Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease.

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

KIRA8 attenuates non-alcoholic steatohepatitis through inhibition of the IRE1α/XBP1 signalling pathway.

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH.

Antibiotic-Induced Primary Biles Inhibit SARS-CoV-2 Endoribonuclease Nsp15 Activity in Mouse Gut.

The gut microbiome profile of COVID-19 patients was found to correlate with a viral load of SARS-CoV-2, COVID-19 severity, and dysfunctional immune responses, suggesting that gut microbiota may be involved in anti-infection. In order to investigate the role of gut microbiota in anti-infection against SARS-CoV-2, we established a high-throughput in vitro screening system for COVID-19 therapeutics by targeting the endoribonuclease (Nsp15). We also evaluated the activity inhibition of the target by substances of intestinal origin, using a mouse model in an attempt to explore the interactions between gut microbiota and SARS-CoV-2. The results unexpectedly revealed that antibiotic treatment induced the appearance of substances with Nsp15 activity inhibition in the intestine of mice. Comprehensive analysis based on functional profiling of the fecal metagenomes and endoribonuclease assay of antibiotic-enriched bacteria and metabolites demonstrated that the Nsp15 inhibitors were the primary bile acids that accumulated in the gut as a result of antibiotic-induced deficiency of bile acid metabolizing microbes. This study provides a new perspective on the development of COVID-19 therapeutics using primary bile acids.

Inhibiting the IRE1α Axis of the Unfolded Protein Response Enhances the Antitumor Effect of AZD1775 in TP53 Mutant Ovarian Cancer.

Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA-like ER kinase (PERK) and inositol-required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF-κB mediated senescence-associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP-homologous protein (CHOP), while IRE1α-induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α-XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.

Inhibition of the hexamerization of SARS-CoV-2 endoribonuclease and modeling of RNA structures bound to the hexamer.

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.

Identification of potential target endoribonuclease NSP15 inhibitors of SARS-COV-2 from natural products through high-throughput virtual screening and molecular dynamics simulation.

SARS-CoV-2 wreaks havoc around the world, triggering the COVID-19 pandemic. It has been confirmed that the endoribonuclease NSP15 is crucial to the viral replication, and thus identified as a potential drug target against COVID-19. The NSP15 protein was used as the target to conduct high-throughput virtual screening on 30,926 natural products from the NPASS database to identify potential NSP15 inhibitors. And 100 ns molecular dynamics simulations were performed on the NSP15 and NSP15-NPC198199 system. In all, 10 natural products with high docking scores with NSP15 protein were obtained, among which compound NPC198199 scored the highest. The analysis of the binding mode between NPC198199 and NSP15 found that NPC198199 would form H-bond interactions with multiple key residues at the catalytic site. Subsequently, a series of post-dynamics simulation analyses (including RMSD, RMSF, PCA, DCCM, RIN, binding free energy, and H-bond occupancy) were performed to further explore inhibitory mechanism of compound NPC198199 on NSP15 protein at the molecular level. The research strongly indicates that the 10 natural compounds screened can be used as potential inhibitors of NSP15, and provides valuable information for the subsequent drug discovery of anti-SARS-CoV-2. PRACTICAL APPLICATIONS: Natural products play an important role in the treatment of many difficult diseases. In this study, high-throughput virtual screening technology was used to screen the natural product database to obtain potential inhibitors against endoribonuclease NSP15. The binding mechanism between natural products and NSP15 was investigated at the molecular level by molecular dynamics technology so that it is expected to become candidate drugs for the treatment of SARS-CoV-2. We hope that our research can provide new clue to combat COVID-19 and overcome the epidemic situation as soon as possible.

Inhibition of IRE1 suppresses the catabolic effect of IL-1ß on nucleus pulposus cell and prevents intervertebral disc degeneration in vivo.

Neck pain and low back pain are two of the major diseases, which causes patients a low quantify of life and a heavy economic burden, intervertebral disc degeneration (IDD) contributes to them, and the mechanism is not totally clear. The increased inflammatory cytokines including interleukin (IL)-1ß and tumor necrosis factor (TNF)α and downstream signaling pathways are involved. Inositol requiring enzyme 1 (IRE1) is a crucial enzyme that regulates endoplasmic reticulum (ER) stress. It is reported that IRE1 plays an important role in the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. Considering this, we performed a series of experiments in vitro and in vivo to evaluate the role of IRE1 in the progress of IDD. We demonstrated that IRE1 pathway was induced by IL-1ß, inhibition of IRE1 suppressed the matrix degeneration of NP cells and ameliorated IDD grade in the punctured rat model. Further results indicated that inhibition of IRE1 suppressed H2O2 induced cell senescence, IL-1ß-induced cellular reactive oxygen species (ROS) level and the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. It also played a crucial role in the apoptosis of NP cells and the progress of macrophage polarization. Our findings demonstrated that inhibition of IRE1 could suppress the degeneration of NP cells and prevent IDD in vivo. IRE1 may be a potential target for IDD treatment.

