Glycyrrhizic acid inhibits DNA damage repair and enhances cisplatin-induced apoptosis of melanoma cells.

This research was designed to prospect the mechanism and impact of glycyrrhizic acid (GA) on DNA damage repair and cisplatin (CP)-induced apoptosis of melanoma cells. First, human melanoma cell SK-MEL-28 was stimulated using GA for 24, 48, and 72 h. Then, the optimal treatment time and dosage were selected. After that, cell counting kit-8 (CCK-8) was employed for testing the cell viability, flow cytometry for the apoptosis, comet assay for the DNA damage of cells, and western blot for the cleaved-Caspase3, Caspase3, Bcl-2, and γH2AX protein expression levels. The experimental outcomes exhibited that as the GA concentration climbed up, the SK-MEL-28 cell viability dropped largely, while the apoptosis level raised significantly, especially at the concentration of 100 µm. In addition, compared with GA or CPtreatment only, CP combined with GA notably suppressed the viability of melanoma cells and promoted cell apoptosis at the cytological level. At the protein level, the combined treatment notably downregulated the Bcl-2 and Caspase3 expression levels, while significantly upregulated the cleaved-Caspase3 and γH2AX expression levels. Besides, CP + GA treatment promoted DNA damage at the DNA molecular level. Collectively, both GA and CP can inhibit DNA damage repair and enhance the apoptosis of SK-MEL-28 cells, and the synergistic treatment of both exhibits better efficacy.

Research and Development Progression of Oridonin for Hematological Malignancies Therapy.

Oridonin is a tetracyclic diterpenoid compound extracted from the medicinal herb Isodon and related species. Since 1976, studies have reported the significant anti-tumor activity of oridonin in vivo. Recently, an increasing number of studies have confirmed the anti-tumor effects of oridonin in various types of cancers, and its effect on hematological malignancies stands out. Herein, we have systematically reviewed the anti-- tumor effects of oridonin and its specific mechanisms in hematological malignancies, including the regulation of cancer proteins, activation of intrinsic and extrinsic apoptosis signaling pathways, accumulation of reactive oxygen species (ROS), modulation of chaperone proteins and miRNA expression, combination therapy with chemotherapeutic drugs, and the development of its derivatives. Taken together, oridonin exhibits multiple anti-tumor activities and serves as a multi-target agent, making it worthy of further investigation.

Transcriptional insights of citrus defense response against Diaporthe citri.

Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6'H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism.

Multi-omics analysis reveals interferon-stimulated gene OAS1 as a prognostic and immunological biomarker in pan-cancer.

Introduction: OAS1(2'-5'-oligoadenylate synthetase 1) is a member of the Interferon-Stimulated Genes which plays an important role in the antiviral process. In recent years, the role of OAS1 in tumors has attracted attention, and it was found to be associated with prognosis in several tumors. However, the mechanism by which OAS1 affects tumors is unclear and pan-cancer study of OAS1 is necessary to better understand its implication in cancers. Methods: The expression, prognostic value, genetic alteration, alternative splicing events of OAS1 in pan-cancers were analyzed using TCGA, GTEx, HPA, GEPIA and OncoSplicing databases. OAS1 associated immune cell infiltration was evaluated using the ESTIMATE, xCell, CIBERSORT and QUANTISEQ algorithm. Single cell transcriptome data download using TISH database. Finally, the roles of the OAS1 on apoptosis, migration and invasion were investigated in two pancreatic cancer cells. Results: Our results revealed significant differences in OAS1 expression among various tumors, which had prognostic implications. In addition, we investigated the impact of OAS1 on genomic stability, methylation status, and other factors across different types of cancer, and the effects of these factors on prognosis. Notably, our study also demonstrated that OAS1 overexpression can contribute to CTL dysfunction and macrophage M2 polarization. In addition, cell experiments showed that the knockdown of OAS1 could reduce the invasive ability and increased the apoptosis rate of PAAD cells. Discussion: These results confirmed that OAS1 could be a prognostic biomarker and therapeutic target for its potential role in CTL dysfunction and macrophage M2 polarization.

