Project description:The repertoire of coronavirus disease 2019 (COVID-19)-mediated adverse health outcomes has continued to expand in infected patients, including the susceptibility to developing long-COVID; however, the molecular underpinnings at the cellular level are poorly defined. In this study, we report that SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection triggers host cell genome instability by modulating the expression of molecules of DNA repair and mutagenic translesion synthesis. Further, SARS-CoV-2 infection causes genetic alterations, such as increased mutagenesis, telomere dysregulation, and elevated microsatellite instability (MSI). The MSI phenotype was coupled to reduced MLH1, MSH6, and MSH2 in infected cells. Strikingly, pre-treatment of cells with the REV1-targeting translesion DNA synthesis inhibitor, JH-RE-06, suppresses SARS-CoV-2 proliferation and dramatically represses the SARS-CoV-2-dependent genome instability. Mechanistically, JH-RE-06 treatment induces autophagy, which we hypothesize limits SARS-CoV-2 proliferation and, therefore, the hijacking of host-cell genome instability pathways. These results have implications for understanding the pathobiological consequences of COVID-19.
Project description:The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.
Project description:The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10-20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).
Project description:Host cell-encoded deaminases act as antiviral restriction factors to impair viral replication and production through introducing mutations in the viral genome. We sought to understand whether deaminases are involved in SARS-CoV-2 mutation and replication, and how the viral factors interact with deaminases to trigger these processes. Here, we show that APOBEC and ADAR deaminases act as the driving forces for SARS-CoV-2 mutagenesis, thereby blocking viral infection and production. Mechanistically, SARS-CoV-2 nucleocapsid (N) protein, which is responsible for packaging viral genomic RNA, interacts with host deaminases and colocalizes with them at stress granules to facilitate viral RNA mutagenesis. N proteins from several coronaviruses interact with host deaminases at RNA granules in a manner dependent on its F17 residue, suggesting a conserved role in modulation of viral mutagenesis in other coronaviruses. Furthermore, mutant N protein bearing a F17A substitution cannot localize to deaminase-containing RNA granules and leads to reduced mutagenesis of viral RNA, providing support for its function in enhancing deaminase-dependent viral RNA editing. Our study thus provides further insight into virus-host cell interactions mediating SARS-CoV-2 evolution.
Project description:Construction of transcriptome sequencing library and transcriptome sequencing was completed by LC-Bio Technology Co., Ltd. (Hangzhou, China). The expression profile of Machado-joseph deubiquitinases (MJDs) family in heart tissues of Ang II mice.
Project description:Non-structural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication and is considered an attractive target for the development of novel antiviral drugs. We were initially interested in the interplay between nsp13 and interferon signaling, and found that nsp13 inhibited reporter signal of IFN-β promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-β promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted expression of foreign genes originated from plasmid transfection, but failed to inhibit them after chromosome integration. These data together with results from run-off transcription assay and RNA sequencing suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to episomal DNA transcriptional inhibition, And this restriction required direct interaction with episomal DNA. Further, we developed a high-throughput nsp13 drug screening method based on the correlation between the helicase activity and nsp13 inhibition on episomal DNA. This method evaluates the inhibitory effect of compounds on nsp13 by detecting the expression of reporter plasmids after co-transfection with nsp13 plasmids, which is economical and convenient compared with conventional methods. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs.
Project description:Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions.
Project description:In this review, we reveal the latest developments at the interface between SARS-CoV-2 and the host cell surface. In particular, we evaluate the current and potential mechanisms of binding, fusion and the conformational changes of the spike (S) protein to host cell surface receptors, especially the human angiotensin-converting enzyme 2 (ACE2) receptor. For instance, upon the initial attachment, the receptor binding domain of the S protein forms primarily hydrogen bonds with the protease domain of ACE2 resulting in conformational changes within the secondary structure. These surface interactions are of paramount importance and have been therapeutically exploited for antiviral design, such as monoclonal antibodies. Additionally, we provide an insight into novel therapies that target viral non-structural proteins, such as viral RNA polymerase. An example of which is remdesivir which has now been approved for use in COVID-19 patients by the US Food and Drug Administration. Establishing further understanding of the molecular details at the cell surface will undoubtably aid the development of more efficacious and selectively targeted therapies to reduce the burden of COVID-19.
Project description:MCL1 is a pivot member of the anti-apoptotic BCL-2 family proteins. While a distinctive feature of MCL1 resides in its efficient ubiquitination and destruction, the deubiquitinase USP9X has been implicated in the preservation of MCL1 expression by removing the polyubiquitin chains. Here we perform an unbiased siRNA screen and identify that the second deubiquitinase, USP13, regulates MCL1 stability in lung and ovarian cancer cells. Mechanistically, USP13 interacts with and stabilizes MCL1 via deubiquitination. As a result, USP13 depletion using CRISPR/Cas9 nuclease system inhibits tumor growth in xenografted nude mice. We further report that genetic or pharmacological inhibition of USP13 considerably reduces MCL1 protein abundance and significantly increases tumor cell sensitivity to BH3 mimetic inhibitors targeting BCL-2 and BCL-XL. Collectively, we nominate USP13 as a novel deubiquitinase which regulates MCL1 turnover in diverse solid tumors and propose that USP13 may be a potential therapeutic target for the treatment of various malignancies.