A label-free LC/MS-based enzymatic activity assay for the detection of PDE5A inhibitors

Phosphodiesterase type 5 (PDE5), a cyclic nucleotide phosphodiesterase, controls the duration of the cyclic guanosine monophosphate (cGMP) signal by hydrolyzing cGMP to GMP. Inhibiting the activity of PDE5A has proven to be an effective strategy for treating pulmonary arterial hypertension and erect...

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Published inFrontiers in chemistry Vol. 11; p. 1097027
Main Authors Ma, Yufeng, Zhang, Fengsen, Zhong, Yijing, Huang, Yongchun, Yixizhuoma, Jia, Qiangqiang, Zhang, Shoude
Format Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 13.02.2023
Subjects
cGMP
Chemistry
enzyme activity
inhibitor
LC/MS
PDE5A
LC/MS
cGMP
PDE5A
inhibitor
enzyme activity
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Summary:Phosphodiesterase type 5 (PDE5), a cyclic nucleotide phosphodiesterase, controls the duration of the cyclic guanosine monophosphate (cGMP) signal by hydrolyzing cGMP to GMP. Inhibiting the activity of PDE5A has proven to be an effective strategy for treating pulmonary arterial hypertension and erectile dysfunction. Current enzymatic activity assay methods for PDE5A mainly use fluorescent or isotope-labeled substrates, which are expensive and inconvenient. Here, we developed an LC/MS-based enzymatic activity assay for PDE5A without labeling, which detects the enzymatic activity of PDE5A by quantifying the substrate cGMP and product GMP at a concentration of 100 nM. The accuracy of this method was verified by a fluorescently labeled substrate. Moreover, a new inhibitor of PDE5A was identified by this method and virtual screening. It inhibited PDE5A with an IC value of 870 nM. Overall, the proposed strategy provides a new method for screening PDE5A inhibitors.
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Edited by: Xuetao Xu, Wuyi University, China
Edson Roberto Silva, University of São Paulo, Brazil
These authors have contributed equally to this work
This article was submitted to Medicinal and Pharmaceutical Chemistry, a section of the journal Frontiers in Chemistry
Reviewed by: Xinhui Su, Zhejiang University, China
ISSN:2296-2646
2296-2646
DOI:10.3389/fchem.2023.1097027