Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease.

The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes.

Inhibitory interactions of the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold with Bunyavirales cap-snatching endonucleases expose relevant drug design features.

The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower µM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 µM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.

Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3.

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.

Repurposing N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human cytomegalovirus pUL89 endonuclease: Synthesis and biological characterization.

The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low µM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.

Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.

Discovery and optimization of new 6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline derivatives as potent influenza virus PAN inhibitors.

Annual unpredictable efficacy of vaccines, coupled with emerging drug resistance, underlines the development of new antiviral drugs to treat influenza infections. The N-terminal domain of the PA (PAN) endonuclease is both highly conserved across influenza strains and serotypes and is indispensable for the viral lifecycle, making it an attractive target for new antiviral therapies. Here, we describe the discovery of a new class of PAN inhibitors derived from recently identified, highly active hits for PAN endonuclease inhibition. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a fragment growing strategy, giving rise to a series of 1, 3-cis-2-substituted-1-(3, 4-dihydroxybenzyl)-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid derivatives as potent PAN inhibitors. This approach ultimately resulted in the development of a new lead compound 13e, which exhibited an EC50 value of 4.50 µM against H1N1 influenza virus in MDCK cells.

Screening and identification of Lassa virus endonuclease-targeting inhibitors from a fragment-based drug discovery library.

Lassa virus (LASV) belongs to the Old World genus Mammarenavirus, family Arenaviridae, and order Bunyavirales. Arenavirus contains a segmented negative-sense RNA genome, which is in line with the bunyavirus and orthomyxoviruses. The segmented negative-sense RNA viruses utilize a cap-snatching strategy to provide primers cleavaged from the host capped mRNA for viral mRNA transcription. As a similar strategy and the conformational conservation shared with these viruses, the endonuclease (EN) would serve as an attractive target for developing broad-spectrum inhibitors. Using the LASV minigenome (MG) system, we screened a fragment-based drug discovery library and found that two hits, F1204 and F1781, inhibited LASV MG activity. Both hits also inhibited the prototype arenavirus Lymphocytic choriomeningitis virus (LCMV) MG activity. Furthermore, both hits effectively inhibited authentic LCMV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections. Similarly, both hits could inhibit the activity of LASV, LCMV, and SFTSV EN. The combination of either compound with an arenavirus entry inhibitor had significant synergistic antiviral effects. Moreover, both hits were found to be capable of binding to LASV EN with a binding affinity at the micromolar level. These findings provide a basis for developing the hits as potential candidates for the treatment of segmented negative-sense RNA virus infections.

Identification of Influenza PAN Endonuclease Inhibitors via 3D-QSAR Modeling and Docking-Based Virtual Screening.

Structural and biochemical studies elucidate that PAN may contribute to the host protein shutdown observed during influenza A infection. Thus, inhibition of the endonuclease activity of viral RdRP is an attractive approach for novel antiviral therapy. In order to envisage structurally diverse novel compounds with better efficacy as PAN endonuclease inhibitors, a ligand-based-pharmacophore model was developed using 3D-QSAR pharmacophore generation (HypoGen algorithm) methodology in Discovery Studio. As the training set, 25 compounds were taken to generate a significant pharmacophore model. The selected pharmacophore Hypo1 was further validated by 12 compounds in the test set and was used as a query model for further screening of 1916 compounds containing 71 HIV-1 integrase inhibitors, 37 antibacterial inhibitors, 131 antiviral inhibitors and other 1677 approved drugs by the FDA. Then, six compounds (Hit01-Hit06) with estimated activity values less than 10 µM were subjected to ADMET study and toxicity assessment. Only one potential inhibitory 'hit' molecule (Hit01, raltegravir's derivative) was further scrutinized by molecular docking analysis on the active site of PAN endonuclease (PDB ID: 6E6W). Hit01 was utilized for designing novel potential PAN endonuclease inhibitors through lead optimization, and then compounds were screened by pharmacophore Hypo1 and docking studies. Six raltegravir's derivatives with significant estimated activity values and docking scores were obtained. Further, these results certainly do not confirm or indicate the seven compounds (Hit01, Hit07, Hit08, Hit09, Hit10, Hit11 and Hit12) have antiviral activity, and extensive wet-laboratory experimentation is needed to transmute these compounds into clinical drugs.

Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor.

Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PAN) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 µM and 26.81 ± 1.2 µM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T's active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

Targeting MUS81 promotes the anticancer effect of WEE1 inhibitor and immune checkpoint blocking combination therapy via activating cGAS/STING signaling in gastric cancer cells.

