Biology subjects

Liu, Q.

Publications and source records attributed to Liu, Q..

At least 433 records · Page 24Linked to original sources

bioRxiv2022

Markers of Fungal Translocation Are Elevated During Post-Acute Sequelae of SARS-CoV-2 Infection and Induce NF-κB Triggered Inflammation.

Long COVID, a type of Post-Acute Sequelae of SARS CoV-2 infection (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the pathophysiological mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 (COVID-19) could be exacerbated by microbial translocation (from the gut and/or lung) to the blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We found higher levels of fungal translocation - measured as {beta}-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher {beta}-glucan correlated with higher levels of markers of inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, {beta}-glucan can directly induce inflammation by binding to myeloid cells (via the Dectin-1 receptor) and activating Syk/NF-{kappa}B signaling. Using an in vitro Dectin-1/NF-{kappa}B reporter model, we found that plasma from individuals experiencing PASC induced higher NF-{kappa}B signaling compared to plasma from SARS-CoV-2 negative controls. This higher NF-{kappa}B signaling was abrogated by the Syk inhibitor Piceatannol. These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.

microbiology↗

bioRxiv2022

Multimodal single cell data integration challenge: results and lessons learned

Biology has become a data-intensive science. Recent technological advances in single-cell genomics have enabled the measurement of multiple facets of cellular state, producing datasets with millions of single-cell observations. While these data hold great promise for understanding molecular mechanisms in health and disease, analysis challenges arising from sparsity, technical and biological variability, and high dimensionality of the data hinder the derivation of such mechanistic insights. To promote the innovation of algorithms for analysis of multimodal single-cell data, we organized a competition at NeurIPS 2021 applying the Common Task Framework to multimodal single-cell data integration. For this competition we generated the first multimodal benchmarking dataset for single-cell biology and defined three tasks in this domain: prediction of missing modalities, aligning modalities, and learning a joint representation across modalities. We further specified evaluation metrics and developed a cloud-based algorithm evaluation pipeline. Using this setup, 280 competitors submitted over 2600 proposed solutions within a 3 month period, showcasing substantial innovation especially in the modality alignment task. Here, we present the results, describe trends of well performing approaches, and discuss challenges associated with running the competition.

bioinformatics↗

bioRxiv2022

Tuberculosis treatment failure associated with evolution of antibiotic resilience

Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

microbiology↗

bioRxiv2022

A potent SARS-CoV-2 antibody neutralizes Omicron variant by disassembling the spike trimer

The continuous emergence of novel SARS-CoV-2 variants poses new challenges to the fight against the COVID-19 pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of antibody targeting conserved and universal epitope is urgently needed. A subset neutralizing antibody(nAbs) against COVID-19 from convalescent patients were isolated in our previous study. Here in this study, we investigated the accommodation of these nAbs to SARS-CoV-2 variants of concerns (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of SARS-CoV-2 Omicron variant. In addition, we determined the cryo-EM structure of SARS-CoV-2 spike complexed with three antibodies targeting different epitopes, including 553-49, 553-15 and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes virus by disassembling spike trimers. 553-15, an antibody that neutralizes all the other VOCs except omicron, cross-links two spike trimers to form trimer dimer, demonstrating that 553-15 neutralizes virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 49 and other antibody targeting this highly conserved epitope as promising cocktail therapeutics reagent for COVID-19. ImportanceThe newly emergence of Omicron strain caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 Omicron neutralizing antibody 553-49, which neutralizes Omicron variant by targeting a completely conserved novel epitope. Besides, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by crosslinking virions and 553-60 functions by blocking receptor binding. Comparison of different RBD epitopes revealed that the epitope of 553-49 is hidden in the S trimer and keeps high conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutics reagent to fight against the emerging Omicron and future variant of SARS-CoV-2.

microbiology↗

bioRxiv2022

Prime-boost vaccinations with two serologically distinct chimpanzee adenovirus vectors expressing SARS-CoV-2 spike or nucleocapsid tested in a hamster COVID-19 model

Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing the spike (S) or nucleocapsid (N) proteins of an early SARS-CoV-2 isolate were tested individually or as a mixture in a hamster COVID-19 challenge model. The N protein, which was expressed as a fusion protein within herpes simplex virus glycoprotein D (gD) stimulated antibodies and CD8+ T cells. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gDN fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable that the S protein may potentiate and prolong protection achieved by the currently used genetic COVID-19 vaccines.

