Guanosine Nucleotides Research Articles

Localization and expression of CRSH transcript, level of calcium ions, and cell cycle activity during Brassica napus L. seed development

Seeds are used as human food and animal feed, but the seed is also the onset of a new life. Therefore, seeds are a crucial agricultural product for global food security. Among several mechanisms involved in seed formation and maturation, the stringent response seems to be of great but rather overlooked significance. Plant homologs of bacterial RelA/SpoT proteins, called RSH, metabolize hyperphosphorylated regulatory nucleotides guanosine tetra- and pentaphosphate ((p)ppGpp, alarmones) which are involved in varied aspects of plant growth and development. The role of plant RSHs and alarmones in seed development remains elusive. In this study, the possible function of Ca2+-dependent RSH (CRSH) proteins during seed formation and maturation was verified. In silico analysis of plant CRSH proteins showed that EF-hand calcium-binding motif was highly conserved in monocotyledonous and dicotyledonous plants. Remarkably, this motif is present only in plant CRSH and has not been found in bacterial homologs. In developing Brassica napus (canola) seeds we analyzed the level and the localization of BnCRSH mRNAs by RT-qPCR and in situ localization, respectively. Further, we examined the cell cycle activity via flow cytometry, and the level of calcium ions using scanning electron microscopy. Our results showed that DNA replication intensity was at the highest level in seeds at the early stages of development, and then constantly decreased to the minimal level reached 70 days after flowering. In contrast, the level of calcium ions and BnCRSH transcript increased during canola seed maturation. The results of this study strongly suggest that calcium-dependent stringent response plays a significant role during canola seed development.

Understanding the Stringent Response: Experimental Context Matters.

As rapidly growing bacteria begin to exhaust essential nutrients, they enter a state of reduced growth, ultimately leading to stasis or quiescence. Investigation of the response to nutrient limitation has focused largely on the consequences of amino acid starvation, known as the "stringent response." Here, an uncharged tRNA in the A-site of the ribosome stimulates the ribosome-associated protein RelA to synthesize the hyperphosphorylated guanosine nucleotides (p)ppGpp that mediate a global slowdown of growth and biosynthesis. Investigations of the stringent response typically employ experimental methodologies that rapidly stimulate (p)ppGpp synthesis by abruptly increasing the fraction of uncharged tRNAs, either by explicit amino starvation or by inhibition of tRNA charging. Consequently, these methodologies inhibit protein translation, thereby interfering with the cellular pathways that respond to nutrient limitation. Thus, complete and/or rapid starvation is a problematic experimental paradigm for investigating bacterial responses to physiologically relevant nutrient-limited states.

Loss of ARHGAP15 affects the directional control of migrating interneurons in the embryonic cortex and increases susceptibility to epilepsy.

GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.

New perspectives on the molecular mechanisms of stress signalling by the nucleotide guanosine tetraphosphate (ppGpp), an emerging regulator of photosynthesis in plants and algae.

The nucleotides guanosine tetraphosphate and guanosine pentaphosphate (together (p)ppGpp) are found in a wide range of prokaryotic and eukaryotic organisms where they are associated with stress signalling. In this review, we will discuss recent research highlighting the role of (p)ppGpp signalling as a conserved regulator of photosynthetic activity in the chloroplasts of plants and algae, and the latest discoveries that open up new perspectives on the emerging roles of (p)ppGpp in acclimation to environmental stress. We explore how rapid advances in the study of (p)ppGpp signalling in prokaryotes are now revealing large gaps in our understanding of the molecular mechanisms of signalling by (p)ppGpp and related nucleotides in plants and algae. Filling in these gaps is likely to lead to the discovery of conserved as well as new plant- and algal-specific (p)ppGpp signalling mechanisms that will offer new insights into the taming of the chloroplast and the regulation of stress tolerance.

Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis.

