Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impacton normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Orthosteric and allosteric modulation of human HCAR2 signaling complex.

Hydroxycarboxylic acids are crucial metabolic intermediates involved in various physiological and pathological processes, some of which are recognized by specific hydroxycarboxylic acid receptors (HCARs). HCAR2 is one such receptor, activated by endogenous ß-hydroxybutyrate (3-HB) and butyrate, and is the target for Niacin. Interest in HCAR2 has been driven by its potential as a therapeutic target in cardiovascular and neuroinflammatory diseases. However, the limited understanding of how ligands bind to this receptor has hindered the development of alternative drugs able to avoid the common flushing side-effects associated with Niacin therapy. Here, we present three high-resolution structures of HCAR2-Gi1 complexes bound to four different ligands, one potent synthetic agonist (MK-6892) bound alone, and the two structures bound to the allosteric agonist compound 9n in conjunction with either the endogenous ligand 3-HB or niacin. These structures coupled with our functional and computational analyses further our understanding of ligand recognition, allosteric modulation, and activation of HCAR2 and pave the way for the development of high-efficiency drugs with reduced side-effects.

Effect of Chestnut (Castanea Mollissima Blume) Bur Polyphenol Extract on Shigella dysenteriae: Antibacterial Activity and the Mechanism.

Shigella dysenteriae is a highly pathogenic microorganism that can cause human bacillary dysentery by contaminating food and drinking water. This study investigated the antibacterial activity of chestnut bur polyphenol extract (CBPE) on S. dysenteriae and the underlying mechanism. The results showed that the minimum inhibitory concentration (MIC) of CBPE for S. dysenteriae was 0.4 mg/mL, and the minimum bactericidal concentration (MBC) was 1.6 mg/mL. CBPE treatment irreversibly disrupted cell morphology, decreased cell activity, and increased cell membrane permeability, cell membrane depolarization, and cell content leakage of S. dysenteriae, indicating that CBPE has obvious destructive effects on the cell membrane and cell wall of S. dysenteriae. Combined transcriptomic and metabolomics analysis revealed that CBPE inhibits S. dysenteriae by interfering with ABC protein transport, sulfur metabolism, purine metabolism, amino acid metabolism, glycerophospholipid metabolism, and some other pathways. These findings provide a theoretical basis for the prevention and treatment of S. dysenteriae infection with extract from chestnut burs.

Deconstructing the role of MALAT1 in MAPK-signaling in melanoma: insights from antisense oligonucleotide treatment.

The long non-coding RNA (lncRNA) MALAT1 is a regulator of oncogenesis and cancer progression. MAPK-pathway upregulation is the main event in the development and progression of human cancer, including melanoma and recent studies have shown that MALAT1 has a significant impact on the regulation of gene and protein expression in the MAPK pathway. However, the role of MALAT1 in regulation of gene and protein expression of the MAPK-pathway kinases RAS, RAF, MEK and ERK in melanoma is largely unknown. We demonstrate the impacts of antisense oligonucleotide (ASO)-based MALAT1-inhibition on MAPK-pathway gene regulation in melanoma. Our results showed that MALAT1-ASO treatment decreased BRAF RNA expression and protein levels, and MALAT1 had increased correlation with MAPK-pathway associated genes in melanoma patient samples compared to healthy skin. Additionally, drug-induced MAPK inhibition upregulated MALAT1-expression, a finding that resonates with a paradigm of MALAT1-expression presented in this work: MALAT1 is downregulated in melanoma and other cancer types in which MALAT1 seems to be associated with MAPK-signaling, while MALAT1-ASO treatment strongly reduced the growth of melanoma cell lines, even in cases of resistance to MEK inhibition. MALAT1-ASO treatment significantly inhibited colony formation in vitro and reduced tumor growth in an NRAS-mutant melanoma xenograft mouse model in vivo, while showing no aberrant toxic side effects. Our findings demonstrate new insights into MALAT1-mediated MAPK-pathway gene regulation and a paradigm of MALAT1 expression in MAPK-signaling-dependent cancer types. MALAT1 maintains essential oncogenic functions, despite being downregulated.

