RÉSUMÉ
Molnupiravir, an orally administered prodrug of ß-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.
RÉSUMÉ
Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.
Sujet(s)
COVID-19 , Cystéine , Humains , SARS-CoV-2 , Conception de médicament , OxydoréductionRÉSUMÉ
The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.
Sujet(s)
COVID-19 , Anticorps à domaine unique , Animaux , Cricetinae , Humains , SARS-CoV-2 , Gouttelettes et aérosols respiratoires , Glycoprotéine de spicule des coronavirus , Techniques de culture cellulaire , Anticorps neutralisants , Anticorps antivirauxRÉSUMÉ
N4-hydroxycytidine (NHC), the active compound of the drug Molnupiravir, is incorporated into SARS-CoV-2 RNA, causing false base pairing. The desired result is an "error catastrophe," but this bears the risk of mutated virus progeny. To address this experimentally, we propagated the initial SARS-CoV-2 strain in the presence of NHC. Deep sequencing revealed numerous NHC-induced mutations and host-cell-adapted virus variants. The presence of the neutralizing nanobody Re5D06 selected for immune escape mutations, in particular p.E484K and p.F490S, which are key mutations of the Beta/Gamma and Omicron-XBB strains, respectively. With NHC treatment, nanobody resistance occurred two passages earlier than without. Thus, within the limitations of this purely in vitro study, we conclude that the combined action of Molnupiravir and a spike-neutralizing antagonist leads to the rapid emergence of escape mutants. We propose caution use and supervision when using Molnupiravir, especially when patients are still at risk of spreading virus.
RÉSUMÉ
Cancer chemotherapy relies on a high ratio of toxicity toward cancer cells vs. nonmalignant cells, making it desirable to protect normal cells. Among the nonmalignant cells, epithelia of the gut belong to the most vulnerable ones toward chemotherapeutics. Here, we use a murine intestinal organoid model to assess a strategy for protecting such epithelia against chemotherapy. Cell cycle progression was first stalled by palbociclib, a clinically established cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Washout of the drug allowed subsequent outgrowth of gut organoids. This transient cell cycle arrest conferred near-complete protection of the cells toward the nucleoside analogue gemcitabine. Moreover, pre-treatment with palbociclib protected the organoids against SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which is otherwise known for its severe gastrointestinal toxicities. In contrast, RB1-mutated cancer cells were not protected against gemcitabine or SN-38 when pre-treated with palbociclib. Taken together, these results outline a strategy for protecting nonmalignant cells against the toxicities of chemotherapeutics commonly used to treat advanced colorectal and pancreatic cancer. We propose that this strategy is particularly promising to protect the gut when treating RB1-deficient tumors that fail to arrest the cell cycle in response to CDK4/6 inhibitors. [Figure: see text].
Sujet(s)
Kinase-6 cycline-dépendante , , Animaux , Souris , Irinotécan/pharmacologie , Kinase-4 cycline-dépendante/métabolisme , Points de contrôle du cycle cellulaire , Cycle cellulaire , Inhibiteurs de protéines kinases/pharmacologieRÉSUMÉ
The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks, even in the absence of functional p53. Here, we show that MDM2 binds, inhibits, ubiquitinates, and destabilizes poly(ADP-ribose) polymerase 1 (PARP1). When cellular MDM2 levels are increased, this leads to accelerated progression of DNA replication forks, much like pharmacological inhibition of PARP1. Conversely, overexpressed PARP1 restores normal fork progression despite elevated MDM2. Strikingly, MDM2 profoundly reduces the frequency of fork reversal, revealed as four-way junctions through electron microscopy. Depletion of RECQ1 or the primase/polymerase (PRIMPOL) reverses the MDM2-mediated acceleration of the nascent DNA elongation rate. MDM2 also increases the occurrence of micronuclei, and it exacerbates camptothecin-induced cell death. In conclusion, high MDM2 levels phenocopy PARP inhibition in modulation of fork restart, representing a potential vulnerability of cancer cells.
Sujet(s)
Réplication de l'ADN , Protéine p53 suppresseur de tumeur , ADN/génétique , Altération de l'ADN , DNA primase/métabolisme , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.
RÉSUMÉ
Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.
Sujet(s)
Anticorps neutralisants/immunologie , Anticorps antiviraux/immunologie , COVID-19/immunologie , Mutation/immunologie , SARS-CoV-2/immunologie , Anticorps à domaine unique/immunologie , Animaux , COVID-19/virologie , Camélidés du Nouveau Monde/immunologie , Camélidés du Nouveau Monde/virologie , Lignée cellulaire , Escherichia coli/virologie , Femelle , Humains , Glycoprotéine de spicule des coronavirus/immunologieRÉSUMÉ
The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 µM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) and ~100-fold reductions in Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 100 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.
