In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylch...In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.展开更多
α_(1)-Adrenergic receptor(AR)blockers can be effective for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms(BPH/LUTS),their usage is limited by cardiovascular-related side effects that are c...α_(1)-Adrenergic receptor(AR)blockers can be effective for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms(BPH/LUTS),their usage is limited by cardiovascular-related side effects that are caused by the subtype nonselective nature or low selectivity of many current drugs.We previously reported that phenylpiperazine analogues with amide and propane linker were moderateα_(1D/1A)adrenoceptor antagonists and exhibited better anti-BPH effect than lead compound naftopidil(NAF)in vivo,however,with modestα_(1D/1A)-subtype selectivity.Herein,we replaced propane moiety with2-hydroxypropanol linker and synthesized twenty-seven racemic derivatives with modified aromatic and hetero aromatic groups.Of these new compounds,quinoline surrogate 17 exhibited extremely weak antagonistic affinity onα_(1B)in both cell-based calcium assay and tissue-based functional assay,so that elicited significantα_(1A/1B)andα_(1D/1B)selectivity.Intriguingly,the R enantiomer of 17 preferentially displayed superior anti-BPH effect in rat model compared with S-17,supporting ligand regulates the receptor in a highly stereospecific manner.Finally,the computer-aided modelling research was also performed in order to deeply understand the unique binding mode of R-17 in complex withα_(1A)and the subtype receptor selectivity for R-17 was also rationalized in this study.Taken together,our work enriched the diversity of phenylpiperazines for the treatment of BPH/LUTS,and provided a basis for discovery ofα_(1D/1A)-selective ligands.展开更多
OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncerta...OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.展开更多
Uridine diphosphate(UDP)-glucuronosyltransferases(UGTs)are enzymes involved in the biotransformation of important endogenous compounds such as steroids,bile acids,and hormones as well as exogenous substances including...Uridine diphosphate(UDP)-glucuronosyltransferases(UGTs)are enzymes involved in the biotransformation of important endogenous compounds such as steroids,bile acids,and hormones as well as exogenous substances including drugs,environmental toxicants,and carcinogens.Here,a novel fluorescent probe BDMP was developed based on boron-dipyrromethene(BODIPY)with high sensitivity for the detection of UGT1A8.The glucuronidation of BDMP not only exhibited a redemission wavelength(lex/lem=500/580 nm),but also displayed an excellent UGT1A8-dependent fluorescence signal with a good linear relationship with UGT1A8 concentration.Based on this perfect biocompatibility and cell permeability,BDMP was successfully used to image endogenous UGT1A8 in human cancer cell lines(LoVo and HCT15)in real time.In addition,BDMP could also be used to visualize UGT1A8 in tumor tissues.These results suggested that BDMP is a promising molecular tool for the investigation of UGT1A8-mediated physiological function in humans.展开更多
基金National Natural Science Foundation of China (Grant No. 30672445)
文摘In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.
基金supported by Natural Science Foundation of Guangdong Province(Nos.2021A1515010101,2021A1515011372,2023A1515011895)National Natural Science Foundation of China(Nos.21807017,82273759,32371529)Guangzhou Medical University Scientific Research Capacity Improvement Project(No.02-410-2405104)。
文摘α_(1)-Adrenergic receptor(AR)blockers can be effective for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms(BPH/LUTS),their usage is limited by cardiovascular-related side effects that are caused by the subtype nonselective nature or low selectivity of many current drugs.We previously reported that phenylpiperazine analogues with amide and propane linker were moderateα_(1D/1A)adrenoceptor antagonists and exhibited better anti-BPH effect than lead compound naftopidil(NAF)in vivo,however,with modestα_(1D/1A)-subtype selectivity.Herein,we replaced propane moiety with2-hydroxypropanol linker and synthesized twenty-seven racemic derivatives with modified aromatic and hetero aromatic groups.Of these new compounds,quinoline surrogate 17 exhibited extremely weak antagonistic affinity onα_(1B)in both cell-based calcium assay and tissue-based functional assay,so that elicited significantα_(1A/1B)andα_(1D/1B)selectivity.Intriguingly,the R enantiomer of 17 preferentially displayed superior anti-BPH effect in rat model compared with S-17,supporting ligand regulates the receptor in a highly stereospecific manner.Finally,the computer-aided modelling research was also performed in order to deeply understand the unique binding mode of R-17 in complex withα_(1A)and the subtype receptor selectivity for R-17 was also rationalized in this study.Taken together,our work enriched the diversity of phenylpiperazines for the treatment of BPH/LUTS,and provided a basis for discovery ofα_(1D/1A)-selective ligands.
基金supported by National Institute of Neurological Disorders and Stroke Intramural Research Program
文摘OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.
基金the Natural Science Foundation of Liaoning Province 2020-MS-252the National Key R&D Program of China(Grant No.2018YFC1603001).
文摘Uridine diphosphate(UDP)-glucuronosyltransferases(UGTs)are enzymes involved in the biotransformation of important endogenous compounds such as steroids,bile acids,and hormones as well as exogenous substances including drugs,environmental toxicants,and carcinogens.Here,a novel fluorescent probe BDMP was developed based on boron-dipyrromethene(BODIPY)with high sensitivity for the detection of UGT1A8.The glucuronidation of BDMP not only exhibited a redemission wavelength(lex/lem=500/580 nm),but also displayed an excellent UGT1A8-dependent fluorescence signal with a good linear relationship with UGT1A8 concentration.Based on this perfect biocompatibility and cell permeability,BDMP was successfully used to image endogenous UGT1A8 in human cancer cell lines(LoVo and HCT15)in real time.In addition,BDMP could also be used to visualize UGT1A8 in tumor tissues.These results suggested that BDMP is a promising molecular tool for the investigation of UGT1A8-mediated physiological function in humans.
