Orthogonal activation of metabotropic glutamate receptor using coordination chemogenetics

Cell-surface receptors play a pivotal role as transducers of extracellular input. Although different cell types express the same receptor, the physiological roles of the receptor are highly dependent on cell type. To understand each role, tactics for cell-specific activation of the target receptor are in high demand. Herein, we developed an orthogonal activation method targeting metabotropic glutamate receptor 1 (mGlu1), a G-protein coupled receptor. In this method, direct activation via coordination-based chemogenetics (dA-CBC) was adopted, where activation of mGlu1 was artificially induced by a protein conformational change in response to the coordination of a metal ion or metal-ion complex. Our structure-based protein design and screening approach identified mGlu1 mutants that were directly activated by the coordination of Cu2+ or Zn2+, in addition to our previous Pd-complex-sensitive mGlu1 mutant. Notably, the activation of the mutants was mutually orthogonal, resulting in cell-type selective activation in a model system using HEK293 cells

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The analysis of AMPA-type glutamate receptor (AMPAR) trafficking is essential for understanding molecular mechanisms of learning and memory, but the analytical tools are currently limited. Here, the authors report a method that combines affinity-based receptor labeling and bioorthogonal click chemistry to quantify AMPAR distribution and trafficking under physiological conditions

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

【研究成果】脳組織において狙った細胞の神経伝達物質受容体の活性化に成功 --記憶・学習のメカニズム解明に期待--. 京都大学プレスリリース. 2022-06-17.Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner

A double-blind randomized comparative clinical trial to evaluate the safety and efficacy of dendritic cell vaccine loaded with WT1 peptides (TLP0-001) in combination with S-1 in patients with advanced pancreatic cancer refractory to standard chemotherapy

Abstract Background Pancreatic cancer is a refractory malignancy, and the development of a new effective treatment strategy is needed. We generated a dendritic cell vaccine by culturing monocytes obtained by apheresis of blood from each patient, inducing their differentiation into dendritic cells, and pulsing with tumor antigen peptides. However, the clinical efficacy of the vaccine has not been established. We therefore decided to conduct an exploratory clinical trial of dendritic cell vaccine loaded with Wilms’ tumor gene 1 peptides (TLP0-001) as a potential new treatment for patients with advanced pancreatic cancer refractory to standard chemotherapy. Methods This is an investigator-initiated, double-blind, comparative trial. The patients were allocated to two groups in a 1:1 ratio through a central registration by dynamic allocation. A total of 185 patients with inoperable or metastatic pancreatic cancer who were refractory or intolerant to standard primary chemotherapy with gemcitabine plus nab-paclitaxel will be allocated to secondary treatment either with placebo in combination with S-1 (the control group) or TLP0-001 in combination with S-1 (the investigational product group). The primary objective of this trial is to evaluate the safety and efficacy (as measured by overall survival) of the investigational product by comparing the two groups. This clinical trial will be performed in accordance with Japanese Good Clinical Practice guidelines. Discussion Clinical trials of the standard regimen, including gemcitabine, for advanced pancreatic cancer are ongoing worldwide. However, a strategy for after the primary treatment has not been established. We therefore decided to conduct this study to evaluate the safety and efficacy of TLP0-001 as a secondary treatment for pancreatic cancer in anticipation of the approval of this new drug in Japan. This trial is conducted with full consideration of safety, as it is the first-in-human clinical trial of TLP0-001; thus, the trial will be conducted only at the Second Department of Surgery at Wakayama Medical University until the safety is confirmed by interim analysis. We plan to conduct a multicenter trial at 18 institutions in Japan after confirmation of the safety. Trial registration University Hospital Medical Information Network Clinical Trials Registry, UMIN000027179. Registered on 9 April 2017