Search

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery. • Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed • Conformational changes in nsp8 and its interaction with the exiting RNA are observed • Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated • Transition model from primase complex to polymerase complex is proposed Cryo-EM structures of the SARS-CoV-2 RNA polymerase in complexes with RNA, before and after RNA translocation, reveals structural rearrangements that the RNA-dependent RNA polymerase (RdRp) nsp12 and its co-factors (nsp7 and nsp8) undergo to accommodate nucleic acid binding. Further insights into how the complex is inhibited by remdesivir, and into the primase to polymerase transition, are also presented. [ABSTRACT FROM AUTHOR]

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G i , as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role. • 3D structures of CB2-AM12033-G i , CB1-AM841-G i , and CB2-AM12033 are determined • Structural evidence of G protein selectivity by CB1 and CB2 is identified • MD simulations reveal the distinct binding behavior of HU308 in CB2 and CB1 • Cholesterol molecule as an endogenous allosteric modulator of CB1 is uncovered Structure and simulations of cannabinoid receptors CB2 and CB1 in their inactive, active-like, and activated signaling states reveal residue differences that may provide G protein selectivity, the distinct binding behavior of CB2 agonists in CB2 and CB1, as well as evidence for modulation of CB1 by cholesterol binding. [ABSTRACT FROM AUTHOR]