Tong-Yi Dou

A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

Assessment of the inhibitory effects of pyrethroids against human carboxylesterases

ABSTRACT Pyrethroids are broad‐spectrum insecticides that widely used in many countries, while humans may be exposed to these toxins by drinking or eating pesticide‐contaminated foods. This study aimed to investigate the inhibitory effects of six commonly used pyrethroids against two major human carboxylesterases (CES) including CES1 and CES2. Three optical probe substrates for CES1 (DME, BMBT and DMCB) and a fluorescent probe substrate for CES2 (DDAB) were used to characterize the inhibitory effects of these pyrethroids. The results demonstrated that most of the tested pyrethroids showed moderate to weak inhibitory effects against both CES1 and CES2, but deltamethrin displayed strong inhibition towards CES1. The IC50 values of deltamethrin against CES1‐mediated BMBT, DME, and DMCB hydrolysis were determined as 1.58 &mgr;M, 2.39 &mgr;M, and 3.3 &mgr;M, respectively. Moreover, deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in living cells. Further investigation revealed that deltamethrin inhibited CES1‐mediated BMBT hydrolysis via competitive manner but noncompetitively inhibited DME or DMCB hydrolysis. The inhibition behaviors of deltamethrin against CES1 were also studied by molecular docking simulation. The results demonstrated that CES1 had at least two different ligand‐binding sites, one was the DME site and another was the BMBT site which was identical to the binding site of deltamethrin. In summary, deltamethrin was a strong reversible inhibitor against CES1 and it could tightly bind on CES1 at the same ligand‐binding site as BMBT. These findings are helpful for the deep understanding of the interactions between xenobiotics and CES1. HIGHLIGHTSThe inhibitory effects of six commonly used pyrethroids on human carboxylesterases were investigated.Deltamethrin displayed strong inhibitory effects against human carboxylesterase 1 (CES1).Deltamethrin was cell membrane permeable and could inhibit intracellular CES1 in living cells.Both experimental and docking studies demonstrated that CES1 had at least two different ligand‐binding sites.

An Optimized Two-Photon Fluorescent Probe for Biological Sensing and Imaging of Catechol-O-Methyltransferase

A practical two-photon fluorescent probe was developed for highly sensitive and selective sensing of the activities of catechol-O-methyltransferase (COMT) in complex biological samples. To this end, a series of 3-substituted 7,8-dihydroxycoumarins were designed and synthesized. Among them, 3-BTD displayed the best combination of selectivity, sensitivity, reactivity, and fluorescence response following COMT-catalyzed 8-O-methylation. The newly developed two-photon fluorescent probe 3-BTD can be used for determining the activities of COMT in complex biological samples and bio-imaging of endogenous COMT in living cells and tissue slices with good cell permeability, low cytotoxicity, and high imaging resolution. All these findings suggest that 3-BTD holds great promise for developing therapeutic molecules that target COMT, as well as for exploring COMT-associated biological processes and its biological functions in living systems. Furthermore, the strategy also sheds new light on the development of fluorescent probes for other conjugative enzymes.

Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel.

Cellulosome is a kind of multienzyme complex that displays high activity, selectivity, and stability. Here, we report a novel, non-cellulolytic, cellulosome-like multienzyme complex that produced by the Cellulosimicrobium cellulans wild-type strain F16 isolated from soil microflora. This multienzyme complex, with excellent catalytic efficiency of kcat 13.2 s−1 to remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (10-DAXP), has an outstanding tolerance against organic solvents and an excellent general stability, with the long half-life of 214 hours. This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones. The key catalytic subunit responsible for the β-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family. The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

Structure-Activity Relationships of Pentacyclic Triterpenoids as Potent and Selective Inhibitors against Human Carboxylesterase 1

Human carboxylesterase 1 (hCE1), one of the most important serine hydrolases distributed in liver and adipocytes, plays key roles in endobiotic homeostasis and xenobiotic metabolism. This study aimed to find potent and selective inhibitors against hCE1 from phytochemicals and their derivatives. To this end, a series of natural triterpenoids were collected and their inhibitory effects against human carboxylesterases (hCEs) were assayed using D-Luciferin methyl ester (DME) and 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB) as specific optical substrate for hCE1, and hCE2, respectively. Following screening of a series of natural triterpenoids, oleanolic acid (OA), and ursolic acid (UA) were found with strong inhibitory effects on hCE1 and relative high selectivity over hCE2. In order to get the highly selective and potent inhibitors of hCE1, a series of OA and UA derivatives were synthesized from OA and UA by chemical modifications including oxidation, reduction, esterification, and amidation. The inhibitory effects of these derivatives on hCEs were assayed and the structure-activity relationships of tested triterpenoids as hCE1 inhibitors were carefully investigated. The results demonstrated that the carbonyl group at the C-28 site is essential for hCE1 inhibition, the modifications of OA or UA at this site including esters, amides and alcohols are unbeneficial for hCE1 inhibition. In contrast, the structural modifications on OA and UA at other sites, such as converting the C-3 hydroxy group to 3-O-β-carboxypropionyl (compounds 20 and 22), led to a dramatically increase of the inhibitory effects against hCE1 and very high selectivity over hCE2. 3D-QSAR analysis of all tested triterpenoids including OA and UA derivatives provide new insights into the fine relationships linking between the inhibitory effects on hCE1 and the steric-electrostatic properties of triterpenoids. Furthermore, both inhibition kinetic analyses and docking simulations demonstrated that compound 22 was a potent competitive inhibitor against hCE1-mediated DME hydrolysis. All these findings are very helpful for medicinal chemists to design and develop highly selective and more potent hCE1 inhibitors for biomedical applications.

