Biology subjects

Zapatero-Belinchon, F. J.

Publications and source records attributed to Zapatero-Belinchon, F. J..

7 recordsLinked to original sources

bioRxiv2025

Programmable translational inhibition by a molecular glue-oligonucleotide conjugate

Selective inhibition of mRNA translation is a promising strategy for modulating the activity of disease-associated genes, yet achieving both high potency and specificity remains challenging. Rocaglamide A (RocA), a molecular glue, inhibits translation by clamping eIF4A onto polypurine motifs found in many transcripts, thereby limiting RocAs specificity. Here, we developed RocASO, a chemical conjugate that links RocA to an antisense oligonucleotide (ASO) capable of base-pairing with defined mRNA sequences, thus directing RocAs clamping mechanism to chosen targets and enhancing overall potency and specificity. We show that RocASOs are compatible with various types of ASO modalities, including gapmers that induce the degradation of target RNAs. RocASOs were designed to effectively knock down endogenous genes (PTGES3, HSPA1B) and SARS-CoV-2 viral RNA, the latter conferring potent antiviral activity in cells. These findings establish RocASO as a versatile platform for programmable translational inhibition with therapeutic potential.

genomics↗

bioRxiv2025

NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of anti-oxidative and detoxifying cell responses. In addition, it plays important roles in host cell defenses against pathogenic viruses, and small molecules that activate NRF2 signaling can exert potent antiviral effects. We recently found that the NRF2 activators 4-octyl itaconate (4OI), bardoxolone (BARD), and sulforaphane (SFN) interfere with influenza A virus replication by blocking the nuclear export factor exportin 1 (XPO1), which did not require NRF2 signaling. Here, we have assessed their potential to inhibit highly pathogenic (SARS-CoV-2) and seasonal (hCoV-229E) coronaviruses and begun to elucidate the involved mechanisms of action. Using human cell lines and iPSC-derived vascular endothelial cells, we find that NRF2 knock-out or knock-down enhances infection by both viruses, indicating that physiologic NRF2 signaling restricts human coronavirus infection. 4OI, BARD, SFN, as well as the XPO1 blocker Selinexor (SEL), greatly limit infection by both viruses, but in an NRF2-independent manner. Strikingly, the compounds (particularly 4OI) downregulate ACE2 and TMPRSS2 mRNA and protein in Calu3 cells, leading to a >10-fold reduction in viral cell entry by 4OI and SEL, as assessed using SARS-CoV-1 and -2 spike protein VSV pseudotypes. A cycloheximide chase experiment revealed that 4OI dramatically reduces ACE2 half-life, which requires the E3 ligases NEDD4L and MCM1, suggesting that 4OI targets ACE2 for destruction by the proteasome. Moreover, 4OI and SEL reduce XPO1 protein levels, and all compounds reduce XPO1 mRNA levels. Co-incubation experiments of 4OI and the transcription blocker actinomycin D in A549 cells suggest that 4OI acts primarily by interfering with transcription of the XPO1 gene. XPO1 knock-down markedly reduces 229E replication. All four compounds interfere with 229E infection, but do not alter expression of ANPEP, the cellular receptor for this virus. Their anti-229E efficacy depends on expression of XPO1 in host cells in the order of SEL (most dependent) >4OI >SFN >BARD (least dependent), suggesting that especially BARD interferes with 229E infectivity via yet another, unknown, target. Taken together, these results suggest that "NRF2 activators" act as potent antivirals against human coronaviruses by targeting diverse host factors which are critical for viral infectivity. Author summaryHost-directed antiviral compounds act by a variety of mechanisms. For instance, they stimulate cellular antiviral immune responses and target host cell factors which are required for the viral life cycle. Pharmacologic activation of the NRF2 signaling pathway is a particularly attractive antiviral strategy, as this pathway restricts replication of a variety of viruses and also protects cells from excessive inflammation and oxidative stress resulting from accumulation of reactive oxygen species. In our previous study of the NRF2 activators bardoxolone, sulforaphane, and 4-octyl itaconate as host-directed treatments for influenza A virus infection, we found that these compounds interfered with replication of the virus. Unexpectedly, this antiviral activity was completely independent of NRF2 signaling, but resulted from blocking the nuclear export factor XPO1. In the present study, we find that these compounds limit infection by SARS-CoV-2 and hCoV-229E and that, again, this antiviral effect is NRF-independent. Instead, it depends to a large extent on downregulating ACE2 and TMPRSS2 (the major host cell receptors for SARS-CoV-1 and 2) and blocking/downregulating XPO1. Our results underscore the potential of "NRF2 activators" as adjunct treatments for viral infections, as they protect the host by anti-oxidative, anti-inflammatory, and cytoprotective mechanisms and also interfere with diverse host factors required for the viral life cycle.

