TRPC5 Channel Inhibition Protects Podocytes in Puromycin-Aminonucleoside Induced Nephrosis Models

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.

PIP2 regulation of TRPC5 channel activation and desensitization

Transient receptor potential canonical type 5 (TRPC5) channels are expressed in the brain and kidney, and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter. They are activated by elevated levels of extracellular Ca2+ or application of lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol bis-phosphate (PIP2) hydrolysis leads to protein kinase C- (PKC-) mediated phosphorylation of TRPC5 channels and desensitization of their activity. Even though PIP2 regulation of TRP channels is being widely studied, the roles of PIP2 in maintaining TRPC5 channel activity, the PIP2 involvement in channel stimulation by its hydrolysis product diacyl glycerol (DAG), or the desensitization of activity by DAG-stimulated PKC activity remain unclear. Here, we show that PIP2 controls both the PKC-mediated inhibition of TRPC5 currents as well as the activation by DAG and lanthanides and that it accomplishes this through control of gating rather than channel cell surface density. The mechanistic insights achieved by the present work promise to aid in the development of more selective and precise molecules to block TRPC5 channel activity and illuminate new therapeutic opportunities for targeted therapies for a group of diseases for which there is currently a great unmet need.

Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels

Diazirine-containing photoaffinity probes, based on the potent and selective TRPC1/4/5 channel inhibitor Pico145, allowed the development of an assay to probe cellular interactions between TRPC5 protein and xanthine-based TRPC5 channel modulators.