immunology↗

bioRxiv2022

Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-{kappa}B signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-{kappa}B-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

immunology↗

bioRxiv2022

Ingestible capsule for detecting labile inflammatory biomarkers in situ

Transient molecules in the gastrointestinal (GI) tract, such as nitric oxide and hydrogen sulfide, are key signals and mediators of inflammatory bowel disease (IBD). Because these molecules are extremely short-lived in the body, they are difficult to detect. To track these reactive molecules in the GI tract, we have developed a miniaturized device that integrates genetically-engineered probiotic biosensors with a custom-designed photodetector and readout chip. Leveraging the molecular specificity of living sensors, we genetically encoded bacteria to respond to IBD-associated molecules by luminescing. Low-power electronic readout circuits (nanowatt power) integrated into the device convert the light from just 1 L of bacterial culture into a wireless signal. We demonstrate biosensor monitoring in the GI tract of small and large animal models and integration of all components into a sub-1.4 cm3 ingestible form factor capable of supporting wireless communication. The wireless detection of short-lived, disease-associated molecules could support earlier diagnosis of disease than is currently possible, more accurate tracking of disease progression, and more timely communication between patient and their care team supporting remote personalized care.

synthetic biology↗

bioRxiv2022

Chromosome-scale genome assembly of the diploid oat Avena longiglumis reveals the landscape of repetitive sequences, genes and chromosome evolution in grasses

BackgroundOat (Avena sativa, 2n=6x=42) is an important crop, and with its wild relatives including A. longiglumis (ALO, 2n=6x=14), has advantageous agronomic and nutritional traits. A de-novo chromosome-level ALO genome assembly was made to investigate diversity and structural genome variation between Avena species and other Poaceae in an evolutionary context, and develop genomic resources to identify the pangenome and economic traits within Pooideae. ResultsThe 3.85 gigabase ALO genome (seven pseudo-chromosomes), contained 40,845 protein-coding genes and 87% repetitive sequences (84.21% transposable elements). An LTR retrotransposon family was abundant at all chromosome centromeres, and genes were distributed without major terminal clusters. Comparisons of synteny with A. eriantha and A. strigosa showed evolutionary translocations of terminal segments including many genes. Comparison with rice (x=12) and the ancestral grass karyotype showed synteny and features of chromosome evolution including fusions, translocations and insertions of syntenic blocks across Pooideae species. With a genome size 10 times larger than rice, ALO showed relatively uniform expansion along the chromosome arms, with few gene-poor regions along arms, and no major duplications nor deletions. Linked gene networks were identified (mixed-linkage glucans and cellulose synthase genes), and CYP450 genes may be related to salt-tolerance. ConclusionsThe high-continuity genome assembly shows gene, chromosomal structural and copy number variation, providing a reference for the Avena pangenome, defining the full spectrum of diversity. Chromosomal rearrangements and genome expansion demonstrate features of evolution across the genus and grass BOP-clade, contributing to exploitation of gene and genome diversity through precision breeding.

genomics↗

bioRxiv2022

Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies

The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.

microbiology↗

bioRxiv2022

Aberrant mechanosensitive signaling underlies activation of vascular endothelial xanthine oxidoreductase that promotes aortic aneurysm formation in Marfan syndrome

Marfan syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 gene encoding fibrillin-1, a matrix component of extracellular microfibrils. The main cause of morbidity and mortality in MFS is thoracic aortic aneurysm and dissection, but the underlying mechanisms remain undetermined. We found a significant increase in reactive oxygen species (ROS) generation in ascending aorta of MFS patients and MFS mice harboring the Fbn1 mutation (C1039G), which was associated with up-regulation of xanthine oxidoreductase (XOR) protein in aortic endothelial cells (ECs). Mechanosensitive signaling involving focal adhesion kinase (FAK)-p38 mitogen-activated protein kinase (MAPK) and early growth response-1 (Egr- 1) was aberrantly activated in ascending aorta of Fbn1C1039G/+ mice, and mechanical stress on human aortic ECs up-regulated XOR expression through FAK-p38 MAPK activation and Egr-1 up-regulation. Inhibition of XOR function by ECs-specific disruption of Xdh gene or by systemic administration of XOR inhibitor febuxostat in Fbn1C1039G/+ mice suppressed ROS generation, FAK-p38 MAPK activation, and Egr-1 up-regulation, leading to attenuation of aortic aneurysm formation. These findings unveil aberrant mechanosensitive signaling in vascular ECs triggering endothelial XOR activation and ROS generation as a culprit underlying the pathogenesis of aortic aneurysm formation in MFS, and highlight a drug repositioning approach using a uric acid lowering drug febuxostat as a potential therapy for MFS.

