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Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasomedependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.

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.

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.

SARS-CoV-2 can activate the inflammasome, which, when unbridled, contributes to pathogenic inflammatory responses and to severe COVID-19. Several SARS-CoV-2 components have been shown to participate in this activation. Here, we have systematically assayed SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7,ORF8, ORF9b, ORF9c and ORF10) for their ability to modulate inflammasome activity. We have found that among all accessory proteins, only ORF9b, a protein that locates in mitochondria, triggers a strong activation of caspase-1 activity and cytokine release in A549 lung epithelial cells and THP-1 monocyte-macrophage cells. This induction is observed both by transducing ORF9b alone or upon concomitant transduction of all accessory proteins. Based on the solved structure of ORF9b, we have conducted an in silico drug discovery effort to identify small molecules capable of disrupting the ORF9b homodimer and to attenuate its observed activity. Iterative steps of blind massive docking and molecular dynamics led to the identification of small molecules predicted to prevent ORF9b homodimeric interactions. This prediction was experimentally validated by means of surface plasmon resonance, yielding two active molecules. These molecules showed a potent inhibition of ORF9b-induced caspase-1 activation and cytokine release, and caused a remarkable eviction of ORF9b from mitochondria. These novel first-in-class ORF9b inhibitors are currently being tested for their ability to mitigate viral cytopathogenic effects.

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of COVID-19. However, the impacts of individual accessory proteins on host cell metabolic pathways are unknown.Here, SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 were individually transduced into A549 lung carcinoma cells. Furthermore, by combining transcriptomic analysis with functional and metabolic data in accessory protein-specific GSMMs, several alterations were identified that may point to a putative target for investigating novel therapies. In this study, we showed that these accessory proteins induced a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical role in mitochondria function and morphology. On the other hand, while all four ORFs altered mitochondrial dynamics and function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features shared across all accessory proteins and specific ones for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. Next, qMTA identified gene knock downs (KDs) that would have the potential to revert the metabolic reprogramming induced by each individual accessory protein, especially in ORF3a and ORF10. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

[Background]: Immune check-point blockade (ICB) has shown clinical beneft in mismatch repair-defcient/microsatellite instability high metastatic colorectal cancer (mCRC) but not in mismatch repair-profcient/microsatellite stable patients. Cancer vaccines with autologous dendritic cells (ADC) could be a complementary therapeutic approach to ICB as this combination has the potential to achieve synergistic efects. [Methods]: This was a Phase I/II multicentric study with translational sub-studies, to evaluate the safety, pharmacodynamics and anti-tumor efects of Avelumab plus ADC vaccine in heavily pre-treated MSS mCRC patients. Primary objective was to determine the maximum tolerated dose and the efcacy of the combination. The primary end-point was 40% progressionfree survival at 6 months with a 2 Simon Stage. [Results]: A total of 28 patients were screened and 19 pts were included. Combined therapy was safe and well tolerated. An interim analysis (Simon design frst-stage) recommended early termination because only 2/19 (11%) patients were disease free at 6 months. Median PFS was 3.1 months [2.1–5.3 months] and overall survival was 12.2 months [3.2–23.2 months]. Stimulation of immune system was observed in vitro but not clinically. The evaluation of basal RNA-seq noted signifcant changes between pre and post-therapy liver biopsies related to lipid metabolism and transport, infammation and oxidative stress pathways. [Conclusions]: The combination of Avelumab plus ADC vaccine is safe and well tolerated but exhibited modest clinical activity. Our study describes, for the frst-time, a de novo post-therapy metabolic rewiring, that could represent novel immunotherapyinduced tumor vulnerabilities.

Compelling evidence has accumulated on the role of oxidative stress on the endothelial cell (EC) dysfunction in acute coronary syndrome. Unveiling the underlying metabolic determinants has been hampered by the scarcity of appropriate cell models to address cell-autonomous mechanisms of EC dysfunction. We have generated endothelial cells derived from thrombectomy specimens from patients affected with acute myocardial infarction (AMI) and conducted phenotypical and metabolic characterizations. AMI-derived endothelial cells (AMIECs) display impaired growth, migration, and tubulogenesis. Metabolically, AMIECs displayed augmented ROS and glutathione intracellular content, with a diminished glucose consumption coupled to high lactate production. In AMIECs, while PFKFB3 protein levels of were downregulated, PFKFB4 levels were upregulated, suggesting a shunting of glycolysis towards the pentose phosphate pathway, supported by upregulation of G6PD. Furthermore, the glutaminolytic enzyme GLS was upregulated in AMIECs, providing an explanation for the increase in glutathione content. Finally, AMIECs displayed a significantly higher mitochondrial membrane potential than control ECs, which, together with high ROS levels, suggests a coupled mitochondrial activity. We suggest that high mitochondrial proton coupling underlies the high production of ROS, balanced by PPP- and glutaminolysis-driven synthesis of glutathione, as a primary, cell-autonomous abnormality driving EC dysfunction in AMI.

