The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral ...RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.
Abstract
The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CL
pro
) ...is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CL
pro
. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CL
pro
s, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.
Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the ...SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.
Favipiravir was first synthesized from an inexpensive and commercially available starting material, 2-aminopyrazine. The preferred route embedded within Scheme
4
consisted of seven steps, and was ...highlighted by the novel and efficient synthesis of 3,6-dichloropyrazine-2-carbonitrile
8
. This intermediate was prepared in four successive steps which were regioselective chlorination of the pyrazine ring, bromination, Pd-catalyzed cyanation, and Sandmeyer diazotization/chlorination. This protocol eliminated the hazardous POCl
3
of previous synthetic methods and offered a better yield (48%) which was 1.3-fold higher than a recently published procedure. From intermediate
8
, the subsequent nucleophilic fluorination, nitrile hydration and hydroxyl substitution efficiently afforded the target product. Another synthetic approach with the same starting material was also investigated to bypass the allergy-causing dichloro intermediate
8
. However, the key step of monofluorination at the pyrazine C6 position of intermediate
19
or
22
was not achieved.
Abstract
The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for ...developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CL
pro
) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CL
pro
with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CL
pro
inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.
•Different dose ketamine induced different behavior at 24 h after treatment.•Low-dose ketamine induced antidepressant and anxiolytic effects.•High-dose ketamine induced cognitive impairment and ...pro-depression behavior.•The antidepressant effects of ketamine involving BDNF levels in hippocampus.
Clinical and preclinical researches have shown that sub-anesthetic ketamine elicits sustained antidepressant effects for up to 1–2 weeks. Pharmacokinetics studies (t1/2 = 23 min) in mice showed no ketamine residue at 24 h after sub-anesthetic or anesthetic ketamine administration. Therefore, this study aims to reveal the mechanism underlying these different biological functions at 24 h after sub-anesthetic and anesthetic ketamine treatment. First, at the animal behavioral level, we found that sub-anesthetic ketamine induced antidepressant and anxiolytic effects while anesthetic ketamine induced depressive-like phenotypes and cognitive impairment. Second, we examined the correlation between behavior phenotype and protein expression, and found that the Brain-derived neurotrophic factor (BDNF) level is oppositely regulated by sub-anesthetic and anesthetic ketamine. Sub-anesthetic ketamine significantly increased the BDNF level, correlating to antidepressant effects; whereas anesthetic dose reduced BDNF expression in the hippocampus, correlating to depressive-like behaviors, anxiety-like behaviors and cognitive impairment. Third, the antidepressant effects of sub-anesthetic ketamine were prevented by pre-treatment of ANA-12, a Tropomyosin receptor kinase B (TrkB) inhibitor. Thus, we conclude that BDNF may be the key factor underlying antidepressant and anxiolytic effects of sub-anesthetic ketamine at 24 h after treatment. These results may shed light on future studies and the development of long-lasting anti-depressant drugs and therapies.
Polycomb Repressive Complex 2 (PRC2) modulates the chromatin structure and transcriptional repression by trimethylation lysine 27 of histone H3 (H3K27me3), a process that necessitates the ...protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED. Deregulated PRC2 is intimately involved in tumorigenesis and progression, making it an invaluable target for epigenetic cancer therapy. However, until now, there have been no reported small molecule compounds targeting the EZH2-EED interactions. In the present study, we identified astemizole, an FDA-approved drug, as a small molecule inhibitor of the EZH2-EED interaction of PRC2. The disruption of the EZH2-EED interaction by astemizole destabilizes the PRC2 complex and inhibits its methyltransferase activity in cancer cells. Multiple lines of evidence have demonstrated that astemizole arrests the proliferation of PRC2-driven lymphomas primarily by disabling the PRC2 complex. Our findings demonstrate the chemical tractability of the difficult PPI target by a small molecule compound, highlighting the therapeutic promise for PRC2-driven human cancers via targeted destruction of the EZH2-EED complex.
Human inflammatory disease, multiple sclerosis (MS), is a demyelinating disease of central nervous system (CNS). The experimental autoimmune encephalomyelitis (EAE) is the most commonly used as ...experimental model because of its key pathological features’ approximation of MS. The interaction between complex elements in immune system and in the CNS determines the MS pathogenesis. However, there is no cure for MS and the treatment for MS still encounters great challenges. Thus, finding a more effective disease-modifying treatment is imminent. In the present study, we investigated whether 9,10-Anhydrodehydroartemisin (ADART), a compound derived from artemisinin, could decrease demyelination in EAE and the underlying mechanisms. In established EAE mice, 100 mg/kg 9,10-Anhydrodehydroartemisinin (ADART) effectively reduced CNS and peripheral immune system infiltration inflammatory cells including CD4
+
IFN-γ
+
Th1 cells and CD4
+
IL-17A
+
Th17 cells. Correspondingly, the serum level of IFN-γ and IL-17A was also reduced.
In vitro
, ADART almost completely inhibited Th17 differentiation, and partially inhibited Th1 differentiation in 10 μM. This research revealed that ADART could be a great promising avenue among current therapies for MS.
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