Ki value lower than 20 µM, presumably through allosteric modulation.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

Spatial magnetic imaging of non-axially symmetric vortex domains in cylindrical nanowire by transmission X-ray microscopy.

The spatial magnetization texture of a cylindrical nanowire has been determined by Transmission X-ray Microscopy (TXM) and X-ray magnetic circular dichroism (XMCD). For this purpose, nanowires with designed geometry, consisting of CoNi/Ni periodic segments, have been grown by designed electrodeposition into alumina templates. Experimental data allow one to conclude the presence of mono- and trivortex magnetic domains in CoNi segments but, unusually, these states are characterized by an asymmetric XMCD contrast across the nanowire's section. Micromagnetic modelling shows non-trivial three-dimensional structures with ellipsoidal vortex cores and non-axially symmetric magnetization along the nanowire direction. The modelled TXM contrast of micromagnetic structures allows to correlate the experimental asymmetric XMCD contrast to the easy axis direction of the uniaxial magnetocrystalline anisotropy.

Establishing the selective phospholipid membrane coordination, permeation and lysis properties for a series of 'druggable' supramolecular self-associating antimicrobial amphiphiles.

The rise of antimicrobial resistance remains one of the greatest global health threats facing humanity. Furthermore, the development of novel antibiotics has all but ground to a halt due to a collision of intersectional pressures. Herein we determine the antimicrobial efficacy for 14 structurally related supramolecular self-associating amphiphiles against clinically relevant Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Escherichia coli. We establish the ability of these agents to selectively target phospholipid membranes of differing compositions, through a combination of computational host:guest complex formation simulations, synthetic vesicle lysis, adhesion and membrane fluidity experiments, alongside our novel 1H NMR CPMG nanodisc coordination assays, to verify a potential mode of action for this class of compounds and enable the production of evermore effective next-generation antimicrobial agents. Finally, we select a 7-compound subset, showing two lead compounds to exhibit 'druggable' profiles through completion of a variety of in vivo and in vitro DMPK studies.

A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels.

The chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl- efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl- efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl- efflux.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.

Distinguishing Local Demagnetization Contribution to the Magnetization Process in Multisegmented Nanowires.

Cylindrical magnetic nanowires are promising materials that have the potential to be used in a wide range of applications. The versatility of these nanostructures is based on the tunability of their magnetic properties, which is achieved by appropriately selecting their composition and morphology. In addition, stochastic behavior has attracted attention in the development of neuromorphic devices relying on probabilistic magnetization switching. Here, we present a study of the magnetization reversal process in multisegmented CoNi/Cu nanowires. Nonstandard 2D magnetic maps, recorded under an in-plane magnetic field, produce datasets that correlate with magnetoresistance measurements and micromagnetic simulations. From this process, the contribution of the individual segments to the demagnetization process can be distinguished. The results show that the magnetization reversal in these nanowires does not occur through a single Barkhausen jump, but rather by multistep switching, as individual CoNi segments in the NW undergo a magnetization reversal. The existence of vortex states is confirmed by their footprint in the magnetoresistance and 2D MFM maps. In addition, the stochasticity of the magnetization reversal is analysed. On the one hand, we observe different switching fields among the segments due to a slight variation in geometrical parameters or magnetic anisotropy. On the other hand, the stochasticity is observed in a series of repetitions of the magnetization reversal processes for the same NW under the same conditions.

Cross-cultural adaptation and psychometric validation of a Spanish version of the revised Patients' Attitudes Towards Deprescribing (rPATD) questionnaire.

OBJECTIVES: Successful deprescribing depends largely on factors related to the patient. The revised Patients' Attitudes Towards Deprescribing (rPATD) questionnaire was developed with the objective of evaluating the beliefs and attitudes of older adults and caregivers towards deprescribing. The present study was designed to validate a Spanish version of the rPATD questionnaire, both the versions for older adults and for caregivers, through a qualitative validation phase and the analysis of its psychometric properties. DESIGN: Cross-sectional validation study. SETTING: Primary care settings in Málaga (Spain). PARTICIPANTS: A sample of 120 subjects (60 patients with polypharmacy and 60 caregivers of patients with polypharmacy) were enrolled in the study. MAIN OUTCOME MEASURES: In the qualitative validation stage, the rPATD questionnaire was translated/back-translated and subjected to a cross-cultural adaptation to evaluate its face validity and feasibility. Next, its psychometric properties were assessed. Confirmatory factor analysis was used to evaluate construct validity. Internal consistency was determined using Cronbach's alpha test. Criterion validity through pre-established hypotheses from the Beliefs about Medicines Questionnaire (BMQ) Specific-Concerns Scale, and test-retest reliability were analysed. RESULTS: Confirmatory factor analysis verified the four-factor structure of the original rPATD questionnaire, with items loading into four factors: involvement, burden, appropriateness and concerns about stopping. The Cronbach's alpha coefficient of the factors ranged from 0.683 to 0.879. The burden, appropriateness and concerns about stopping factors were significantly correlated with the BMQ Specific-Concerns Score, except for the concerns about stopping factor in the older adults' version. The consistency of the items between administration times (test-retest reliability) showed weighted Cohen's kappa values ranging from moderate (>0.4) to very good (>0.8). CONCLUSIONS: The Spanish version of the rPATD questionnaire is a feasible, valid and reliable instrument to evaluate attitudes towards deprescribing in Spanish-speaking patients and caregivers.