In-silico screening of plant-derived antivirals against main protease, 3CLpro and endoribonuclease, NSP15 proteins of SARS-CoV-2.

Novel Coronavirus or SARS-CoV-2 outbreak has developed a pandemic condition all over the world. The virus is highly infectious and spreads by human to human local transmission mode. Till date, there is no vaccination or drugs been approved for the treatment by the World Health Organisation. Henceforth, the discovery of the potential drugs is an urgent and utmost requirement for the medical fraternity. Since, the side effects of plant-derived compounds will be lower compared to synthetic/chemical drugs. The Main protease (3CLpro or NSP5) and endoribonuclease (NSP15) proteins are necessity for viral replication and its survival in the host cell. In the present study, in-silico approach of drug development was used to search for potential antiviral plant-derived compounds as inhibitors against SARS-CoV-2 replication proteins. Eight plant-derived compounds of which the antiviral activity was known and available, and two reported drugs against SARS-CoV-2 selected for the molecular docking analysis. The docking results suggested that bisdemethoxycurcumin, demethoxycurcumin, scutellarin, quercetin and myricetin showed least binding energy, i.e., greater than -6.5 Kcal/mol against 3CLpro and endoribonuclease of SARS-CoV-2. Further studies of ADME-Tox and bioavailability of drugs were also performed that exhibited efficient parameters of drug likeness. Molecular dynamics simulation calculations were performed for the most negative binding affinity of the compound to evaluate the dynamic behavior,and stability of protein-ligand complex. Our findings suggest that these compounds could be potential inhibitors of SARS-CoV-2 main protease and endoribonuclease. However, further in-vitro and pre-clinical experiments would validate the potential inhibitors of SARS-CoV-2 proteins.

Prediction of potential inhibitors against SARS-CoV-2 endoribonuclease: RNA immunity sensing.

The World Health Organization has classified the COVID-19 outbreak a pandemic which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and declared it a global health emergency. Repurposing drugs with minimum side effects are one approach to quickly respond in attempt to prevent the spread of COVID-19. SARS-CoV-2 encodes several RNA processing enzymes that are unusual and unique for single-stranded RNA viruses, including Nsp15, a hexameric endoribonuclease that discriminatory cleaves immediately 3' of uridines. The structure of SARS-CoV-2 Nsp15 is reported to be homologous to that of the Nsp15 endoribonucleases of SARS-CoV and MERS-CoV, but it exhibits differences that may contribute to the greater virulence of SARS-CoV-2. This study aimed to identify drugs that targeted SARS-COV-2 Nsp15 using a molecular docking-based virtual screening of a library containing 10,000 approved and experimental drugs. The molecular docking results revealed 19 medications that demonstrated a good ability to inhibit Nsp15. Among all the candidated 19 drugs only five FDA approved drugs were used for further investigation by molecular dynamics simulation, the stability of Nsp15-ligand system was evaluated by calculating the RMSD, RMSF, radius of gyration and hydrogen bond profile. Furthermore, MM-PBSA method was employed to validate the binding affinity. According to the obtained results of MD, the complex of Olaparib was showed more stability and lower binding free energy than the control inhibitor during MD simulation time. Finally, we suggest that Olaparib is a potential drug for treating patients infected with SARS-CoV-2 and provide insight into the host immune response to viral RNA.Communicated by Ramaswamy H. Sarma.

Identification of Small Molecule Inhibitors of RNase L by Fragment-Based Drug Discovery.

The pseudokinase-endoribonuclease RNase L plays important roles in antiviral innate immunity and is also implicated in many other cellular activities. The inhibition of RNase L showed therapeutic potential for Aicardi-Goutières syndrome (AGS). Thus, RNase L is a promising drug target. In this study, using an enzyme assay and NMR screening, we discovered 13 inhibitory fragments against RNase L. Cocrystal structures of RNase L separately complexed with two different fragments were determined in which both fragments bound to the ATP-binding pocket of the pseudokinase domain. Myricetin, vitexin, and hyperoside, three natural products sharing similar scaffolds with the fragment AC40357, demonstrated a potent inhibitory activity in vitro. In addition, myricetin has a promising cellular inhibitory activity. A cocrystal structure of RNase L with myricetin provided a structural basis for inhibitor design by allosterically modulating the ribonuclease activity. Our findings demonstrate that fragment screening can lead to the discovery of natural product inhibitors of RNase L.