pharmacology and toxicology↗

bioRxiv2024

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation enhanced host lipid droplet consumption by NSP6 without impacting its known ER-zippering function or double-membrane vesicle morphology. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6s key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications. Author SummaryAs SARS-CoV-2 continues to spread and adapt in humans, viral variants with enhanced spread and immune evasion have emerged throughout the COVID-19 pandemic. While most of the mutations occur in the Spike protein and have been extensively studied, non-Spike mutations have been accumulating and are not as well understood. Here, we constructed Spike-defective genomes of recent Omicron variants and systematically compared their RNA replication capabilities independently from Spike. We also performed single-round infection assays with various Omicron variant Spike proteins to quantify viral entry capabilities. Interestingly, viral entry and RNA replication were negatively correlated, suggesting that as variants evolved reduced entry functions under growing immune pressure on Spike, RNA replication increased as a compensatory mechanism. We focused on a viral protein NSP6 that acquired mutations that significantly enhanced RNA replication. We validated that a frequently accessed L260F mutation in NSP6 enhanced viral infection in cells and increased pathogenesis in mice. While the mutation did not alter NSP6s role in maintaining a membranous network of viral replication factories, the mutation enhanced viral hijacking of the host lipid droplet machinery. Collectively, we highlight the important role of non-Spike mutations in the evolutionary trajectories of SARS-CoV-2 variants with possible monitoring and therapeutic implications.

microbiology↗

bioRxiv2024

The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Standard cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner. However, a stronger antiviral effect for AVI-4206 is observed in human airway organoids and peripheral blood monocyte-derived macrophages. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.

immunology↗

bioRxiv2023

Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase

An under-explored target for SARS-CoV-2 is non-structural protein 14 (Nsp14), a crucial enzyme for viral replication that catalyzes the methylation of N7-guanosine of the viral RNA at 5'-end; this enables the virus to evade the host immune response by mimicking the eukaryotic post-transcriptional modification mechanism. We sought new inhibitors of the S-adenosyl methionine (SAM)-dependent methyltransferase (MTase) activity of Nsp14 with three large library docking strategies. First, up to 1.1 billion make-on-demand ("tangible") lead-like molecules were docked against the enzymes SAM site, seeking reversible inhibitors. On de novo synthesis and testing, three inhibitors emerged with IC50 values ranging from 6 to 43 M, each with novel chemotypes. Structure-guided optimization and in vitro characterization supported their non-covalent mechanism. In a second strategy, docking a library of 16 million tangible fragments revealed nine new inhibitors with IC50 values ranging from 12 to 341 M and ligand efficiencies from 0.29 to 0.42. In a third strategy, a newly created library of 25 million tangible, virtual electrophiles were docked to covalently modify Cys387 in the SAM binding site. Seven inhibitors emerged with IC50 values ranging from 3.2 to 39 M, the most potent being a reversible aldehyde. Initial optimization of a second series yielded a 7 M acrylamide inhibitor. Three inhibitors characteristic of the new series were tested for selectivity against 30 human protein and RNA MTases, with one showing partial selectivity and one showing high selectivity. Overall, 32 inhibitors encompassing eleven chemotypes had IC50 values <50 M and 5 inhibitors in four chemotypes had IC50 values <10 M. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing multiple starting points for optimizing towards antiviral activity.

biophysics↗

bioRxiv2022

Innate immune response to SARS-1 CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons

Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection causes neurological disease in some patients suggesting that infection can affect both the peripheral and central nervous system (PNS and CNS, respectively). It is not clear whether the outcome of SARS-CoV-2 infection of PNS and CNS neurons is similar, and which are the key factors that cause neurological disease: SARS-CoV-2 infection or the subsequent immune response. Here, we addressed these questions by infecting human induced-pluripotent stem cell-derived CNS and PNS neurons with the {beta} strain of SARS-CoV-2. Our results show that SARS-CoV-2 infects PNS neurons more efficiently than CNS neurons, despite lower expression levels of angiotensin converting enzyme 2. Infected PNS neurons produced interferon {lambda}1, several interferon stimulated genes and proinflammatory cytokines. They also displayed neurodegenerative-like alterations, as indicated by increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and -synuclein and lower levels of nicotinamide mononucleotide adenylyltransferase 2 and {beta}-III-tubulin. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neurodegeneration, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS.

microbiology↗

bioRxiv2022

Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.

microbiology↗