A GPX4 non-enzymatic domain and MDM2 targeting peptide PROTAC for acute lymphoid leukemia therapy through ferroptosis induction.

Ferroptosis, an emerging form of programmed cell death, has garnered substantial attention as a potential target for cancer therapy. However, despite the potential promise, no ferroptosis-related therapies have progressed to clinical trials. Identifying disease types sensitive to ferroptosis and developing specific ferroptosis-targeting drugs are critical focal points in the field of ferroptosis-based treatment. In this study, we conducted a comprehensive database analysis and presented compelling evidence indicating a high expression of GPX4 in patients with acute lymphoblastic leukemia (ALL), significantly correlating with poor prognosis. Notably, elevated GPX4 expression is closely associated with ALL relapse, a major challenge in the treatment of this disease. Building upon these findings, we devised a novel peptide-based Proteolysis Targeting Chimeras (PROTAC) drug targeting GPX4 through computer-aided design. In contrast to existing drugs that target the conjugative enzyme active site, our design focused on a peptide drug targeting the non-active site of GPX4. Furthermore, we strategically selected MDM2, an E3 ligase highly expressed in ALL, for the PROTAC drug design. This deliberate choice amplifies the drug's effect on cancer cells while minimizing its impact on normal cells, achieving desirable selectivity for cancer cells. Leveraging nanogold delivery, we successfully facilitated intracellular action of the GPX4-targeting peptide PROTAC drug, denoted as Au-PGPD (peptide GPX4 PROTAC drug). Au-PGPD effectively induced GPX4 degradation and inhibited ALL cell proliferation. Remarkably, Au-PGPD exhibited significantly less efficacy on normal cells, underscoring the selectivity and safety of our design.

Radiation-Induced Bystander Effect on the Genome of Bone Marrow Mesenchymal Stem Cells in Lung Cancer.

Aims: Radiation by-radiation effect (RIBE) can induce the genomic instability of bone marrow mesenchymal stem cells (BMSCs) adjacent to lung cancer, and this effect not only exists in the short-term, but also accompanies it in the long-term, but its specific mechanism is not clear. Our goal is to explore the similarities and differences in the mechanism of genomic damage in tumor-associated BMSCs induced by short-term and long-term RIBE, and to provide a theoretical basis for adjuvant drugs for protection against RIBE at different clinical time periods. Results: We found that both short- and long-term RIBE induced genomic instability. We could show a high expression of TGF-ß1, TNF-α, and HIF-1α in tumor-associated BMSCs after short-term RIBE whereas only TNF-α and HIF-1α expression was increased in long-term RIBE. We further confirmed that genomic instability is associated with the activation of the HIF-1α pathway and that this is mediated by TNF-α and TGF-ß1. In addition, we found differences in the mechanisms of genomic instability in the considered RIBE windows of analysis. In short-term RIBE, both TNF-α and TGF-ß1 play a role, whereas only TNF-α plays a decisive role in long-term RIBE. In addition, there were differences in BMSC recruitment and genomic instability of different tissues with a more pronounced expression in tumor and bone marrow than compared to lung. Innovation and Conclusion: We could show dynamic changes in the expression of the cytokines TGF-ß1 and TNF-α during short- and long-term RIBE. The differential expression of the two is the key to causing the genomic damage of tumor-associated BMSCs in the considered windows of analysis. Therefore, these results may serve as a guideline for the administration of radiation protection adjuvant drugs at different clinical stages. Antioxid. Redox Signal. 38, 747-767.

De Novo design of a humanized antiCD33 antibody-oridonin conjugate for acute myeloid leukemia therapy.