Characterization and structure-activity relationship studies of flavonoids as inhibitors against human carboxylesterase 2

Human carboxylesterases (hCEs) are key enzymes from the serine hydrolase superfamily. Among all identified hCEs, human carboxylesterase 2 (hCE2) plays crucial roles in the metabolic activation of ester drugs including irinotecan and flutamide. Selective and potent hCE2 inhibitors could be used to alleviate the toxicity induced by hCE2-substrate drugs. In this study, more than fifty flavonoids were collected to assay their inhibitory effects against hCE2 using a fluorescence-based method. The results demonstrated that C3 and C6 hydroxy groups were essential for hCE2 inhibition, while O-glycosylation or C-glycosylation would lead to the loss of hCE2 inhibition. Among all tested flavonoids, 5,6-dihydroxyflavone displayed the most potent inhibitory effect against hCE2 with the IC50 value of 3.50 μM. The inhibition mechanism of 5,6-dihydroxyflavone was further investigated by both experimental and docking simulations. All these findings are very helpful for the medicinal chemists to design and develop more potent and highly selective flavonoid-type hCE2 inhibitors.

A Naturally Occurring Isoform-Specific Probe for Highly Selective and Sensitive Detection of Human Cytochrome P450 3A5

Cytochrome P450 (CYP) 3A5 characterized with polymorphic and extensive expression in multiple tissues is the most important P450 enzyme among the minor CYP3A isoforms. However, a selective and sensitive probe for CYP3A5 remains unavailable. In this study, we identified and characterized a naturally occurring lignan 12 (schisantherin E) as an isoform-specific probe for selective detection of CYP3A5 activity in complex biological samples. With thorough characterization including LC-MS and NMR, we found that 12 can be metabolized by CYP3A5 to generate a major metabolite 2-O-demethylated 12. Meanwhile, both reaction phenotyping and chemical inhibition experiments further revealed that CYP3A5 selectively catalyzed the 2-O-demethylation of 12. Specifically, the interactions between the Phe210 residue of CYP3A5 and methyl benzoate of 12 might play key roles in 12-O-demethylation, which was revealed by docking simulation and site-directed mutagenesis studies. These findings are beneficial for exploring the role of CYP3A5 in drug metabolism and pathologic process.

In vitro characterization of the glucuronidation pathways of licochalcone A mediated by human UDP-glucuronosyltransferases

Abstract This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM). HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM. LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4′-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4′-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4′-O-glucuronide. UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4′-O-glucuronidation.

Interspecies comparison in the COMT-mediated methylation of 3-BTD

Catechol-O-methyltransferase (COMT) is a druggable biological target and COMT modulators have been widely applied in the treatment of various central and peripheral nervous system disorders. The interspecies differences of COMT were carefully investigated using 3-BTD (a newly developed fluorescent probe of COMT) methylation as the probe reaction, and liver S9 from humans and seven experimental animals including monkeys, dogs, mice, rats, minipigs, guinea pigs and New Zealand rabbits as the enzyme source. Metabolite profiling demonstrated that all the tested liver S9 samples from the different animals could catalyse 3-BTD methylation but displayed significant differences in reaction rate. Also, the differential effects of tolcapone (a potent inhibitor against COMT) on 3-BTD methylation among various species were observed. The apparent kinetic parameters and the maximum intrinsic clearances (Clint) for 3-BTD methylation in liver S9 from the different animals were determined, and the order of the Clint values for the formation of 3-BTD was RLS9 > DLS9 ≈ PLS9 > MLS9 > CyLS9 > RaLS9 > GpLS9 > HLS9. These findings are helpful for further exploring COMT-associated biological processes in animal models, as well as for developing therapeutic molecules that target COMT.

Synthesis and Structure-Activity Relationship of Daphnetin Derivatives as Potent Antioxidant Agents

In this study, daphnetin 1 was chosen as the lead compound, and C-3 or C-4-substituted daphnetins were designed and synthesized to explore the potential relationship between the antioxidant activities and the chemical structures of daphnetin derivatives. The antioxidant activities of the generated compounds were evaluated utilizing the free radical scavenging effect on 2,2′-diphenyl-1-picrylhydrazyl, 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation, and the ferric reducing power assays, and were then compared with those of the standard antioxidant Trolox. The results showed that the catechol group was the key pharmacophore for the antioxidant activity of the daphnetins. The introduction of an electron-withdrawing hydrophilic group at the C-4 position of daphnetin enhanced the antioxidative capacity, but this trend was not observed for C-3 substitution. In addition, introduction of a a hydrophobic phenyl group exerted negative effects on the antioxidant activity in both the C-3 and C-4 substitutions. Among all of the derivatives tested, the most powerful antioxidant was 4-carboxymethyl daphnetin (compound 9), for which the strongest antioxidant activity was observed in all of the assays. In addition, compound 9 also displayed strong pharmaceutical properties in the form of metabolic stability. To summarize, compound 9 holds great potential to be developed as an antioxidant agent with excellent antioxidant activity and proper pharmacokinetic behavior.