Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors.

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dual agonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their multiplexed pharmacological actions over monoagonists such as semaglutide, we determine cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR. The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the near-atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility of tirzepatide and peptide 20.

Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol.

BACKGROUND: As a bladder-preserving therapy, radiation therapy (RT) has been widely used in the treatment of bladder cancer (BCa) and made great progress in the past few decades. However, some BCa patients have low RT responsiveness and local recurrence rate after RT could reach 50%. Acquired radio-resistance (ARR) is one of the important reasons for the failure of RT. Unfortunately, these ARR cells also lack sensitivity to chemotherapy and cause tumor recurrence and metastasis. PURPOSE: To build ARR-phenotype BCa cell model, discuss the possible molecular mechanism of ARR and find effective target molecules to overcome ARR. MATERIALS AND METHODS: Five thousand six hundred and thirty-seven cells were subjected 30 times to 2 Gy of γ-rays and the surviving cells were called 5637R. Colony formation and MTT assay were applied to evaluate cells sensitivity to ionizing radiation (IR) and anti-neoplastic agents, respectively. Cells abilities of migration and invasion were determined using transwell method. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) were respectively utilized to compare the difference of gene and protein expression between 5637 and 5637R cells. Molecule inhibitors and small interfering RNA (siRNA) systems were employed to decrease the expression of target proteins, respectively. RESULTS: BCa cells survived from fractionated irradiation (FI) exhibited tolerance to both IR and chemotherapy drugs. These ARR cells (5637R) had elevated migration and invasion abilities, accompanied by increased expression of epithelial mesenchymal transition (EMT)-related transcription factors (ZEB1/Snail/Twist). Moreover, 5637R cells showed enhanced cancer stem cell (CSC)-like characteristics with activated KMT1A-GATA3-STAT3 circuit, a newly reported self-renewal pathway of human bladder cancer stem cell (BCSC). Combined with Kaplan-Meier's analysis, we speculated that GATA3/MMP9/STAT3 could be an effective molecular panel predicting poor prognosis of BCa. In order to enhance the sensitivity of resistant cells to radiation, we introduced ERK inhibitor (FR 180204) and STAT3 inhibitor (S3I-201). However, both of them could not enhance ARR cells response to IR. On the other hand, siRNAs were respectively implemented to inhibit the expression of endogenous Beclin1 and Atg5, two important autophagy-related genes, in BCa cells, which significantly increased 5637R cells death upon taxol exposing. Similarly, chloroquine (CQ), a classic autophagy inhibitor, enhanced the cytotoxicity of taxol only on 5637R cells. CONCLUSIONS: Long-term FI treatment is an effective method to establish the ARR-phenotype BCa cell model, by enriching BCSCs and enhancing cells migration and invasion. Both inhibiting the expression of autophagy-related proteins and using autophagy inhibitor can increase the sensitivity of ARR cells to taxol, suggesting that autophagy may play an important role in ARR cells chemical tolerance.

Structural basis of Gs and Gi recognition by the human glucagon receptor.

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.

ATM mediates DAB2IP-deficient bladder cancer cell resistance to ionizing radiation through the p38MAPK and NF-κB signaling pathway.

Although surgery remains the standard therapy for the treatment of bladder cancer (BCa), the data from previous clinical studies suggest that there is an increase in the number of patients with a preference for bladder preservation strategies, including radiotherapy, to improve their life quality. Our preliminary results showed that disabled homolog 2 interactive protein (DAB2IP), a putative tumor suppressor gene, is often downregulated in BCa with a radioresistant phenotype. Subsequent investigations revealed that elevated expression of ataxia­telangiectasia mutated (ATM) induced by DAB2IP­knockdown may be the key event in BCa cell resistance to ionizing radiation (IR). However, how ATM is involved in the survival of DAB2IP­deficient cells exposed to IR remains to be fully elucidated. The present study knocked down the expression of ATM in DAB2IP­deficient BCa cells using RNA interference technology. Activation of mitogen­activated protein kinase (MAPK) and nuclear factor­κB (NF­κB) signaling pathways were detected by western blot analysis and immunofluorescence assay, respectively. It was demonstrated that knockdown of ATM enhanced the response of DAB2IP­deficient BCa cells to IR, which may have resulted from delayed DNA double­strand break repair kinetics, compromised nuclear factor­κB translocation, inhibited phosphorylation of p38 and the induced activation of c­Jun N­terminal kinase. Taken together, these findings suggested that ATM may be an effective target in the radiotherapy of patients with DAB2IP-deficient BCa.

Upregulating DAB2IP expression via EGR-1 inhibition, a new approach for overcoming fractionated-irradiation-induced cross-tolerance to ionizing radiation and mitomycin C in tumor cells.

PURPOSE: To evaluate the effect of fractionated irradiation (FI) on tumor cells' sensitivity to ionizing radiation (IR) and antineoplastic drugs, and examine the potential of early growth response-1 (EGR-1) inhibition to sensitize tumor cells to IR. MATERIALS AND METHODS: PC3 and HepG2 cells were subjected 10 times to γ-rays at 2 Gy. The surviving cells were named PC3/R and HepG2/R, respectively. The cells' sensitivity to irradiation and chemotherapeutic drugs, including cisplatin (PT), doxorubicin (DOX), mitomycin C (MMC) and 5-fluorouracil (5-FU), were identified by colony formation assay and MMT method, respectively. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was utilized to compare the difference of gene expression between radioresistant cells and parental cells. The small interfering RNA system was implemented to inhibit endogenous EGR-1 expression in radiation-resistant cells. Western blot was employed to identify the possible mechanism by which EGR-1 regulates cells' radiosensitivity. RESULTS: FI induced cross-resistant to IR and MMC in tumor cells. Along with the reduction of ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP) expression, EGR-1 gene was upregulated in FI-treated cells. On the other hand, downregulation of EGR-1 gene expression sensitized radioresistant cells to IR accompanied by DAB2IP overexpression and STAT3 inactivation. In addition, NF-κB inhibitor, BAY11-7082 enhanced resistant cells' radiosensitivity and chemosensitivity. CONCLUSIONS: Conventionally FI has a higher risk of forming acquired radioresistance (ARR) in vitro. EGR-1 gene-targeted drug design could be an effective strategy to overcome DAB2IP-dysregulation-induced ARR in tumor patients.