Association between CORIN promoter methylation and stroke: Results from two independent samples of Chinese adults.

Objective: As the physical activator of natriuretic peptides, corin has been associated with stroke, but the underlying mechanism is not very clear. Here, we examined whether the CORIN promoter's methylation, an epigenetic DNA modification, was associated with the risk of stroke in two independent samples. Methods: A total of 1771 participants including 853 stroke cases and 918 healthy controls were included as a discovery sample and 2,498 community members with 10 years of follow-up were included as a replication sample. DNA methylation of the CORIN promoter was quantified by target bisulfite sequencing in both samples. We first examined the single CpG association, followed by a gene-based analysis of the joint association between multiple CpG methylation and stroke, adjusting for conventional risk factors. Results: The single CpG association analysis found that hypermethylation at all of the 9 CpG sites assayed was significantly associated with lower odds of prevalent stroke in the discovery sample (all p < 0.05), and three of them located at Chr4:47840038 (HR = 0.74, p = 0.015), Chr4:47839941 (HR = 0.80, p = 0.047), and Chr4:47839933 (HR = 0.82, p = 0.050) were also significantly associated with incident stroke in the replication sample. The gene-based association analysis found that DNA methylation of the 9 CpG sites at the CORIN promoter was jointly associated with stroke in both samples (all p < 0.05). Conclusion: DNA methylation levels of the CORIN gene promoter were lower in stroke patients and predicted a higher risk of incident stroke in Chinese adults. The underlying causality warranted further investigation.

Association between soluble neprilysin and diabetes: Findings from a prospective longitudinal study.

Background: The potential role of neprilysin (NEP) in glucose metabolism has been found by basic studies but lacks population evidence. The objective of this study was to examine the association between serum NEP and diabetes in Chinese adults. Methods: In a prospective longitudinal cohort study - the Gusu cohort (n=2,286, mean age: 52 years, 61.5% females), the cross-sectional, longitudinal, and prospective associations between serum NEP and diabetes were systemically examined by logistic regression adjusting for conventional risk factors. Serum NEP was measured at baseline using commercial ELISA assays. Fasting glucose was repeatedly measured 4 years apart. Results: The cross-sectional analysis found a positive association between serum NEP and fasting glucose at baseline (ß=0.08, P=0.004 for log-transformed NEP). This association persisted after controlling for the dynamic risk profiles during follow-up (ß=0.10, P=0.023 for log-transformed NEP). The prospective analysis found that a higher level of serum NEP at baseline was associated with a higher risk of diabetes during follow-up (OR=1.79, P=0.039 for log-transformed NEP). Conclusions: Serum NEP was not only associated with prevalent diabetes but also predicted the future risk of diabetes development in Chinese adults, independent of many behavioral and metabolic factors. Serum NEP may be a predictor and even a new therapeutic target for diabetes. However, the casualty and mechanisms of NEP in the development of diabetes require further investigation.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry coupled with three-step data post-processing techniques for comprehensive profiling of the multiple components in Fufang Xianzhuli Ye.

INTRODUCTION: Fufang Xianzhuli (FXZL) Ye, a classical formula of traditional Chinese medicine, is composed of Succus Bambusae, Houttuyniae herba, Pinelliae Rhizoma, Zingiberis Rhizoma Recens, Eriobotryae Folium, Platycodonis Radix, and peppermint oil. For many years, FXZL has been primarily utilised in China to treat cough and phlegm. The chemical composition of FXZL has not been reported, which seriously affects the safety of the clinical application. OBJECTIVE: To establish a systematic method for rapidly classifying and recognising the chemical constituents in the FXZL for the safety of the clinical application. METHODS: An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry coupled with a three-step data post-processing strategy was developed to screen the chemical constituents of FXZL. RESULTS: In this experiment, the diagnostic ions in FXZL were classified into six main compounds. A total of 106 compounds were unambiguously identified in FXZL based on their retention times, accurate masses, and tandem mass spectrometry data. These include 11 chlorogenic acids, three flavonoids, eight sesquiterpenoids, six organic acids, 65 triterpenoid saponins, and 13 other compounds. CONCLUSION: The chemical composition of FXZL was identified and summarised, providing useful information for quality control and a basis for further exploration of its active ingredients in vivo.