[EN] The present invention provides a method for deciding a check-point inhibitor (ICI)-based therapy, for a patient suffering from cancer, the method comprising the steps: a) measuring the expression level of the following markers: TGFB1, ZEB1, FAP, ZEB2, GLUL, ENTPD1, GOT1, LDHA, TWIST1, and GLS, in an isolated cancerous biological tissue sample from the patient; b) determining which markers are differentially expressed; and c) classifying the sample, depending on the markers differentially expressed, in one of the following clusters: cluster 1: when ZEB1, ZEB2, ENTPD1, FAP and GLUL are up-regulated; cluster 2: when GLS is upregulated; and LDHA, GOT1, ZEB1, ZEB2 and ENTPD1 are downregulated; and cluster 3: when GOT1 is upregulated and TGFB1 and TWIST 1 are downregulated., [FR] La présente invention concerne un procédé permettant de décider d'une thérapie à base d'inhibiteurs de points de contrôle (ICI), pour un patient souffrant d'un cancer, le procédé comprenant les étapes suivantes : a) mesure du niveau d'expression des marqueurs suivants : TGFB1, ZEB1, FAP, ZEB2, GLUL, ENTPD1, GOT1, LDHA, TWIST1 et GLS, dans un échantillon de tissu biologique cancéreux isolé provenant du patient ; b) détermination des marqueurs exprimés de manière différentielle ; et c) classification de l'échantillon, en fonction des marqueurs exprimés de manière différentielle, dans l'un des groupes suivants : groupe 1 : lorsque ZEB1, ZEB2, ENTPD1, FAP et GLUL sont fortement exprimées ; groupe 2 : lorsque GLS est fortement exprimée ; et LDHA, GOT1, ZEB1, ZEB2 et ENTPD1 sont faiblement exprimées ; et groupe 3 : lorsque GOT1 est fortement exprimée et TGFB1 et TWIST 1 sont faiblement exprimées.

Drug development is a complex, risky, expensive and time-consuming process that requires the accurate execution of multiple stages, from identification and selection of collections of potentially druggable molecules to proof-of-concept validation. Prior to embarking on a drug discovery project, a detailed strategic plan must be designed that includes hundreds of critical considerations such as source of compounds for screening, feasibility of their synthetic pathways, target selection (molecules, cells, organisms), type of output (binding, function, phenotype), throughput, nature, layers and iterations of the screening process, scoring systems, lead optimization approaches or model systems for validation (biochemical activity, cellular function, organismal properties) and, crucially, good contingency plans. The need to discover new drugs rests on practical matters of human progress, rather than mere market considerations. For example, the global emergence of multidrug resistant pathogens makes it a research target to find alternative antibiotics; current cancer drugs, including advanced biologicals, face drug resistance; many parasitic diseases lack effective drug treatments; highly prevalent neurodegenerative diseases and rare diseases, which collectively affect significant numbers of people, are essentially drug orphan; vital agricultural crops are plagued by fungal and parasitic diseases that have evolved to become increasingly resistant to currently available chemicals. The current COVID-19 pandemic illustrates how pharmaceutically unprepared humanity is (vaccines aside) to confront the sudden emergence of a novel, deadly and highly transmissible pathogen. To date, only a handful of repurposed drugs display demonstrable therapeutic efficacy against infection and disease by the causing agent, SARS-CoV-2 (Lui and Guaraldi, 2023; Sandulescu et al., 2023). Despite an unprecedented parallel effort by hundreds of thousands of industrial and academic scientists worldwi

Infection with SARS-CoV-2 portends a broad range of outcomes, from a majority of asymptomatic cases or mild clinical courses to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty and co-morbidities such as obesity, diabetes or cardiovascular disease. A precise knowledge is still lacking of the molecular and biological mechanisms that may explain the association of severe disease with male sex. Here, we show that testosterone trajectories are highly accurate individual predictors (AUC of ROC = 0.928, p < 0.0001) of survival in male COVID-19 patients. Longitudinal determinations of blood levels of luteinizing hormone (LH) and androstenedione suggest an early modest inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by either full recovery in survivors or a peripheral failure in lethal cases. Moreover, failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and decrease of non-classical monocytes. The strong association of recovery or failure to reinstate testosterone levels with survival or death from COVID-19 in male patients is suggestive of a significant role of testosterone status in the immune responses to COVID-19.