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

Monitoring the Dynamic Regulation of the Mitochondrial GTP‐to‐GDP Ratio with a Genetically Encoded Fluorescent Biosensor

AbstractThe interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is known to be integral to a wide variety of biological cellular activities, yet to date there are no analytical methods available to directly detect the ratio of intracellular GTP to GDP. Herein, we report GRISerHR, a genetically encoded fluorescent biosensor to monitor the GTP : GDP ratio in multiple cell types and in various organelles under metabolic perturbation. Additionally, we characterized the differential mitochondrial GTP : GDP ratios resulting from genetic modulation of two isoforms of a tricarboxylic acid (TCA) cycle enzyme (succinyl‐CoA synthetase; SCS‐ATP and SCS‐GTP) and of a phosphoenolpyruvate (PEP) cycle enzyme (PEPCK‐M). Thus, our GRISerHR sensor achieves spatiotemporally precise detection of dynamic changes in the endogenous GTP : GDP ratio in living cells and can help deepen our understanding about the energy metabolic contributions of guanosine nucleotides in biology.

Monitoring the Dynamic Regulation of the Mitochondrial GTP-to-GDP Ratio with a Genetically Encoded Fluorescent Biosensor.

The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is known to be integral to a wide variety of biological cellular activities, yet to date there are no analytical methods available to directly detect the ratio of intracellular GTP to GDP. Herein, we report GRISerHR, a genetically encoded fluorescent biosensor to monitor the GTP : GDP ratio in multiple cell types and in various organelles under metabolic perturbation. Additionally, we characterized the differential mitochondrial GTP : GDP ratios resulting from genetic modulation of two isoforms of a tricarboxylic acid (TCA) cycle enzyme (succinyl-CoA synthetase; SCS-ATP and SCS-GTP) and of a phosphoenolpyruvate (PEP) cycle enzyme (PEPCK-M). Thus, our GRISerHR sensor achieves spatiotemporally precise detection of dynamic changes in the endogenous GTP : GDP ratio in living cells and can help deepen our understanding about the energy metabolic contributions of guanosine nucleotides in biology.

Dynamically Self-Assembled Supramolecular Probes in Liposomes

Liposomes are artificial vesicles, in which an aqueous inner compartment is separated from its environment by a phospholipid membrane. They have been extensively studied as cell membrane models and offer the possibility to confine molecules and chemical reactions to a small sub-micrometer-sized volume. This short review provides an overview of liposome-encapsulated, dynamically self-assembled, supramolecular structures, in which the assembly and disassembly of the supramolecular structures can be followed by optical spectroscopic methods. This includes self-quenched fluorescent dyes and dye/quencher pairs, helical stacks of guanosine nucleotides, dynamic covalent boronate esters, and supramolecular host–guest complexes. The resulting liposomes are typically used to study membrane transport processes, but the results summarized herein also serve as a potential blueprint for studying dynamic self-assembly in confined spaces by optical spectroscopic methods.Table of content:1 Introduction2 Probes Based on Fluorescence Quenching3 Chirogenic G-Quartet Probes4 Chromogenic Probes Using Dynamic Covalent Bonds5 Self-Assembled Host–Dye Reporter Pairs6 Conclusions and Outlook

Structural variations between small alarmone hydrolase dimers support different modes of regulation of the stringent response

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelHCg) and Leptospira levettii (RelHLl) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation.

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.

Spatially resolved in silico modeling of NKG2D signaling kinetics suggests a key role of NKG2D and Vav1 Co-clustering in generating natural killer cell activation.

Natural Killer (NK) cells provide key resistance against viral infections and tumors. A diverse set of activating and inhibitory NK cell receptors (NKRs) interact with cognate ligands presented by target host cells, where integration of dueling signals initiated by the ligand-NKR interactions determines NK cell activation or tolerance. Imaging experiments over decades have shown micron and sub-micron scale spatial clustering of activating and inhibitory NKRs. The mechanistic roles of these clusters in affecting downstream signaling and activation are often unclear. To this end, we developed a predictive in silico framework by combining spatially resolved mechanistic agent based modeling, published TIRF imaging data, and parameter estimation to determine mechanisms by which formation and spatial movements of activating NKG2D microclusters affect early time NKG2D signaling kinetics in a human cell line NKL. We show co-clustering of NKG2D and the guanosine nucleotide exchange factor Vav1 in NKG2D microclusters plays a dominant role over ligand (ULBP3) rebinding in increasing production of phospho-Vav1(pVav1), an activation marker of early NKG2D signaling. The in silico model successfully predicts several scenarios of inhibition of NKG2D signaling and time course of NKG2D spatial clustering over a short (~3 min) interval. Modeling shows the presence of a spatial positive feedback relating formation and centripetal movements of NKG2D microclusters, and pVav1 production offers flexibility towards suppression of activating signals by inhibitory KIR ligands organized in inhomogeneous spatial patterns (e.g., a ring). Our in silico framework marks a major improvement in developing spatiotemporal signaling models with quantitatively estimated model parameters using imaging data.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

Targeted disruption of Rab1a causes early embryonic lethality.

Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5-14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a β-galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for β-galactosidase to determine the tissue-specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild-type and heterozygous Rab1a+/− mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.

AT-752, a double prodrug of a guanosine nucleotide analog, inhibits yellow fever virus in a hamster model.

Yellow fever virus (YFV) is a zoonotic pathogen re-emerging in parts of the world, causing a viral hemorrhagic fever associated with high mortality rates. While an effective vaccine is available, having an effective antiviral against YFV is critical against unexpected outbreaks, or when vaccination is not recommended. We have previously identified AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, as a potent inhibitor of YFV in vitro, with a 50% effective concentration (EC50) of 0.31 μM. In hamsters infected with YFV (Jimenez strain), viremia rose about 4 log10-fold and serum alanine aminotransferase (ALT) 2-fold compared to sham-infected animals. Treatment with 1000 mg/kg AT-752 for 7 days, initiated 4 h prior to viral challenge, reduced viremia to below the limit of detection by day 4 post infection (pi) and returned ALT to normal levels by day 6 pi. When treatment with AT-752 was initiated 2 days pi, the virus titer and ALT dropped >2 log10 and 53% by day 4 and 6 pi, respectively. In addition, at 21 days pi, 70–100% of the infected animals in the treatment groups survived compared to 0% of the untreated group (p<0.001). Moreover, in vivo formation of the active triphosphate metabolite AT-9010 was measured in the animal tissues, with the highest concentrations in liver and kidney, organs that are vulnerable to the virus. The demonstrated in vivo activity of AT-752 suggests that it is a promising compound for clinical development in the treatment of YFV infection.

Nuclear Magnetic Resonance Spectra and AMBER OL3 and ROC-RNA Simulations of UCUCGU Reveal Force Field Strengths and Weaknesses for Single-Stranded RNA.

Single-stranded regions of RNA are important for folding of sequences into 3D structures and for design of therapeutics targeting RNA. Prediction of ensembles of 3D structures for single-stranded regions often involves classical mechanical approximations of interactions defined by quantum mechanical calculations on small model systems. Nuclear magnetic resonance (NMR) spectra and molecular dynamics (MD) simulations of short single strands provide tests for how well the approximations model many of the interactions. Here, the NMR spectra for UCUCGU at 2, 15, and 30 °C are compared to simulations with the AMBER force fields, OL3 and ROC-RNA. This is the first such comparison to an oligoribonucleotide containing an internal guanosine nucleotide (G). G is particularly interesting because of its many H-bonding groups, large dipole moment, and proclivity for both syn and anti conformations. Results reveal formation of a G amino to phosphate non-bridging oxygen H-bond. The results also demonstrate dramatic differences in details of the predicted structures. The variations emphasize the dependence of predictions on individual parameters and their balance with the rest of the force field. The NMR data can serve as a benchmark for future force fields.

How Structural Biology Has Directly Impacted Our Understanding of P2X Receptor Function and Gating.