Sujet(s)
AMP/analogues et dérivés , Alanine/analogues et dérivés , Antiviraux/pharmacologie , Traitements médicamenteux de la COVID-19 , Antifoliques/pharmacologie , Méthotrexate/pharmacologie , SARS-CoV-2/effets des médicaments et des substances chimiques , AMP/pharmacologie , Alanine/pharmacologie , Animaux , COVID-19/virologie , Techniques de culture cellulaire , Chlorocebus aethiops , Humains , ARN viral/génétique , SARS-CoV-2/physiologie , Cellules Vero , Réplication virale/effets des médicaments et des substances chimiquesRÉSUMÉ
Psoriasis is a frequent, inflammatory skin disease characterized by keratinocyte hyperproliferation and a disease-related infiltration of immune cells. Here, we identified a novel proinflammatory signaling pathway driven by cyclin-dependent kinase 4 (CDK4) and CDK6 and the methyltransferase EZH2 as a valid target for psoriasis therapy. Delineation of the pathway revealed that CDK4/6 phosphorylated EZH2 in keratinocytes, thereby triggering a methylation-induced activation of STAT3. Subsequently, active STAT3 resulted in the induction of IκBζ, which is a key proinflammatory transcription factor required for cytokine synthesis in psoriasis. Pharmacological or genetic inhibition of CDK4/6 or EZH2 abrogated psoriasis-related proinflammatory gene expression by suppressing IκBζ induction in keratinocytes. Importantly, topical application of CDK4/6 or EZH2 inhibitors on the skin was sufficient to fully prevent the development of psoriasis in various mouse models by suppressing STAT3-mediated IκBζ expression. Moreover, we found a hyperactivation of the CDK4/6-EZH2 pathway in human and mouse psoriatic skin lesions. Thus, this study not only identifies a novel psoriasis-relevant proinflammatory pathway, but also proposes the repurposing of CDK4/6 or EZH2 inhibitors as a new therapeutic option for patients with psoriasis.
Sujet(s)
Kinase-4 cycline-dépendante/métabolisme , Kinase-6 cycline-dépendante/métabolisme , Protéine-2 homologue de l'activateur de Zeste/métabolisme , Kératinocytes/métabolisme , Psoriasis/métabolisme , Transduction du signal , Animaux , Kinase-4 cycline-dépendante/génétique , Kinase-6 cycline-dépendante/génétique , Modèles animaux de maladie humaine , Protéine-2 homologue de l'activateur de Zeste/génétique , Cellules HEK293 , Humains , Kératinocytes/anatomopathologie , Souris , Psoriasis/génétique , Psoriasis/anatomopathologie , Psoriasis/thérapieRÉSUMÉ
The genes encoding MDM2 and CDK4 are frequently co-amplified in sarcomas, and inhibitors to both targets are approved or clinically tested for therapy. However, we show that inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells. CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition. Moreover, the p53 response was also attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown. The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associated with each other, suggesting direct regulation of p53 by CDK4. Interestingly, CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters, but rather attenuated the subsequent recruitment of RNA Polymerase II. Taken together, our results suggest that caution must be used when considering combined CDK4 and MDM2 inhibition for patient treatment. Moreover, they uncover a hitherto unknown role for CDK4 and Cyclin D1 in sustaining p53 activity.
Sujet(s)
Cycline D1/métabolisme , Kinase-4 cycline-dépendante/antagonistes et inhibiteurs , Protéines proto-oncogènes c-mdm2/antagonistes et inhibiteurs , Sarcomes/anatomopathologie , Protéine p53 suppresseur de tumeur/métabolisme , Aminopyridines/pharmacologie , Antinéoplasiques/pharmacologie , Benzimidazoles/pharmacologie , Lignée cellulaire tumorale , Prolifération cellulaire/génétique , Kinase-4 cycline-dépendante/métabolisme , Humains , Pipérazines/pharmacologie , Protéines proto-oncogènes c-mdm2/métabolisme , Purines/pharmacologie , Pyridines/pharmacologie , Interférence par ARN , Petit ARN interférent/génétique , Sarcomes/génétiqueRÉSUMÉ
The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.