Association Between Sleep Quality and Hypertension in Chinese Adults: A Cross-Sectional Analysis in the Tianning Cohort.

Background: Poor sleep quality is becoming very common in a developed society and relates to many health disorders. However, the association between sleep quality and hypertension has not been well studied in Chinese adults. Methods: Blood pressure was measured and sleep quality was assessed by the Pittsburgh Sleep Quality Index (PSQI) for 5167 participants (mean aged 51±15 years, 41.5% males) in the Tianning Cohort. A logistic regression model was constructed to examine the association between sleep quality, as well as its components, and hypertension, adjusting for age, sex, education level, current smoking, current drinking, physical activity, obesity, glucose, and blood lipids. Results: After multivariate adjustment, a higher score Of the PSQI was significantly associated with an increased risk of prevalent hypertension (OR=1.03, P=0.018). Compared to participants with normal sleep (the PSQI score <5), those with a poor sleep quality (the PSQI score ≥5) had a 17% increased risk of prevalent hypertension (OR=1.17, P=0.042). Three of the seven components of sleep quality, such as subjective sleep quality (OR=1.17, P=0.001), sleep latency (OR=1.11, P=0.010), and sleep disturbances (OR=1.19, P=0.004), were also significantly associated with prevalent hypertension. Conclusion: Poor sleep quality is increasingly prevalent in developed societies and may be related to an increased risk of hypertension in Chinese adults. The underlying causality is waiting to be studied.

A Prediction Model for Rapid Identification of Ischemic Stroke: Application of Serum Soluble Corin.

Objective: Rapid identification is critical for ischemic stroke due to the very narrow therapeutic time window. The objective of this study was to construct a diagnostic model for the rapid identification of ischemic stroke. Methods: A mixture population constituted of patients with ischemic stroke (n = 481), patients with hemorrhagic stroke (n = 116), and healthy individuals from communities (n = 2498) were randomly resampled into training (n = 1547, mean age: 55 years, 44% males) and testing (n = 1548, mean age: 54 years, 43% males) samples. Serum corin was assayed using commercial ELISA kits. Potential risk factors including age, sex, education level, cigarette smoking, alcohol consumption, obesity, blood pressure, lipids, glucose, and medical history were obtained as candidate predictors. The diagnostic model of ischemic stroke was developed using a backward stepwise logistic regression model in the training sample and validated in the testing sample. Results: The final diagnostic model included age, sex, cigarette smoking, family history of stroke, history of hypertension, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, fasting glucose, and serum corin. The diagnostic model exhibited good discrimination in both training (AUC: 0.910, 95% CI: 0.884-0.936) and testing (AUC: 0.907, 95% CI: 0.881-0.934) samples. Calibration curves showed good concordance between the observed and predicted probability of ischemic stroke in both samples (all P>0.05). Conclusion: We developed a simple diagnostic model with routinely available variables to assist rapid identification of ischemic stroke. The effectiveness and efficiency of this model warranted further investigation.

Soluble Corin Predicts the Risk of Cardiovascular Disease: A 10-Year Follow-Up Study.