P2X receptors are ATP-gated ion channels expressed in a wide variety of eukaryotic cells. They play key roles in diverse processes such as platelet activation, smooth muscle contraction, synaptic transmission, nociception, cell proliferation, and inflammation making this receptor family an important pharmacological target. Structures of P2X receptors solved by X-ray crystallography have been instrumental in helping to define mechanisms of molecular P2X receptor function. In 2009, the first X-ray structure of the P2X4 receptor subtype confirmed a trimeric stoichiometry and revealed the overall architecture of the functional ion channel. Subsequent X-ray structures have provided the molecular details to define the orthosteric ATP binding pocket, the orthosteric antagonist binding pocket, an allosteric antagonist binding pocket, and the pore architecture in each of the major conformational states of the receptor gating cycle. Moreover, the unique gating mechanism by which P2X receptor subtypes desensitize at differing rates, referred to as the helical recoil model of receptor desensitization, was discovered directly from X-ray structures of the P2X3 receptor. However, structures of P2X receptors solved by X-ray crystallography have only been able to provide limited information on the cytoplasmic domain of this receptor family, as this domain was always truncated to varying degrees in order to facilitate crystallization. Because the P2X7 receptor subtype has a significantly larger cytoplasmic domain that has been shown to be necessary for its ability to initiate apoptosis, an absence of structural information on the P2X7 receptor cytoplasmic domain has limited our understanding of its complex signaling pathways as well as its unusual ability to remain open without undergoing desensitization. This absence of cytoplasmic structural information for P2X7 receptors was recently overcome when the first full-length P2X7 receptor structures were solved by single-particle cryogenic electron microscopy. These structures finally provide insight into the large and unique P2X7 receptor cytoplasmic domain and revealed two novel structural elements and several surprising findings: first, a cytoplasmic structural element called the cytoplasmic ballast was identified that contains a dinuclear zinc ion complex and a high affinity guanosine nucleotide binding site and second, a palmitoylated membrane proximal structural element called the C-cys anchor was identified which prevents P2X7 receptor desensitization. This chapter will highlight the major structural and functional aspects of P2X receptors discovered through structural biology, with a key emphasis on the most recent cryogenic electron microscopy structures of the full-length, wild-type P2X7 receptor.

Pharmacogenomics of mycophenolic acid in kidney transplantation: Contribution of immune response-related genes

Mycophenolic acid (MPA) inhibits IMPDH, involved in the guanosine nucleotides synthesis, and prevents DNA replication in immune cells. The repression of cell and humoral immunity by MPA induces allograft tolerance preventing acute rejection in solid organ transplantation. MPA is an effective and safe drug, but genetic and non-genetic factors have been implicated in the interindividual variability of drug response. Several studies have shown the impact of variants of pharmacokinetics or pharmacodynamics-related genes on MPA response in kidney transplantation. This review explored further the influence of genes involved in the immune response on clinical outcomes of kidney recipients on short- or long-term MPA treatment. Variants in genes related to T cell activation (CD28, CTL4, ICOS, PDPC1), pro-inflammatory cytokines (IL2, IL6, IL12A, IL12B, TNF, IFNG), immunomodulatory cytokines (IL4, IL10, TGFB1), and innate immune response (CD14, TLR2, TLR4) were shown to be associated with increased risk of acute rejection, graft function or survival, chronic graft nephropathy, viral infections or MPA-induced myelotoxicity. Some of the significant pharmacogenetic associations were confirmed by meta-analyses of kidney transplantation. These findings are suggestive that variants in immune response-related genes contribute to the variability of MPA response, and have potential application as biomarkers of acute rejection in kidney transplantation.

Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

Evaluation of AT-752, a Double Prodrug of a Guanosine Nucleotide Analog with In Vitro and In Vivo Activity against Dengue and Other Flaviviruses.

ABSTRACTEvery year, millions of people worldwide are infected with dengue virus (DENV), with a significant number developing severe life-threatening disease. There are currently no broadly indicated vaccines or therapeutics available for treatment of DENV infection. Here, we show that AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, was a potent inhibitor of DENV serotypes 2 and 3 in vitro, requiring concentrations of 0.48 and 0.77 μM, respectively, to inhibit viral replication by 50% (EC50) in Huh-7 cells. AT-281 was also a potent inhibitor of all other flaviviruses tested, with EC50 values ranging from 0.19 to 1.41 μM. Little to no cytotoxicity was observed for AT-281 at concentrations up to 170 μM. After oral administration of AT-752, substantial levels of the active triphosphate metabolite AT-9010 were formed in vivo in peripheral blood mononuclear cells of mice, rats, and monkeys. Furthermore, AT-9010 competed with GTP in RNA template-primer elongation assays with DENV2 RNA polymerase, which is essential for viral replication, with incorporation of AT-9010 resulting in termination of RNA synthesis. In AG129 mice infected with DENV D2Y98P, treatment with AT-752 significantly reduced viremia and morbidity and increased survival. The demonstrated in vitro and in vivo activity of AT-752 suggests that it is a promising compound for the treatment of dengue virus infection and is currently under evaluation in clinical studies.