The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity.

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells.

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.

IMPORTANCE: We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction and cellular dNTP levels.

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of an additional SAMHD1-mediated antiviral activity independent of T592 de-phosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet poor correlation of HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.

The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity.

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but minimal efficacy with substantial toxicity in clinical trials. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, these findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Mechanistic Interplay between HIV-1 Reverse Transcriptase Enzyme Kinetics and Host SAMHD1 Protein: Viral Myeloid-Cell Tropism and Genomic Mutagenesis.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been the primary interest among studies on antiviral discovery, viral replication kinetics, drug resistance, and viral evolution. Following infection and entry into target cells, the HIV-1 core disassembles, and the viral RT concomitantly converts the viral RNA into double-stranded proviral DNA, which is integrated into the host genome. The successful completion of the viral life cycle highly depends on the enzymatic DNA polymerase activity of RT. Furthermore, HIV-1 RT has long been known as an error-prone DNA polymerase due to its lack of proofreading exonuclease properties. Indeed, the low fidelity of HIV-1 RT has been considered as one of the key factors in the uniquely high rate of mutagenesis of HIV-1, which leads to efficient viral escape from immune and therapeutic antiviral selective pressures. Interestingly, a series of studies on the replication kinetics of HIV-1 in non-dividing myeloid cells and myeloid specific host restriction factor, SAM domain, and HD domain-containing protein, SAMHD1, suggest that the myeloid cell tropism and high rate of mutagenesis of HIV-1 are mechanistically connected. Here, we review not only HIV-1 RT as a key antiviral target, but also potential evolutionary and mechanistic crosstalk among the unique enzymatic features of HIV-1 RT, the replication kinetics of HIV-1, cell tropism, viral genetic mutation, and host SAMHD1 protein.

Nucleotide Analogues Bearing a C2' or C3'-Stereogenic All-Carbon Quaternary Center as SARS-CoV-2 RdRp Inhibitors.

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2' or C3' is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2' was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.

Elimination of Aicardi-Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication.

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi-Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR-Cas9 gene KO or lentiviral viral protein X-mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2-infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment.

Current systemic treatment options for patients with adrenocortical carcinomas (ACCs) are far from being satisfactory. DNA damage/repair mechanisms, which involve, e.g., ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia/Rad3-related (ATR) protein signaling or ribonucleotide reductase subunits M1/M2 (RRM1/RRM2)-encoded ribonucleotide reductase (RNR) activation, commonly contribute to drug resistance. Moreover, the regulation of RRM2b, the p53-induced alternative to RRM2, is of unclear importance for ACC. Upon extensive drug screening, including a large panel of chemotherapies and molecular targeted inhibitors, we provide strong evidence for the anti-tumoral efficacy of combined gemcitabine (G) and cisplatin (C) treatment against the adrenocortical cell lines NCI-H295R and MUC-1. However, accompanying induction of RRM1, RRM2, and RRM2b expression also indicated developing G resistance, a frequent side effect in clinical patient care. Interestingly, this effect was partially reversed upon addition of C. We confirmed our findings for RRM2 protein, RNR-dependent dATP levels, and modulations of related ATM/ATR signaling. Finally, we screened for complementing inhibitors of the DNA damage/repair system targeting RNR, Wee1, CHK1/2, ATR, and ATM. Notably, the combination of G, C, and the dual RRM1/RRM2 inhibitor COH29 resulted in previously unreached total cell killing. In summary, we provide evidence that RNR-modulating therapies might represent a new therapeutic option for ACC.

A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site.

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

Baicalein and Baicalin Inhibit SARS-CoV-2 RNA-Dependent-RNA Polymerase.

Coronavirus Disease 2019 (COVID-19) is a deadly emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 is easily transmitted through the air and has a relatively long incubation time, COVID-19 has rapidly developed into a global pandemic. As there are no antiviral agents for the prevention and treatment of this severe pathogen except for remdesivir, development of antiviral therapies to treat infected individuals remains highly urgent. Here, we showed that baicalein and baicalin exhibited significant antiviral activity against SARS-CoV-2, the causative agent of COVID-19 through in vitro studies. Our data through cell-based and biochemical studies showed that both compounds act as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors directly and inhibit the activity of the SARS-CoV-2 RdRp, but baicalein was more potent. We also showed specific binding of baicalein to the SARS-CoV-2 RdRp, making it a potential candidate for further studies towards therapeutic development for COVID-19 as a selective non-nucleoside polymerase inhibitor.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

Tetraspanin CD81 regulates HSV-1 infection.

Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.

Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages.

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

Dengue Outbreak during Ongoing Civil War, Taiz, Yemen.

We identified dengue in ≈51% of patients given a clinical diagnosis of suspected dengue in Taiz, Yemen, during 2016. The cosmopolitan genotype of dengue virus type 2 was most common; viruses appeared to have originated in Saudi Arabia. Damage to public health infrastructure during the ongoing civil war might enable dengue to become endemic to Yemen.

Baicalein and baicalin as Zika virus inhibitors.

At present, there is no effective antiviral agent for Zika virus (ZIKV), an arbovirus that is known for its teratogenic effects on newborns. Baicalein and baicalin were found to be capable of downregulating ZIKV replication up to 10 hours postinfection, while prophylactic effects were evident in pre-treated cells. Baicalein exhibited its highest potency during intracellular ZIKV replication, whereas baicalin was most effective against virus entry. Our in silico interaction assays predicted that both compounds exhibited the strongest binding affinities towards ZIKV NS5, while the virus envelope glycoprotein was the least likely target protein. These findings serve as a crucial platform for further in-depth studies to decipher the underlying anti-ZIKV mechanism(s) of each compound.

Deciphering the potential of baicalin as an antiviral agent for Chikungunya virus infection.

The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 µM, ≈ 350 µM and ≈110 µM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 µM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of -9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 µM followed by the inhibition of synthesis of positive-strand genomic (EC50 ≈ 13 µM) and subgenomic RNAs (EC50 ≈ 14 µM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 µM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.

Exploring the in vitro potential of celecoxib derivative AR-12 as an effective antiviral compound against four dengue virus serotypes.

Objectives: With no clinically effective antiviral options available, infections and fatalities associated with dengue virus (DENV) have reached an alarming level worldwide. We have designed this study to evaluate the efficacy of the celecoxib derivative AR-12 against the in vitro replication of all four DENV serotypes. Methods: Each 24-well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR-12. Following 48 h of incubation, inhibitory efficacies of AR-12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT-PCR. The underlying mechanism(s) possibly involved in the compound's inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains. Results: The qRT-PCR data demonstrated that DENV-3 was most potently inhibited by AR-12, followed by DENV-1, DENV-2 and DENV-4. Our molecular docking findings suggested that AR-12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins. Conclusions: These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR-12 as a potential therapeutic option for DENV infection.

Flavonoids: promising natural compounds against viral infections.

Flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. Different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. This review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. We also present future perspectives on therapeutic applications of flavonoids against viral infections.