The COVID-19 pandemic had a delayed onset in South America compared to Asia (outside of China), Europe or North America. In spite of the presumed time advantage for the implementation of preventive measures to help contain its spread, the pandemic in that region followed growth rates that paralleled, and currently exceed, those observed several weeks before in Europe. Indeed, in early August, 2020, many countries in South and Central America presented among the highest rates in the world of COVID-19 confirmed cases and deaths per million inhabitants. Here, we have taken an ecological approach to describe the current state of the pandemic in Peru and its dynamics. Our analysis supports a protective effect of altitude from COVID-19 incidence and mortality. Further, we provide circumstantial evidence that internal migration through a specific land route is a significant factor progressively overriding the protection from COVID-19 afforded by high altitude. Finally, we show that protection by altitude is independent of poverty indexes and is inversely correlated with the prevalence in the population of risk factors associated with severe COVID-19, including hypertension and hypercholesterolemia. We discuss long-term multisystemic adaptations to hypobaric hypoxia as possible mechanisms that may explain the observed protective effect of high altitude from death from COVID-19.

Coronavirus disease 2019 (COVID‐19) is frequently associated with severe systemic consequences, including vasculitis, a hyperinflammatory state and hypercoagulation. The mechanisms leading to these life‐threatening abnormalities are multifactorial. Based on the analysis of publicly available interactomes, we propose that severe acute respiratory syndrome coronavirus‐2 infection directly causes a deficiency in C1 esterase inhibitor, a pathogen‐specific mechanism that may help explain significant systemic abnormalities in patients with COVID‐19.

Background SARS-CoV-2 infection portends a broad range of outcomes, from a majority of asymptomatic cases to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty, and co-morbidities such as obesity, diabetes, and cardiovascular disease. A precise knowledge of the molecular and biological mechanisms that may explain the association of severe disease with male sex is still lacking. Here, we analyzed the relationship of serum testosterone levels and the immune cell skewing with disease severity in male COVID-19 patients. Methods Biochemical and hematological parameters of admission samples in 497 hospitalized male and female COVID-19 patients, analyzed for associations with outcome and sex. Longitudinal (in-hospital course) analyses of a subcohort of 114 male patients were analyzed for associations with outcome. Longitudinal analyses of immune populations by flow cytometry in 24 male patients were studied for associations with outcome. Results We have found quantitative differences in biochemical predictors of disease outcome in male vs. female patients. Longitudinal analyses in a subcohort of male COVID-19 patients identified serum testosterone trajectories as the strongest predictor of survival (AUC of ROC = 92.8%, p < 0.0001) in these patients among all biochemical parameters studied, including single-point admission serum testosterone values. In lethal cases, longitudinal determinations of serum luteinizing hormone (LH) and androstenedione levels did not follow physiological feedback patterns. Failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and augmented circulating classical monocytes. Conclusions Recovery or failure to reinstate testosterone levels is strongly associated with survival or death, respectively, from COVID-19 in male patients. Our data suggest an early inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work., Peer Reviewed, Postprint (author's final draft)

La irrupción del coronavirus SARS-CoV-2 y de la pandemia de covid-19 ha planteado un enorme desafío a la ciencia. Buscar soluciones a un virus que ha causado, hasta el momento, 130 millones de infectados y ha provocado casi 3 millones de muertes. Para desentrañar las claves de esta nueva amenaza global, el Consejo Superior de Investigaciones Científicas, el CSIC, puso en marcha en marzo de 2020 la Plataforma Temática Interdisciplinar Salud Global. Su objetivo es coordinar a cientos de equipos de diversas disciplinas en busca de soluciones a la pandemia, creando nuevas redes de colaboración con organismos públicos de investigación, universidades, la clínica y el sector empresarial. La plataforma ha reunido a expertos de la investigación en biomedicina (virólogos, inmunólogos, genetistas y biotecnólogos), junto a químicos y físicos, además de sociólogos y demógrafos. Su trabajo es determinar las características del virus y de la covid-19, y diseñar estrategias que permitan hacer frente a esta y futuras pandemias. Tras un año de investigación, la Plataforma Salud Global ha obtenido conocimientos fundamentales en todos los aspectos de la pandemia: prevención, contención, diagnóstico, terapia y vacunas, e impacto social.

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.

[Background]: The COVID-19 pandemic had a delayed onset in America. Despite the time advantage for the implementation of preventative measures to contain its spread, the pandemic followed growth rates that paralleled those observed before in Europe., [Objectives]: To analyze the temporal and geographical distribution of the COVID-19 pandemic at district-level in Perú during the full lockdown period in 2020., [Methods]: Analysis of publicly available data sets, stratified by altitude and geographical localization. Correlation tests of COVID-19 case and death rates to population prevalence of comorbidities., [Results]: We observe a strong protective effect of altitude from COVID-19 mortality in populations located above 2,500 m. We provide evidence that internal migration through a specific land route is a significant factor progressively overriding the protection from COVID-19 afforded by high altitude. This protection is independent of poverty indexes and is inversely correlated with the prevalence of hypertension and hypercholesterolemia., [Discussion]: Long-term adaptation to residency at high altitude may be the third general protective factor from COVID-19 severity and death, after young age and female sex. Multisystemic adaptive traits or acclimatization processes in response to chronic hypobaric hypoxia may explain the apparent protective effect of high altitude from COVID-19 death.