Background: As a key enzyme of the natriuretic peptides system, corin may participate in the development of cardiovascular disease (CVD). Its level in circulation predicted CVD recurrence in patients with myocardial infarction and heart failure, but no study examined this prediction in general populations. Objectives: This study sought to examine the prospective association between corin and CVD in a community-based population of Chinese adults. Methods: The Gusu cohort included 2,498 participants (mean age 53 years, 39% men) who were free of CVD at baseline. Serum corin was measured by enzyme-linked immunosorbent assay kits at baseline and CVD events were followed every 2 years for all participants. A competing-risks survival regression model was used to examine the association between serum corin and CVD. Results: During 10 years of follow-up, 210 participants developed CVD including 88 stroke events. A higher serum corin (after log-transformation) at baseline was significantly associated with an increased risk of CVD (HR: 1.88; P = 0.019) and stroke (HR: 3.19; P = 0.014). Analysis using categorical serum corin (in quartiles) showed that participants in the highest quartile had a 62% and 179% increased risk for CVD (HR: 1.62; P = 0.024) and stroke (HR: 2.79; P = 0.004), respectively, compared with those in the lowest quartile. We did not find a significant association between serum corin and coronary heart disease. Conclusions: A higher serum corin at baseline predicted a higher risk of CVD events and stroke, but not coronary heart disease, in Chinese adults, independent of conventional risk factors. Serum corin may be a predictor for stroke but the underlying mechanism needs further investigation.

Structure, Physicochemical Property, and Functional Activity of Dietary Fiber Obtained from Pear Fruit Pomace (Pyrus ussuriensis Maxim) via Different Extraction Methods.

In this study, soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were extracted from Pyrus ussuriensis Maxim pomace via three methods including enzymic extraction (EE), microwave-assisted enzymatic extraction (MEE), and three-phase partitioning (TPP). The effects of different extraction methods on the structure, physicochemical property, and functional activity of the extracted dietary fiber were evaluated. The results showed that different extraction methods had significant effects on the extraction yield, molecular weight distribution, thermal stability, antioxidant activity, and hypoglycemic activity in vitro, but resulted in no difference in the structure and composition of functional groups. It is noteworthy that SDF extracted by TPP has a more complex and porous structure, lower molecular weight, and higher thermal stability, as well as better physicochemical properties and in vitro hypoglycemic activity. IDF extracted by MEE showed the greatest water and oil holding capacity; the highest adsorption capacity for glucose, cholesterol, and nitrite ion; as well as the strongest inhibitory activity on α-amylase. These results suggest that PUP may be a source of cheap natural dietary fiber.

Structural insights into human brain-gut peptide cholecystokinin receptors.

The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain-gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. CCK1R couples to Gs and Gq, whereas CCK2R primarily couples to Gq. Here we report the cryo-electron microscopy (cryo-EM) structures of CCK1R-Gs signaling complexes liganded either by sulfated cholecystokinin octapeptide (CCK-8) or a CCK1R-selective small-molecule SR146131, and CCK2R-Gq complexes stabilized by either sulfated CCK-8 or a CCK2R-selective ligand gastrin-17. Our structures reveal a location-conserved yet charge-distinct pocket discriminating the effects of ligand PTM states on receptor subtype preference, the unique pocket topology underlying selectivity of SR146131 and gastrin-17, the conformational changes in receptor activation, and key residues contributing to G protein subtype specificity, providing multiple structural templates for drug design targeting the brain-gut axis.

Molecular insights into ligand recognition and activation of chemokine receptors CCR2 and CCR3.

Chemokine receptors are a family of G-protein-coupled receptors with key roles in leukocyte migration and inflammatory responses. Here, we present cryo-electron microscopy structures of two human CC chemokine receptor-G-protein complexes: CCR2 bound to its endogenous ligand CCL2, and CCR3 in the apo state. The structure of the CCL2-CCR2-G-protein complex reveals that CCL2 inserts deeply into the extracellular half of the transmembrane domain, and forms substantial interactions with the receptor through the most N-terminal glutamine. Extensive hydrophobic and polar interactions are present between both two chemokine receptors and the Gα-protein, contributing to the constitutive activity of these receptors. Notably, complemented with functional experiments, the interactions around intracellular loop 2 of the receptors are found to be conserved and play a more critical role in G-protein activation than those around intracellular loop 3. Together, our findings provide structural insights into chemokine recognition and receptor activation, shedding lights on drug design targeting chemokine receptors.

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 these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron 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 was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to 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 the 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 nonpeptidic 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.