pathology↗

bioRxiv2022

Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and their Potential Use in Biocontrol of Coffee Wilt Disease

The present study investigated the distribution status and biodiversity of Trichoderma species surveyed from coffee rhizosphere soil samples from Ethiopia and their potential for biocontrol of coffee wilt disease (CWD) caused by Fusarium xylarioides. Trichoderma isolates were identified based on molecular approaches and morphological characteristics followed by biodiversity analysis using different biodiversity indices. The antagonistic potential of Trichoderma isolates was evaluated against F. xylarioides using the dual confrontation technique and agar diffusion bioassays. A relatively high diversity of species was observed including 16 taxa and 11 undescribed isolates. Trichoderma asperellum, T. asperelloides and T. longibrachiatum were classified as abundant species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. Trichoderma asperellum was the most abundant species (comprising 39.6% of all isolates) in all investigated coffee ecosystems. Shannons biodiversity index (H), the evenness (E), Simpsons biodiversity index (D), and the abundance index (J) were calculated for each coffee ecosystem, revealing that species diversity and evenness were highest in the Jimma zone (H =1.97, E = 0.76, D = 0.91, J = 2.73). The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. In vitro confrontation experiments revealed that T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The potential use of these Trichoderma species for disease management of F. xylarioides and to reduce its impact on coffee cultivation is discussed in relation to Ethiopias ongoing coffee wilt disease crisis.

microbiology↗

bioRxiv2022

High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental Hibiscus

Hibiscus rosa-sinensis L. (Hibiscus, Malvaceae) is an ornamental species grown widely in amenity plantings. We collected leaves on an urban roadside pavement (sidewalk) near a market in Guangzhou which showed multiple symptoms of leaf rolling, deformation and chlorosis. Initial evaluation by electron microscopy using negative staining of drip preparations revealed the presence of tobamovirus-like particles. Total RNA was extracted, and, unusually, without any RNA selection based on sequence, was used for cDNA library construction and high-throughput survey sequencing. From the 814 Mb of clean sequence data (from 2,712,161 paired reads of 150 bp) reads representing chloroplast, ribosomal, and mitochondrial genes were filtered out, eliminating 79.1% of reads. 1,135,848 x 150 bp of the sequence was retained and screened for viral sequences. Assembly of these sequences detected nine virus species from seven virus genera comprising three tobamoviruses, namely, Tobacco mosaic virus, Tobacco mild green mosaic virus and Hibiscus latent Singapore virus, Turnip mosaic virus (Potyvirus), Potato virus M (Carlavirus), Hibiscus chlorotic ringspot virus (Betacarmovirus), Fabavirus sp (Fabavirus), Cotton leaf curl Multan virus (Begomovirus) and a putative mitoviruses replicating in mitochondria, Chenopodium quinoa mitovirus 1. Mapping the reads to complete virus reference sequences showed high and uniform coverage of the genomes from 3,729 x coverage for Turnip mosaic virus to 22 x for Cotton leaf curl Multan virus. By comparison, nuclear reference genes actin showed 14 x coverage and polyubiquitin 27 x. Notable variants from reference sequences (SNPs) were identified. With the low cost of sequencing and potential for semi-automated bioinformatic pipelines, the whole-RNA approach has huge potential for identifying multiple undiagnosed viruses in ornamental plants, resulting in the ability to take preventive measures in production facilities against spread and to product quality for the mutual benefit of producers and consumers.

genomics↗

bioRxiv2022

Multiplexed strain phenotyping defines consequences of genetic diversity in Mycobacterium tuberculosis for infection and vaccination outcomes

