Target Enzyme‐Activated Two‐Photon Fluorescent Probes: A Case Study of CYP3A4 Using a Two‐Dimensional Design Strategy

Abstract

The rapid development of fluorescent probes for monitoring target enzymes is still agreat challenge owingtothe lacko fe fficient ways to optimizeaspecific fluorophore. Herein, ap ractical two-dimensional strategy was designed for the development of an isoform-specific probe for CYP3A4, ak ey cytochrome P450 isoform responsible for the oxidation of most clinical drugs.Infirst dimension of the design strategy, ap otential two-photon fluorescent substrate (NN)f or CYP3A4 was effectively selected using ensemble-based virtual screening.Inthe second dimension, various substituent groups were introduced into NN to optimize the isoform-selectivity and reactivity.F inally,w ith ideal selectivity and sensitivity, NEN was successfully applied to the real-time detection of CYP3A4 in living cells and zebrafish. These findings suggested that our strategy is practical for developing an isoform-specific probe for atarget enzyme.Cytochrome P450 monooxygenase (CYP) is as uperfamily of oxidative enzymes that metabolizes thousands of endogenous and exogenous substances through alkyl carbon and aromatic ring hydroxylation, O-a nd N-dealkylation, and epoxidation. [1] CYP3A4 is regarded as the most important CYP isoform in humans owing to its high abundance in liver and broad substrate spectrum, which contributes to the metabolism of more than 50 %o fclinical drugs. [2,3] Unfortunately,C YP3A4 activity can be modulated by many clinical drugs,t hereby frequently causing unfavorable drug-drug interactions (DDI), further leading to altered clinical outcomes or even life-threatening adverse reactions. [4] Additionally,asignificant inter-individual variability of CYP3A4 activity is frequently reported, arising from an umber of sources including genetic polymorphism and the response to environmental influences. [5] These factors have greatly limited the understanding of the precise role of CYP3A4 in drug metabolism and DDI, as aresult, negatively impacted clinical medication safety and effectiveness.To accurately characterize CYP3A4 activity,s ensitive technologies capable of the real-time monitoring of CYP3A4 activity are urgently needed. Tr aditional detection methods mainly rely upon mass spectrometry or high-performance liquid chromatography, [6,7] which are not compatible with living cell or in vivo applications.T wo-photon (TP) fluorescence microscopy,byvirtue of its higher sensitivity,real-time high spatial resolution imaging,a nd amenability to deeptissue bioimaging,h as shed new light on the monitoring of target enzyme activity in complex systems. [8, 9] However,n o TP fluorescent probe has been developed for the real-time and selective imaging of endogenous CYP3A4 activity in living systems.Previous attempts to develop af luorescent probe for CYP3A4 were based on adealkylation mechanism to release ad etectable moiety.T he O-alkyl derivatives of coumarin, resorufin, and fluorescein were designed and synthesized but met with limited success in isoform selectivity. [10] Theleading cause for the poor specificity of these probes is th...

Systems pharmacological study illustrates the immune regulation, anti-infection, anti-inflammation, and multi-organ protection mechanism of Qing-Fei-Pai-Du decoction in the treatment of COVID-19

“…The probe substrates for cytochrome P450 enzymes, including 7-ethoxyresorufin (for CYP1A1) and N-ethyl-1,8-naphthalimide (NEN, for CYP3A4) were synthesized by the authors according to previously reported literatures ( Ning et al., 2019 ; Yamaori et al., 2010 ). D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP+, resorufin were obtained from meilunbio (Dalian, China).…”

Optical substrates for drug-metabolizing enzymes: Recent advances and future perspectives

“…2 ). In general, O -dealkylation is a preferential reaction for CYP1A and other CYPs rather than CYP3A4, while CYP3A4 has the extraordinary ability to mediate the aromatic hydroxylation 36 . Notably, the exception is the development of hydroxylation-based fluorescent probe of CYP3A4 (CYP3A4-F1) recently reported by Ning et al.…”

“…Notably, the exception is the development of hydroxylation-based fluorescent probe of CYP3A4 (CYP3A4-F1) recently reported by Ning et al. 36 . This probe was designed by introducing the alkyl substituent groups to the non-reaction site of 1,8-naphthalimide, resulting the aromatic hydroxylation reaction of CYP3A4-F1.…”

“…for 3A5 16.0 c for 3A4 N.A. for 3A5 490/520 187 CYP3A-F3 1.03 for 3A4 2.17 for 3A5 0.55 for 3A4 1.28 for 3A5 490/520 34 CYP3A4 CYP3A4-F1 59.8 2.18 a 450/558 36 CYP3A4-F2 70 3.39 b 420/530 37 CYP3A4-F3 26 61.2 c 410/510 182 CYP3A4-F4 7.8 1.6 490/520 187 CYP3A4-F5 1.4 1.1 550/590 181 CYP3A4-F6 4.6 0.02 b 410/510 37 CYP1A1 Luciferin-1A1 3.0 N.A. 560 188 CYP1A2 Luciferin-1A2 6.0 N.A.…”

A NIR fluorescent probe for Vanin-1 and its applications in imaging, kidney injury diagnosis, and the development of inhibitor

“…Traditional analysis methods for Vanin-1 including radioactive isotope labeling and spectrophotometric assay are chiefly based on the quantification of cysteamine 15 , 16 , which are not applicable for in situ dynamic determining enzyme's bioactivity, due to insufficient sensitivity and laborious measurement procedure. Recently, fluorescence/bioluminescence-based analytical technology has received considerable attention for the advantages of simplicity, superb selectivity/sensitivity, real-time visualization, and non-invasive detecting 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 . For Vanin-1, few probes have been designed and developed for detecting Vanin-1 in cells and mice recently.…”