Acute myeloid leukemia (AML) is the most common blood cancer in adults. Patients' 5-year overall survival is less than 30% thus having a poor prognosis. To date, the development of novel target therapies is still necessary to ameliorate patients' survival. Antibody-drug conjugates (ADCs) represent a promising class of drugs for the treatment of AML. CD33 is highly expressed on AML cells, and the FDA-approved CD33-targeted ADC drug-gemtuzumab ozogamicin (GO) has proved the feasibility of CD33-targeted ADC drug design. In this study, we constructed a novel CD33-targeted ADC drug composed of a humanized anti-CD33 antibody and oridonin as a payload with a cleaved chemical linker. Oridonin is a natural product that has great cancer therapy potential while its poor bioavailability and targeting ability limited its clinical use. Herein, we demonstrated that antiCD33-oridonin specifically delivered oridonin in AML cells improved AML cells killing ability of oridonin. Meanwhile, it did not show any non-specific toxicity on CD33 negative cells. In summary, we developed a novel AML targeting ADC with clinical application potential, and therefore provided a new solution for the druggability improvement of oridonin.

Potential molecular mechanism of Guiqi Baizhu Decoction in radiation-induced intestinal edema by regulating HIF-1a, AQP4 and Na+/K+-ATPase.

BACKGROUND: Guiqi Baizhu Decoction (GQBZD) has a good protective effect on radiation-induced intestinal edema (RIIE). However, the underlying molecular mechanisms need further elucidation. PURPOSE: To reveal the potential mechanism of RIIE and GQBZD treatment. METHODS: SD rats were irradiated with 6Gy X-ray to establish RIIE model. The general condition of the rats was observed; the dry/wet weight ratio of colon tissue was detected; the morphological changes of colon tissue were observed by HE staining; the expressions of ROS, HIF-1α and AQP4 in colon tissue were detected by confocal laser scanning; the expression of edema-related proteins was detected by Western blot. In addition, human colon epithelial cells (NCM460) was irradiated with 2Gy X-ray, and HIF-1α expression in NCM460 was knocked down by small interfering RNA (siRNA) transfection, and the activity of Na+/K+-ATPase was detected by enzyme activity kit; the ROS expression was detected by flow cytometer; the AQP4 expression was detected by laser confocal microscopy; and the expression of edema-related proteins were detected by Western blot. RESULTS: We found that after irradiation, the colon tissue of rats was significantly edema, mainly manifested as mucosal and submucosal edema, and the ultrastructure was reflected in the structural damage of nucleus and mitochondria. ROS, HIF-1α and AQP4 were significantly expressed, and Na+/K+-ATPase expression/activity was decreased. After the intervention of GQBZD, the edema of the colon tissue of the rats was improved, the expressions of ROS, HIF-1α and AQP4 were decreased, and the expression/activity of Na+/K+-ATPase was increased. CONCLUSION: Ionizing radiation (IR) can cause significant intestinal edema. AQP4 and Na+/K+-ATPase are the key factors of RIIE, which are regulated by ROS and HIF-1α. GQBZD can improve hypoxia and oxidative stress, regulate the expression of AQP4 and Na+/K+-ATPase, and achieve a protective effect on RIIE. This study is the first to reveal the mechanism of RIIE.

Kill Two Birds with One Stone: A Multifunctional Dual-Targeting Protein Drug to Overcome Imatinib Resistance in Philadelphia Chromosome-Positive Leukemia.

The Bcr/Abl plays a central role in Philadelphia chromosome-positive (Ph+) leukemia because of the constitutively activated Abl tyrosine kinase and its downstream pathways. Currently, the clinical treatment of imatinib-resistant patients with tyrosine kinase inhibitors is severely limited by drug resistance and adverse effects. Herein, a dual-targeting proteolysis-targeting chimera (PROTAC) protein drug, termed PMI Bcr/Abl-R6, is designed by engrafting an MDM2/p53 inhibition peptide sequence onto the Bcr/Abl tetramerization domain. PMI Bcr/Abl-R6, harboring a Bcr/Abl targeting sequence and an MDM2 binding sequence, acts as a PROTAC drug in Ph+ leukemia cells. Its dual-targeting constitution suggests that PMI Bcr/Abl-R6 designs to target the tetramerization domain instead of the Abl kinase domain, therefore has the potential to overcome drug resistance mutations in the kinase domain. The efficient ability of PMI Bcr/Abl-R6 is demonstrated to simultaneously induce Bcr/Abl degradation and activate the p53 pathway. PMI Bcr/Abl-R6 has the potential to overcome drug resistance in Ph+ leukemias by multiple mechanisms.

