Rhodojaponin Ⅵ indirectly targets Cav2.2 channels via N-ethylmaleimide-sensitive fusion protein to alleviate neuropathic pain

Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients,but currently available treatments are often ineffective.Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed.Rhodojaponin Ⅵ,a grayanotoxin from Rhododendron molle,showed remarkable antinociceptive efficacy in models of neuropathic pain,but its biotargets and mechanisms are unknown.Given the reversible action of rhodojaponin Ⅵ and the narrow range over which its structure can be modified,we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin Ⅵ.N-Ethylmaleimide-sensitive fusion(NSF) was confirmed as the key target of rhodojaponin Ⅵ through biological and biophysical experiments.Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca2+current intensity,whereas rhodojaponin Ⅵ reversed the effects of NSF.In conclusion,rhodojaponin Ⅵ represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.

The toxic natural product tutin causes epileptic seizures in mice by activating calcineurin

Tutin,an established toxic natural product that causes epilepsy in rodents,is often used as a tool to develop animal model of acute epileptic seizures.However,the molecular target and toxic mechanism of tutin were unclear.In this study,for the first time,we conducted experiments to clarify the targets in tutin-induced epilepsy using thermal proteome profiling.Our studies showed that calcineurin(CN)was a target of tutin,and that tutin activated CN,leading to seizures.Binding site studies further established that tutin bound within the active site of CN catalytic subunit.CN inhibitor and calcineurin A(CNA)knockdown experiments in vivo proved that tutin induced epilepsy by activating CN,and produced obvious nerve damage.Together,these findings revealed that tutin caused epileptic seizures by activating CN.Moreover,further mechanism studies found that N-methyl-D-aspartate(NMDA)receptors,gammaaminobutyric acid(GABA)receptors and voltage-and Ca2+-activated K+(BK)channels might be involved in related signaling pathways.Our study fully explains the convulsive mechanism of tutin,which provides new ideas for epilepsy treatment and drug development.

A targeted covalent inhibitor of p97 with proteome-wide selectivity

A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,viral infections and neurodegenerative diseases.Extensive efforts were devoted to the development of p97 inhibitors.The most promising inhibitor of p97 was in phase 1 clinical trials,but failed due to the off-target-induced toxicity,suggesting the selective inhibitors of p97 are highly needed.We report herein a new type of TCIs(i.e.,FL-18)that showed proteome-wide selectivity towards p97.Equipped with a Michael acceptor and a basic imidazole,FL-18 showed potent inhibition towards U87 MG tumor cells,and in proteome-wide profiling,selectively modified endogenous p97 as confirmed by in situ fluorescence scanning,label-free quantitative proteomics and functional validations.FL-18 selectively modified cysteine residues located within the D2 ATP site of p97.This covalent labeling of cysteine residue in p97 was verified by LC-MS/MS-based site-mapping and site-directed mutagenesis.Further structure-activity relationship(SAR)studies with FL-18 analogs were established.Collectively,FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity,thus providing a promising scaffold for cancer therapy.

A fundamental study on the fluorescence-quenching effect of nitro groups in tetraphenylethene AIE dyes with electron-withdrawing groups

A nitro group is a common fluorescence quencher,but its quenching efficiency can be easily affected by the surrounding environment.To date,there has been no systematic study on the effects of electronwithdrawing groups on the quenching efficiency of nitro groups.Herein,by virtue of experimental validation and theoretical calculations,we found that strong electron-withdrawing groups,such as pyridinium and dicyanovinyl groups,are detrimental to the quenching effect of nitro groups.Decreasing the electron-withdrawing ability could restore the nitro group’s quenching effect.

A heme-activatable probe and its application in the high-throughput screening of Plasmodium falciparum ring-stage inhibitors

Dear Editor,The resistance of Plasmodium falciparum to artemisinin derivatives(ARTs)in Asian,African and South American countries is a pressing global concern.Heme is an important biomarker of malaria parasites and is closely related to the mode of action of ARTs,but no heme-activatable fluorescent probe has been developed to study the biology of heme in malaria parasites in a light-up manner.Here,we rationally developed probes from the unique reaction between heme and Yingzhaosu A(YZSA),then we unprecedentedly applied the probe to identify ring-stage inhibitors of P.falciparum.