Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due ...to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs
and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC
) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC
The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same
resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.
Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV
and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.
Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral ...therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC
values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.
GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under ...compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.
The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening ...and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3–14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals Nature 2016, 531, 381−385 . A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood ...outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.
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•Nutritional ketone bodies can promote the advantageous aspects to starvation ketosis•Nutritional ketosis alters the hierarchy of substrate competition for respiration in exercise•Ketosis increases metabolic flexibility during exercise, reducing glycolysis and increasing muscle fat oxidation•Improved performance during cycling time trial suggests ketosis during exercise may be beneficial for some athletes
Cox et al. show the metabolic benefit of ketone metabolism through the administration of a ketone ester-based drink to athletes during exercise. The physiological alterations achieved by acute nutritional ketosis may improve human physical performance in some athletes as indicated by initial endurance test results.
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for ...safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson’s disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease ...progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V
and a A53T–α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T–SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V
mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T–SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a diseasemodifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.
Abstract Alzheimer's disease (AD) involves progressive accumulation of amyloid β-peptide (Aβ) and neurofibrillary pathologies, and glucose hypometabolism in brain regions critical for memory. The ...3xTgAD mouse model was used to test the hypothesis that a ketone ester–based diet can ameliorate AD pathogenesis. Beginning at a presymptomatic age, 2 groups of male 3xTgAD mice were fed a diet containing a physiological enantiomeric precursor of ketone bodies (KET) or an isocaloric carbohydrate diet. The results of behavioral tests performed at 4 and 7 months after diet initiation revealed that KET-fed mice exhibited significantly less anxiety in 2 different tests. 3xTgAD mice on the KET diet also exhibited significant, albeit relatively subtle, improvements in performance on learning and memory tests. Immunohistochemical analyses revealed that KET-fed mice exhibited decreased Aβ deposition in the subiculum, CA1 and CA3 regions of the hippocampus, and the amygdala. KET-fed mice exhibited reduced levels of hyperphosphorylated tau deposition in the same regions of the hippocampus, amygdala, and cortex. Thus, a novel ketone ester can ameliorate proteopathic and behavioral deficits in a mouse AD model.
Epidemiological studies suggest that insulin resistance accelerates progression of age-based cognitive impairment, which neuroimaging has linked to brain glucose hypometabolism. As cellular inputs, ...ketones increase Gibbs free energy change for ATP by 27% compared to glucose. Here we test whether dietary changes are capable of modulating sustained functional communication between brain regions (network stability) by changing their predominant dietary fuel from glucose to ketones. We first established network stability as a biomarker for brain aging using two large-scale (n = 292, ages 20 to 85 y; n = 636, ages 18 to 88 y) 3 T functional MRI (fMRI) datasets. To determine whether diet can influence brain network stability, we additionally scanned 42 adults, age < 50 y, using ultrahigh-field (7 T) ultrafast (802 ms) fMRI optimized for single-participant-level detection sensitivity. One cohort was scanned under standard diet, overnight fasting, and ketogenic diet conditions. To isolate the impact of fuel type, an independent overnight fasted cohort was scanned before and after administration of a calorie-matched glucose and exogenous ketone ester (D-β-hydroxybutyrate) bolus. Across the life span, brain network destabilization correlated with decreased brain activity and cognitive acuity. Effects emerged at 47 y, with the most rapid degeneration occurring at 60 y. Networks were destabilized by glucose and stabilized by ketones, irrespective of whether ketosis was achieved with a ketogenic diet or exogenous ketone ester. Together, our results suggest that brain network destabilization may reflect early signs of hypometabolism, associated with dementia. Dietary interventions resulting in ketone utilization increase available energy and thus may show potential in protecting the aging brain.
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