Review: Effect of Gut Microbiota and Its Metabolite SCFAs on Radiation-Induced Intestinal Injury.

Gut microbiota is regarded as the second human genome and forgotten organ, which is symbiotic with the human host and cannot live and exist alone. The gut microbiota performs multiple physiological functions and plays a pivotal role in host health and intestinal homeostasis. However, the gut microbiota can always be affected by various factors and among them, it is radiotherapy that results in gut microbiota dysbiosis and it is often embodied in a decrease in the abundance and diversity of gut microbiota, an increase in harmful bacteria and a decrease in beneficial bacteria, thereby affecting many disease states, especially intestine diseases. Furthermore, gut microbiota can produce a variety of metabolites, among which short-chain fatty acids (SCFAs) are one of the most abundant and important metabolites. More importantly, SCFAs can be identified as second messengers to promote signal transduction and affect the occurrence and development of diseases. Radiotherapy can lead to the alterations of SCFAs-producing bacteria and cause changes in SCFAs, which is associated with a variety of diseases such as radiation-induced intestinal injury. However, the specific mechanism of its occurrence is not yet clear. Therefore, this review intends to emphasize the alterations of gut microbiota after radiotherapy and highlight the alterations of SCFAs-producing bacteria and SCFAs to explore the mechanisms of radiation-induced intestinal injury from the perspective of gut microbiota and its metabolite SCFAs.

Three Immune-Related Prognostic mRNAs as Therapeutic Targets for Pancreatic Cancer.

Objective: Pancreatic cancer is a highly lethal malignancy globally. This study aimed to probe and validate immune-related prognostic mRNAs as therapeutic targets for pancreatic cancer. Methods: Gene transcriptome data of pancreatic cancer and normal pancreas were retrieved from TCGA-GTEx projects. Two thousand four hundred and ninety-eight immune-related genes were obtained from the IMMUPORT database. Abnormally expressed immune-related genes were then identified. Under univariate and multivariate cox models, a gene signature was constructed. Its predictive efficacy was assessed via ROCs. The interactions between the 21 genes were analyzed by Spearson analysis and PPI network. Using the GEPIA and The Human Protein Atlas databases, their expression and prognostic value were evaluated. The TIMER database was utilized to determine the relationships between MET, OAS1, and OASL mRNAs and immune infiltrates. Finally, their mRNA expression was externally verified in the GSE15471 and GSE62452 datasets. Results: An immune-related 21-gene signature was developed for predicting patients' prognosis. Following verification, this signature exhibited the well predictive performance. There were physical and functional interactions between them. MET, OAS1, and OASL mRNAs were all up-regulated in pancreatic cancer and associated with unfavorable prognosis. They showed strong correlations with tumor progression. Furthermore, the three mRNAs were distinctly associated with immune infiltrates. Their up-regulation was confirmed in the two external datasets. Conclusion: These findings identified three immune-related prognostic mRNAs MET, OAS1, and OASL, which may assist clinicians to choose targets for immunotherapy and make personalized treatment strategy for pancreatic cancer patients.

Classification of heroin, methamphetamine, ketamine and their additives by attenuated total reflection-Fourier transform infrared spectroscopy and chemometrics.

