Identification of the hypertension drug niflumic acid as a glycine receptor inhibitor

Daishi Ito,Motonari Uesugi,Yoshinori Kawazoe,Hiromi Hirata,Ayato Sato

doi:10.1038/s41598-020-70983-2

Daishi Ito, Motonari Uesugi + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-70983-2

Copy DOI

Abstract

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl− conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Highlights

To search for new chemical compounds that block glycine receptor (GlyR), we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules through their ability to cause touch-evoked bilateral muscle contractions in zebrafish embryos
We have previously reported that zebrafish embryos swim when touched at 24 and 48 h postfertilization, whereas strychnine-treated embryos show obvious shrinkage of the body without swimming due to the loss of GlyRmediated reciprocal inhibition[13,19]
We found 10 drugs that caused touchevoked body shrinkage or weird uncoordinated response, the latter with contractions of small amplitude and/ or compromised rhythmicity (Table 1). We purchased these drugs, performed second round of screening at several different concentrations and identified three drugs that caused touch-evoked body shrinkage: strychnine, nifedipine and niflumic acid, which are known as specific GlyR inhibitor, voltage-gated calcium channel inhibitor and cyclooxygenase inhibitor, respectively

Summary

Introduction

To search for new chemical compounds that block GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules through their ability to cause touch-evoked bilateral muscle contractions in zebrafish embryos. We found that picrotoxin affects zebrafish behavior when applied at high concentration. Our electrophysiological recordings using Xenopus oocytes revealed that all of the strychnine, picrotoxin, nifedipine and niflumic acids showed concentration-dependent blockade of glycine-gated currents in both homomeric and heteromeric GlyRs. Our electrophysiological recordings using Xenopus oocytes revealed that all of the strychnine, picrotoxin, nifedipine and niflumic acids showed concentration-dependent blockade of glycine-gated currents in both homomeric and heteromeric GlyRs In both human and zebrafish GlyR cases, the half-maximal inhibitory concentration ( IC50) was strychnine < picrotoxin < nifedipine < niflumic acid

Methods

Results

Conclusion