, Mesfer Al Shahrani 1Highlight
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High-throughput virtual screening (HTVS) was employed to identify potential inhibitors of C5aR1 binding, with C5R-131 emerging as a top candidate based on favorable docking interactions. |
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ADMET-AI predictions indicated C5R-131 exhibited favorable pharmacokinetic properties, including high absorption, optimal distribution, and low toxicity, making it a strong candidate for further development. |
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Molecular Dynamics Simulations confirmed the stable binding of C5R-131 to C5aR1, with minimal deviation in the binding pose and strong hydrogen bonding interactions, suggesting its potential for therapeutic targeting of C5aR1. |
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C5R-131 inhibited C5aR1 dose dependently in cell free enzyme assay. |
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C5R-131 effectively protected HK-2 cells from apoptosis and necrosis induced by high glucose, suggesting its potential to preserve renal cell integrity in DKD. |
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The compound significantly reduced STAT3 phosphorylation in HK-2 cells, demonstrating its ability to modulate inflammatory and fibrotic signaling in DKD. |
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Treatment with C5R-131 restored CD31 expression in HK-2 cells, indicating its ability to modulate endothelial cell activation and potentially prevent endothelial dysfunction in DKD. |
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The novelty of this approach lies in its potential to specifically target the C5a-C5aR1 signaling pathway, which remains underexplored in the context of DKD treatment. |
Abstract
Background: Diabetic Kidney Disease (DKD) is a leading cause of chronic kidney disease (CKD) and end-stage renal failure. The complement component 5a (C5a)-C5a Receptor 1 (C5aR1) signaling axis plays a critical role in these pathological processes by activating inflammatory pathways, including signal transducer and activator of transcription 3 (STAT3) signaling, exacerbating kidney injury. As the current treatments for DKD fail to address the underlying inflammatory and fibrotic processes, targeting the C5a-C5aR1 pathway may be a novel therapeutic approach.
Methods: High-throughput virtual screening (HTVS) was conducted to screen C5a-C5aR1 inhibition. Selected compounds were further validated through molecular dynamics (MD) simulations, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, followed by in vitro target evaluations using a cell-free C5aR1 inhibition assay. Apoptosis, downstream assessment was carried out by flow cytometry using the HK-2 cells.
Results: The in silico screening and ADMET analysis identified C5R-131 as a promising C5aR1 antagonist with favorable pharmacokinetic properties. MD simulations revealed stable binding of C5R-131 to C5aR1, with strong hydrogen bonding interactions, with a binding value of –9.4 kcal/mol. Molecular Mechanics Poisson–Boltzmann Surface Area (MMPBSA)-based binding free energy calculation from the 100 ns simulation trajectories shows C5R-131 has –21.11 kcal/mol when bound to C5aR1. In vitro assays demonstrated that C5R-131 significantly inhibited C5aR1 activity with a half-dose inhibitory concentration (IC50) value of 53.75 nM. The compound reduced apoptosis, necrosis, STAT3 phosphorylation, and modulated CD31 expression in high-glucose-exposed HK-2 cells, indicating its potential to protect against renal cell injury and inflammation in DKD.
Conclusion: Through a combination of in silico screening and in vitro validation, we demonstrate that C5R-131 effectively modulates STAT3 activation, reduces renal cell apoptosis, and protects endothelial cells from dysfunction in high glucose (HG) conditions. These findings offer a new therapeutic strategy for developing targeted therapies for DKD and other diseases associated with C5a receptor activation.
Keywords
- Diabetic Kidney Disease
- C5a-C5aR1 signaling
- C5R-131
- STAT3 phosphorylation
- virtual screening