Drug crime is a prominent issue of concern from pole to pole. In order to seek higher profits, drug gangs often add diluents and adulterants to the drugs to disperse drug products Analysis of these additives would be greatly conducive to determine the origin of drug products for law enforcement departments. A method using attenuated total reflectance-Fourier transform infrared spectroscopy and chemometrics methods to classify the heroin hydrochloride, methamphetamine hydrochloride, ketamine hydrochloride and their five additives (caffeine, phenacetin, starch, glucose, and sucrose), was developed. The Baseline correction, multivariate scatter correction, standard normal variate and Savitzky-Golay algorithm were adopted to pre-process the spectral data. Several supervised pattern recognition methods including decision tree, Bayes discriminant analysis, and support vector machine were considered as algorithms of constructing classifiers. The results reveal that, repetitive and interfering data in original spectrum data could be eliminated by principal component analysis and factor analysis. F-measure, as a comprehensive evaluation index of precision rate and recall rate, was more objective than precision rate and recall rate to reflect the ability of model to distinguish samples. It should be used as one of the indicators to evaluate the model. The CHAID classification tree could be identified as priorities in the decision tree model, while the linear kernel could be considered as the optimal kernel in the support vector machine model. The classification ability of three hydrochloride mixtures based on Bayes discriminant analysis was better than that of another models. Bayes discriminant analysis model was the more useful and practical method for classifying the target drugs of abuse than that of decision trees and support vector machine. The designed approach represents a potentially simple, non-destructive, and rapid method of classifying hydrochloride mixtures.

A tetrameric protein scaffold as a nano-carrier of antitumor peptides for cancer therapy.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMIBcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMIBcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMIBcr/Abl-R6 effectively induced apoptosis of HCT116 p53+/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Dithiocarbamate-inspired side chain stapling chemistry for peptide drug design.

Two major pharmacological hurdles severely limit the widespread use of small peptides as therapeutics: poor proteolytic stability and membrane permeability. Importantly, low aqueous solubility also impedes the development of peptides for clinical use. Various elaborate side chain stapling chemistries have been developed for α-helical peptides to circumvent this problem, with considerable success in spite of inevitable limitations. Here we report a novel peptide stapling strategy based on the dithiocarbamate chemistry linking the side chains of residues Lys(i) and Cys(i + 4) of unprotected peptides and apply it to a series of dodecameric peptide antagonists of the p53-inhibitory oncogenic proteins MDM2 and MDMX. Crystallographic studies of peptide-MDM2/MDMX complexes structurally validated the chemoselectivity of the dithiocarbamate staple bridging Lys and Cys at (i, i + 4) positions. One dithiocarbamate-stapled PMI derivative, DTCPMI, showed a 50-fold stronger binding to MDM2 and MDMX than its linear counterpart. Importantly, in contrast to PMI and its linear derivatives, the DTCPMI peptide actively traversed the cell membrane and killed HCT116 tumor cells in vitro by activating the tumor suppressor protein p53. Compared with other known stapling techniques, our solution-based DTC stapling chemistry is simple, cost-effective, regio-specific and environmentally friendly, promising an important new tool for the development of peptide therapeutics with improved pharmacological properties including aqueous solubility, proteolytic stability and membrane permeability.

Lanthanide-doped nanoparticles conjugated with an anti-CD33 antibody and a p53-activating peptide for acute myeloid leukemia therapy.

Roughly one third of all human cancers are attributable to the functional inhibition of the tumor suppressor protein p53 by its two negative regulators MDM2 and MDMX, making dual-specificity peptide antagonists of MDM2 and MDMX highly attractive drug candidates for anticancer therapy. Two pharmacological barriers, however, remain a major obstacle to the development of peptide therapeutics: susceptibility to proteolytic degradation in vivo and inability to traverse the cell membrane. Here we report the design of a fluorescent lanthanide oxyfluoride nanoparticle (LONp)-based multifunctional peptide drug delivery system for potential treatment of acute myeloid leukemia (AML) that commonly harbors wild type p53, high levels of MDM2 and/or MDMX, and an overexpressed cell surface receptor, CD33. We conjugated to LONp via metal-thiolate bonds a dodecameric peptide antagonist of both MDM2 and MDMX, termed PMI, and a CD33-targeted, humanized monoclonal antibody to allow for AML-specific intracellular delivery of a stabilized PMI. The resultant nanoparticle antiCD33-LONp-PMI, while nontoxic to normal cells, induced apoptosis of AML cell lines and primary leukemic cells isolated from AML patients by antagonizing MDM2 and/or MDMX to activate the p53 pathway. Fluorescent antiCD33-LONp-PMI also enabled real-time visualization of a series of apoptotic events in AML cells, proving a useful tool for possible disease tracking and treatment response monitoring. Our studies shed light on the development of antiCD33-LONp-PMI as a novel class of antitumor agents, which, if further validated, may help targeted molecular therapy of AML.