BACKGROUND: Identification of genomic biomarkers to predict the anticancer effects of indicated drugs is considered a promising strategy for the development of precision medicine. DNA endonuclease MUS81 plays a pivotal role in various biological processes during malignant diseases, mainly in DNA damage repair and replication fork stability. Our previous study reported that MUS81 was highly expressed and linked to tumor metastasis in gastric cancer; however, its therapeutic value has not been fully elucidated. METHODS: Bioinformatics analysis was used to define MUS81-related differential genes, which were further validated in clinical tissue samples. Gain or loss of function MUS81 cell models were constructed to elucidate the effect and mechanism of MUS81 on WEE1 expression. Moreover, the antitumor effect of targeting MUS81 combined with WEE1 inhibitors was verified using in vivo and in vitro assays. Thereafter, the cGAS/STING pathway was evaluated, and the therapeutic value of MUS81 for immunotherapy of gastric cancer was determined. RESULTS: In this study, MUS81 negatively correlated with the expression of cell cycle checkpoint kinase WEE1. Furthermore, we identified that MUS81 regulated the ubiquitination of WEE1 via E-3 ligase ß-TRCP in an enzymatic manner. In addition, MUS81 inhibition could sensitize the anticancer effect of the WEE1 inhibitor MK1775 in gastric cancer in vitro and in vivo. Interestingly, when MUS81 was targeted, it increased the accumulation of cytosolic DNA induced by MK1775 treatment and activated the DNA sensor STING-mediated innate immunity in the gastric cancer cells. Thus, the WEE1 inhibitor MK1775 specifically enhanced the anticancer effect of immune checkpoint blockade therapy in MUS81 deficient gastric cancer cells. CONCLUSIONS: Our data provide rational evidence that targeting MUS81 could elevate the expression of WEE1 by regulating its ubiquitination and could activate the innate immune response, thereby enhancing the anticancer efficacy of WEE1 inhibitor and immune checkpoint blockade combination therapy in gastric cancer cells.

Comparison of Antiviral Agents for Seasonal Influenza Outcomes in Healthy Adults and Children: A Systematic Review and Network Meta-analysis.

Importance: Antiviral treatment of influenza is recommended for patients with influenza-like illness during periods of community cocirculation of influenza viruses and SARS-CoV-2; however, questions remain about which treatment is associated with the best outcomes and fewest adverse events. Objective: To compare the efficacy and safety of neuraminidase inhibitors and the endonuclease inhibitor for the treatment of seasonal influenza among healthy adults and children. Data Sources: Medline, Embase, and the Cochrane Register of Clinical Trials were searched from inception to January 2020 (the last search was updated in October 2020). Study Selection: Included studies were randomized clinical trials conducted among patients of all ages with influenza treated with neuraminidase inhibitors (ie, oseltamivir, peramivir, zanamivir, or laninamivir) or an endonuclease inhibitor (ie, baloxavir) compared with other active agents or placebo. Data Extraction and Synthesis: Two investigators identified studies and independently abstracted data. Frequentist network meta-analyses were performed; relative ranking of agents was conducted using P-score probabilities. Quality of evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations criteria. Data were analyzed in October 2020. Main Outcomes and Measures: The time to alleviation of influenza symptoms (TTAS), complications of influenza, and adverse events (total adverse events, nausea, and vomiting). Results: A total of 26 trials were identified that investigated antiviral drugs at high or low doses; these trials included 11 897 participants, among whom 6294 (52.9%) were men and the mean (SD) age was 32.5 (16.9) years. Of all treatments comparing with placebo in efficacy outcomes, high-quality evidence indicated that zanamivir was associated with the shortest TTAS (hazard ratio, 0.67; 95% CI, 0.58-0.77), while baloxavir was associated with the lowest risk of influenza-related complications (risk ratio [RR], 0.51; 95% CI, 0.32-0.80) based on moderate-quality evidence. In safety outcomes, baloxavir was associated with the lowest risk of total adverse events (RR, 0.84; 95% CI, 0.74-0.96) compared with placebo based on moderate-quality evidence. There was no strong evidence of associations with risk of nausea or vomiting among all comparisons, except for 75 mg oseltamivir, which was associated with greater occurrence of nausea (RR, 1.82; 95% CI, 1.38-2.41) and vomiting (RR, 1.88; 95% CI, 1.47-2.41). Conclusions and Relevance: In this systematic review and network meta-analysis, all 4 antiviral agents assessed were associated with shortening TTAS; zanamivir was associated with the shortest TTAS, and baloxavir was associated with reduced rate of influenza-related complications.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition.

