Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

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.

Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

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 Gi, 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.

Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target.

Despite intensive efforts to discover highly effective treatments to eradicate tuberculosis (TB), it remains as a major threat to global human health. For this reason, new TB drugs directed toward new targets are highly coveted. MmpLs (Mycobacterial membrane proteins Large), which play crucial roles in transporting lipids, polymers and immunomodulators and which also extrude therapeutic drugs, are among the most important therapeutic drug targets to emerge in recent times. Here, crystal structures of mycobacterial MmpL3 alone and in complex with four TB drug candidates, including SQ109 (in Phase 2b-3 clinical trials), are reported. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear to be key facilitators of proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs. This structural data will greatly advance the development of MmpL3 inhibitors as new TB drugs.

Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Crystal Structure of the Human Cannabinoid Receptor CB1.

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Meet the authors: Lizhen Chen, Shasha Chong, and Zhijie "Jason" Liu.

Here, Molecular Cell talks to first and co-corresponding author Lizhen Chen and co-corresponding authors Shasha Chong and Zhijie "Jason" Liu about their paper, ''Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions'' (in this issue of Molecular Cell) and their scientific journeys until now.

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Ligand-dependent enhancer activation regulated by topoisomerase-I activity.

The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking-a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Enhancer activation requires trans-recruitment of a mega transcription factor complex.

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.

Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

A Non-canonical Role of YAP/TEAD Is Required for Activation of Estrogen-Regulated Enhancers in Breast Cancer.

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an in vivo proximity-dependent labeling (BioID) technique, we identified YAP1 and TEAD4 protein as co-regulators of ERα on enhancers. The binding of YAP1/TEAD4 to ERα-bound enhancers is augmented upon E2 stimulation and is required for the induction of E2/ERα target genes and E2-induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. Our data reveal a non-canonical function of YAP1 and TEAD4 as ERα cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERα+ breast cancer.

Structural basis of CXC chemokine receptor 2 activation and signalling.

Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor-chemokine recognition2-4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles.

Glucocorticoid Receptor:MegaTrans Switching Mediates the Repression of an ERα-Regulated Transcriptional Program.

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response.

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Remimazolam has similar anesthetic effect and superior safety compared to propofol in elderly patients: A meta-analysis of randomized controlled trials.

BACKGROUND: The superiority between remimazolam and propofol for anesthesia is controversial in elderly patients (≥60 years). This meta-analysis aimed to systematically compare anesthetic effect and safety profile between remimazolam and propofol in elderly patients under any surgery. METHODS: Cochrane Library, Web of Science, and PubMed were searched until December 25, 2023 for relevant randomized controlled trials. RESULTS: Ten studies with 806 patients receiving remimazolam (experimental group) and 813 patients receiving propofol (control group) were included. Time to loss of consciousness [standard mean difference (SMD) (95% confidence interval (CI): 1.347 (-0.362, 3.055), p = 0.122] and recovery time [SMD (95% CI): -0.022 (-0.300, 0.257), p = 0.879] were similar between experimental and control groups. Mean arterial pressure at baseline minus 1 min after induction [SMD (95% CI): -1.800 (-3.250, -0.349), p = 0.015], heart rate at baseline minus 1 min after induction [SMD (95% CI): -1.041 (-1.537, -0.545), p < 0.001], incidences of hypoxemia [relative risk (RR) (95% CI): 0.247 (0.138, 0.444), p < 0.001], respiratory depression [RR (95% CI): 0.458 (0.300, 0.700), p < 0.001], bradycardia [RR (95% CI): 0.409 (0.176, 0.954), p = 0.043], hypotension [RR (95% CI): 0.415 (0.241, 0.714), p = 0.007], and injection pain [RR (95% CI): 0.172 (0.113, 0.263), p < 0.001] were lower in the experimental group compared to the control group. Postoperative nausea and vomiting was not different between groups [RR (95% CI): 1.194 (0.829, 1.718), p = 0.341]. Moreover, this meta-analysis displayed a low risk of bias, minimal publication bias, and good robustness. CONCLUSION: Remimazolam shows comparative anesthetic effect and better safety profile than propofol in elderly patients under any surgery.