OSMI-1 Enhances TRAIL-Induced Apoptosis through ER Stress and NF-κB Signaling in Colon Cancer Cells.

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.

Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes.

ß-cell ER stress plays an important role in ß-cell dysfunction and death during the pathogenesis of diabetes. Proinsulin misfolding is regarded as one of the primary initiating factors of ER stress and unfolded protein response (UPR) activation in ß-cells. Here, we found that the ER stress sensor inositol-requiring enzyme 1α (IRE1α) was activated in the Akita mice, a mouse model of mutant insulin gene-induced diabetes of youth (MIDY), a monogenic diabetes. Normalization of IRE1α RNase hyperactivity by pharmacological inhibitors significantly ameliorated the hyperglycemic conditions and increased serum insulin levels in Akita mice. These benefits were accompanied by a concomitant protection of functional ß-cell mass, as shown by the suppression of ß-cell apoptosis, increase in mature insulin production and reduction of proinsulin level. At the molecular level, we observed that the expression of genes associated with ß-cell identity and function was significantly up-regulated and ER stress and its associated inflammation and oxidative stress were suppressed in islets from Akita mice treated with IRE1α RNase inhibitors. This study provides the evidence of the in vivo efficacy of IRE1α RNase inhibitors in Akita mice, pointing to the possibility of targeting IRE1α RNase as a therapeutic direction for the treatment of diabetes.

Tomatidine and Patchouli Alcohol as Inhibitors of SARS-CoV-2 Enzymes (3CLpro, PLpro and NSP15) by Molecular Docking and Molecular Dynamics Simulations.

Considering the current dramatic and fatal situation due to the high spreading of SARS-CoV-2 infection, there is an urgent unmet medical need to identify novel and effective approaches for prevention and treatment of Coronavirus disease (COVID 19) by re-evaluating and repurposing of known drugs. For this, tomatidine and patchouli alcohol have been selected as potential drugs for combating the virus. The hit compounds were subsequently docked into the active site and molecular docking analyses revealed that both drugs can bind the active site of SARS-CoV-2 3CLpro, PLpro, NSP15, COX-2 and PLA2 targets with a number of important binding interactions. To further validate the interactions of promising compound tomatidine, Molecular dynamics study of 100 ns was carried out towards 3CLpro, NSP15 and COX-2. This indicated that the protein-ligand complex was stable throughout the simulation period, and minimal backbone fluctuations have ensued in the system. Post dynamic MM-GBSA analysis of molecular dynamics data showed promising mean binding free energy 47.4633 ± 9.28, 51.8064 ± 8.91 and 54.8918 ± 7.55 kcal/mol, respectively. Likewise, in silico ADMET studies of the selected ligands showed excellent pharmacokinetic properties with good absorption, bioavailability and devoid of toxicity. Therefore, patchouli alcohol and especially, tomatidine may provide prospect treatment options against SARS-CoV-2 infection by potentially inhibiting virus duplication though more research is guaranteed and secured.

Targeting the IRE1α/XBP1s pathway suppresses CARM1-expressing ovarian cancer.

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers.

ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR.

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

Targeting the IRE1α/XBP1 Endoplasmic Reticulum Stress Response Pathway in ARID1A-Mutant Ovarian Cancers.

The SWI/SNF chromatin-remodeling complex is frequently altered in human cancers. For example, the SWI/SNF component ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCC), for which effective treatments are lacking. Here, we report that ARID1A transcriptionally represses the IRE1α-XBP1 axis of the endoplasmic reticulum (ER) stress response, which confers sensitivity to inhibition of the IRE1α-XBP1 pathway in ARID1A-mutant OCCC. ARID1A mutational status correlated with response to inhibition of the IRE1α-XBP1 pathway. In a conditional Arid1aflox/flox/Pik3caH1047R genetic mouse model, Xbp1 knockout significantly improved survival of mice bearing OCCCs. Furthermore, the IRE1α inhibitor B-I09 suppressed the growth of ARID1A-inactivated OCCCs in vivo in orthotopic xenograft, patient-derived xenograft, and the genetic mouse models. Finally, B-I09 synergized with inhibition of HDAC6, a known regulator of the ER stress response, in suppressing the growth of ARID1A-inactivated OCCCs. These studies define the IRE1α-XBP1 axis of the ER stress response as a targetable vulnerability for ARID1A-mutant OCCCs, revealing a promising therapeutic approach for treating ARID1A-mutant ovarian cancers. SIGNIFICANCE: These findings indicate that pharmacological inhibition of the IRE1α-XBP1 pathway alone or in combination with HDAC6 inhibition represents an urgently needed therapeutic strategy for ARID1A-mutant ovarian cancers.

Structural and molecular bases to IRE1 activity modulation.

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy.

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 µM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.