Sujet(s)
Assemblage et désassemblage de la chromatine , Cellules souches pluripotentes induites/métabolisme , Cellules souches mésenchymateuses/métabolisme , Cellules souches tumorales/métabolisme , Complexe répresseur Polycomb-2/métabolisme , Protéines proto-oncogènes c-mdm2/métabolisme , Protéine p53 suppresseur de tumeur/métabolisme , Animaux , Différenciation cellulaire , Lignage cellulaire , Prolifération cellulaire , Survie cellulaire , Régulation de l'expression des gènes tumoraux , Cellules HCT116 , Histone/métabolisme , Humains , Cellules MCF-7 , Méthylation , Souris , Ostéogenèse , Phénotype , Complexe répresseur Polycomb-1/métabolisme , Complexe répresseur Polycomb-2/génétique , Protéines proto-oncogènes c-mdm2/génétique , Interférence par ARN , Transduction du signal , Facteurs temps , Transfection , Protéine p53 suppresseur de tumeur/génétique , Ubiquitin-protein ligases/métabolisme , UbiquitinationRÉSUMÉ
The p53 homologue p63/TP73L is required for the proper development of squamous epithelia, mammary glands and limb buds, with some of these tissues also displaying strong canonical Wnt signalling activity. It was previously suggested that DeltaNp63alpha, the predominant isoform of p63 in epithelia, positively regulates beta-Catenin through inhibition of GSK3beta. Results reported in this communication show that, upon transient overexpression, DeltaNp63alpha indeed promotes Wnt-inducible reporter gene activity in human cells, as well as secondary axis formation in Xenopus embryos. However, in apparent contradiction to these observations, siRNA-mediated knockdown of endogenous p63 equally enhanced the expression of Wnt-responsive genes. While p63 knockdown did not detectably affect beta-Catenin levels or phosphorylation, DeltaNp63alpha was found in a complex with members of the TCF/LEF family of Wnt-responsive transcription factors. On The basis of these findings, we propose that DeltaNp63alpha has a function in recruiting transcriptional repressors to Wnt-responsive genes. Overexpression of p63 may lead to sequestration of such repressors (squelching), resulting in a similar effect like siRNA-mediated removal of p63, i.e., activation of Wnt-responsive genes. The role of p63 as a negative Wnt-regulator thus matches with the frequently observed downregulation of p63 during tumor progression, when cancer cells adopt a more mesenchymal, invasive phenotype.
Sujet(s)
Phosphoprotéines/métabolisme , Transactivateurs/métabolisme , Transcription génétique , Protéines suppresseurs de tumeurs/métabolisme , Protéines de type Wingless/métabolisme , Protéines de Xénope/métabolisme , Xenopus/métabolisme , Animaux , Plan d'organisation du corps , Embryon non mammalien/métabolisme , Techniques de knock-down de gènes , Gènes rapporteurs , Humains , Facteur de transcription LEF-1/métabolisme , Modèles biologiques , Phosphorylation , Liaison aux protéines , Petit ARN interférent/métabolisme , Transduction du signal , Facteurs de transcription , Activation de la transcription/génétique , Xenopus/embryologie , bêta-Caténine/métabolismeRÉSUMÉ
Ammonium transporters (Amts) are integral membrane proteins found in all kingdoms of life that fulfill an essential function in the uptake of reduced nitrogen for biosynthetic purposes. Amt-1 is one of three Amts encoded in the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus. The crystal structure of Amt-1 shows a compact trimer with 11 transmembrane helices per monomer and a central channel for substrate conduction in each monomer, similar to the known crystal structure of AmtB from Escherichia coli. Xenon derivatization has been used to identify apolar regions of Amt-1, emphasizing not only the hydrophobicity of the substrate channel but also the unexpected presence of extensive internal cavities that should be detrimental for protein stability. The substrates ammonium and methylammonium have been used for cocrystallization experiments with Amt-1, but the identification of binding sites that are distinct from water positions is not unambiguous. The well ordered cytoplasmic C terminus of the protein in the Amt-1 structure has allowed for the construction of a docking model between Amt-1 and a homology model for its physiological interaction partner, the P(II) protein GlnB-1. In this model, GlnB-1 binds tightly to the cytoplasmic face of the transporter, effectively blocking conduction through the three individual substrate channels.
Sujet(s)
Protéines d'archée/composition chimique , Archaeoglobus fulgidus/composition chimique , Transporteurs de cations/composition chimique , Composés d'ammonium quaternaire/métabolisme , Séquence d'acides aminés , Protéines d'archée/génétique , Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Sites de fixation , Transport biologique , Transporteurs de cations/génétique , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Protéines de régulation du métabolisme azoté/composition chimique , Protéines de régulation du métabolisme azoté/métabolisme , Structure quaternaire des protéines , Structure secondaire des protéines , Structure tertiaire des protéines , Alignement de séquences , Xénon/composition chimiqueRÉSUMÉ
Ammonium transporters (Amts) are a class of membrane-integral transport proteins found in organisms from all kingdoms of life. Their key function is the transport of nitrogen in its reduced bioavailable form, ammonia, across cellular membranes, a crucial step in nitrogen assimilation for biosynthetic purposes. The genome of the hyperthermophilic archaeon Archaeoglobus fulgidus has been annotated with three individual genes for ammonium transporters, amt1-3, the roles of which are as yet unknown. The amt1 gene product has been produced by heterologous overexpression in Escherichia coli and the resulting protein has been purified to electrophoretic homogeneity. Crystals of Amt-1 have been obtained by sitting-drop vapour diffusion and diffraction data have been collected.