Self-Assembled Peptide-Lanthanide Nanoclusters for Safe Tumor Therapy: Overcoming and Utilizing Biological Barriers to Peptide Drug Delivery.

Developing a sophisticated nanomedicine platform to deliver therapeutics effectively and safely into tumor/cancer cells remains challenging in the field of nanomedicine. In particular, reliable peptide drug delivery systems capable of overcoming biological barriers are still lacking. Here, we developed a simple, rapid, and robust strategy to manufacture nanoclusters of ∼90 nm in diameter that are self-assembled from lanthanide-doped nanoparticles (5 nm), two anticancer peptides with different targets (BIM and PMI), and one cyclic peptide iNGR targeted to cancer cells. The peptide-lanthanide nanoclusters (LDC-PMI-BIM-iNGR) enhanced the resistance of peptide drugs to proteolysis, disassembled in response to reductive conditions that are present in the tumor microenvironment and inhibited cancer cell growth in vitro and in vivo. Notably, LDC-PMI-BIM-iNGR exhibited extremely low systemic toxicity and side effects in vivo. Thus, the peptide-lanthanide nanocluster may serve as an ideal multifunctional platform for safe, targeted, and efficient peptide drug delivery in cancer therapy.

Peptide-Induced Self-Assembly of Therapeutics into a Well-Defined Nanoshell with Tumor-Triggered Shape and Charge Switch.

Peptide-tuned self-assembly of macromolecular agents (>500 Da) such as therapeutic peptides offers a strategy to improve the properties and biofunctions of degradable nanomaterials, but the tough requirement of macromolecular therapeutics delivery and a lack of understanding of peptide-based self-assembly design present high barriers for their applications. Herein, we developed a new strategy for nanoengineering macromolecular drugs by an elaborate peptide, termed PSP (VVVVVHHRGDC), capable of directly conjugating with cargo to be a PSP-cargo monomer as building block tending to self-assemble into a well-defined nanoshell with tumor-triggered shape and charge switch. As a proof of concept, conjugation PSP to a D-peptide activator of tumor suppressor p53 termed DPMI (1492.5 Da) generated hollow spheres ~80 nm in diameter named PSP-DPMI that disintegrated only in the acidic microenvironment of tumor tissues, followed by integrin-mediated cellular uptake of PSP-DPMI monomers. Importantly, PSP-based self-assembly successfully endowed the DPMI with long circulation time and high cancer-cell-specific intracellular accumulation. PSP-DPMI nanoshells potently inhibited tumor growth in vitro and in vivo by the p53 restoration, while maintaining a highly favorable in vivo safety profile. Out of conventional encapsulation and conjugation, our study showcases a clinically viable novel method to nanoengineer macromolecular agents such as peptide for anticancer therapy and provides a hazard-free alternative strategy for the theranostics delivery.

Distinct CCK-2 receptor conformations associated with ß-arrestin-2 recruitment or phospholipase-C activation revealed by a biased antagonist.

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2R(G)) or recruiting ß-arrestin2 (CCK2R(ß)) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2R(G) but was nearly inefficient as inhibitor of CCK2R(ß). Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2R(ß) were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2R(ß) state. These data establish structural evidence for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.