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

Synthesis and In Vitro Evaluation of C-7 and C-8 Luteolin Derivatives as Influenza Endonuclease Inhibitors.

The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3',4'-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.

Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex.

The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low µM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.

Developing a Peptide That Inhibits DNA Repair by Blocking the Binding of Artemis and DNA Ligase IV to Enhance Tumor Radiosensitivity.

PURPOSE: Artemis and DNA Ligase IV are 2 critical elements in the nonhomologous end joining pathway of DNA repair, acting as the nuclease and DNA ligase, respectively. Enhanced cellular radiosensitivity by inhibition of either protein contributes to a promising approach to develop molecular targeted radiosensitizers. The interaction between Artemis and DNA Ligase IV is required for the activation of Artemis as nuclease at 3'overhang DNA; thus, we aim to generate an inhibitory peptide targeting the interaction between Artemis and DNA Ligase IV for novel radiosensitizer development. METHODS AND MATERIALS: We synthesized the peptide BAL, which consists of the interaction residues of Artemis to DNA Ligase IV. The radiosensitization effect of BAL was evaluated by colony formation assay. The effects of BAL on radiation-induced DNA repair were evaluated with Western blotting and immunofluorescence. The effects of BAL on cell proliferation, cell cycle arrest, and cell apoptosis were assessed via CCK-8 and flow cytometry assays. The potential synergistic effects of BAL and irradiation in vivo were investigated in a xenograft mouse model. RESULTS: The generated peptide BAL blocking the interaction between Artemis and DNA Ligase IV significantly enhanced the radiosensitivity of GBC-SD and HeLa cell lines. BAL prolonged DNA repair after irradiation; BAL and irradiation showed synergistic effects on cell proliferation, cell cycle, and cell apoptosis, and these functions are all DNA Ligase IV-related. Finally, we confirmed the endogenous radiosensitization effect of BAL in a xenograft mouse model. CONCLUSIONS: The inhibitory peptide BAL targeting the binding of Artemis and DNA Ligase IV successfully functions as a novel radiosensitizer that delays DNA repair and synergizes with irradiation to inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis.

Overexpression of AtBBD1, Arabidopsis Bifunctional Nuclease, Confers Drought Tolerance by Enhancing the Expression of Regulatory Genes in ABA-Mediated Drought Stress Signaling.

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.

The active form of the influenza cap-snatching endonuclease inhibitor baloxavir marboxil is a tight binding inhibitor.

Baloxavir marboxil (BXM) is an FDA-approved antiviral prodrug for the treatment of influenza A and B infection and postexposure prophylaxis. The active form, baloxavir acid (BXA), targets the cap-snatching endonuclease (PA) of the influenza virus polymerase complex. The nuclease activity delivers the primer for transcription, and previous reports have shown that BXA blocks the nuclease activity with high potency. However, biochemical studies on the mechanism of action are lacking. Structural data have shown that BXA chelates the two divalent metal ions at the active site, like inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase or ribonuclease (RNase) H. Here we studied the mechanisms underlying the high potency of BXA and how the I38T mutation confers resistance to the drug. Enzyme kinetics with the recombinant heterotrimeric enzyme (FluB-ht) revealed characteristics of a tight binding inhibitor. The apparent inhibitor constant (Kiapp) is 12 nM, while the I38T mutation increased Kiapp by ∼18-fold. Order-of-addition experiments show that a preformed complex of FluB-ht, Mg2+ ions and BXA is required to observe inhibition, which is consistent with active site binding. Conversely, a preformed complex of FluB-ht and RNA substrate prevents BXA from accessing the active site. Unlike integrase inhibitors that interact with the DNA substrate, BXA behaves like RNase H inhibitors that compete with the nucleic acid at the active site. The collective data support the conclusion that BXA is a tight binding inhibitor and the I38T mutation diminishes these properties.

Diketo acids inhibit the cap-snatching endonuclease of several Bunyavirales.

Several fatal bunyavirus infections lack specific treatment. Here, we show that diketo acids engage a panel of bunyavirus cap-snatching endonucleases, inhibit their catalytic activity and reduce viral replication of a taxonomic representative in vitro. Specifically, the non-salt form of L-742,001 and its derivatives exhibited EC50 values between 5.6 and 6.9 µM against a recombinant BUNV-mCherry virus. Structural analysis and molecular docking simulations identified traits of both the class of chemical entities and the viral target that could help the design of novel, more potent molecules for the development of pan-bunyavirus antivirals.

Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors.

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.