There is growing evidence that genetic diversity in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of Mtb, strains belonging to Lineage 2 (L2) are associated with concerning clinical features including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of L2 has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of Mtb clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse Mtb strains during infection and after vaccination in the mouse model. We found that L2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of L2 strains distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress- and host-response pathways in a representative L2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage.

microbiology↗

bioRxiv2022

Molecular basis for allosteric agonism and G protein subtype selectivity of galanin receptors

Peptide hormones and neuropeptides are complex signaling molecules that predominately function through G protein-coupled receptors (GPCRs). Two fundamental questions remained in the field of peptide-GPCR signaling systems are the basis for the diverse binding mode of peptide ligands and the specificity of G protein coupling. Here we report the structures of a neuropeptide, galanin, bound to its receptors, GAL1R and GAL2R, in complex with their primary G protein subtypes Gi and Gq, respectively. The structures reveal a unique binding pose of galanin, which almost lay flat on the top of the receptor transmembrane domain pocket in an -helical conformation, and acts as an allosteric-like agonist via a distinct signal transduction cascade. The structures also uncover the important features of intracellular loop 2 (ICL2) that mediate specific interactions with Gq, thus determining the selective coupling of Gq to GAL2R. ICL2 replacement in Gi-coupled GAL1R, OR, 5-HT1AR, and Gs-coupled b2AR and D1R with that of GAL2R promotes Gq coupling of these receptors, highlighting the dominant roles of ICL2 in Gq selectivity. Together our results provide important insights into peptide ligand recognition and allosteric activation of galanin receptors and uncover a general structural element for Gq coupling selectivity.

molecular biology↗

bioRxiv2022

Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice

The CRISPR-Cas13 system is an RNA-guided RNA-targeting system, and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. Here, we found that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which was not resulted from the loss of target gene function or off-target effects. Mechanistically, we showed that RfxCas13d exhibited collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAK-JNK/p38-immediate early gene (IEG) pathway. These results provide new mechanistic insights into the collateral activity of RfxCas13d and warn that the biosafety of CRISPR-Cas13 system needs further evaluation before applying it to clinical treatments.

bioengineering↗

bioRxiv2022

Asymmetric Pendrin Homodimer Reveals its Molecular Mechanism as Anion Exchanger

Pendrin SLC26A4 is an anion exchanger expressed in apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder disease associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However, its molecular structure has remained unknown limiting our understanding. Here, we determined the structures of mouse pendrin with symmetric and characteristically asymmetric homodimer conformations by cryo-electron microscopy. The asymmetric homodimer consists of an inward-facing protomer and an intermediate-state protomer, representing the coincident uptake and secretion process, and exhibits the unique state of pendrin as an electroneutral exchanger. This previously unrevealed conformation, together with other conformations we captured, provides an inverted alternate-access mechanism for anion exchange. Furthermore, our structural and functional data disclosed the properties of anion exchange cleft and interpreted the important pathogenetic mutations. These investigations shed light on the pendrin exchange mechanism and extend our structure-guided understanding of pathogenetic mutations. One-Sentence SummaryCryo-EM asymmetric Pendrin homodimer cracks its exchange mechanism and guides our understanding for pathogenetic mutations.

biophysics↗

bioRxiv2022

Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.

cell biology↗

bioRxiv2022

Structural basis of peptidomimetic agonism revealed by small molecule GLP-1R agonists Boc5 and WB4-24

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of them are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric non-peptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryo-electron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3 and 7, one arm of both compounds inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8-D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts showing an interaction feature substantially similar to a previously known small molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by non-peptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine tuning of pharmacological efficacy in the development of future small molecule therapeutics targeting GLP-1R. SignificanceGLP-1R agonists are efficacious in the treatment of type 2 diabetes and obesity. While most clinically used agents require subcutaneous injection, Boc5, as the first orthosteric non-peptidic agonist of GLP-1R, suffers from poor oral bioavailability that hinders its therapeutic development. The cryo-electron microscopy structures of Boc5 and its closely related analog WB4-24 presented here reveal a previously unknown binding pocket located deeper in the transmembrane domain for non-peptidic GLP-1R agonists. Molecular interaction with this site may facilitate a broad spectrum of in vivo agonistic activities, in addition to that with the upper helical bundles presumably responsible for biased signaling. These findings deepen our understanding of peptidomimetic agonism at GLP-1R and may help design better drug leads against this important target.

molecular biology↗