Rationally Engineered CYP3A4 Fluorogenic Substrates for Functional Imaging Analysis and Drug-Drug Interaction Studies.

Cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme-mediated drug metabolism and drug-drug interaction (DDI). Herein, an effective strategy was used to rationally construct a practical two-photon fluorogenic substrate for hCYP3A4. Following two-round structure-based substrate discovery and optimization, we have successfully constructed a hCYP3A4 fluorogenic substrate (F8) with desirable features, including high binding affinity, rapid response, excellent isoform specificity, and low cytotoxicity. Under physiological conditions, F8 is readily metabolized by hCYP3A4 to form a brightly fluorescent product (4-OH F8) that can be easily detected by various fluorescence devices. The practicality of F8 for real-time sensing and functional imaging of hCYP3A4 has been examined in tissue preparations, living cells, and organ slices. F8 also demonstrates good performance for high-throughput screening of hCYP3A4 inhibitors and assessing DDI potentials in vivo. Collectively, this study develops an advanced molecular tool for sensing CYP3A4 activities in biological systems, which strongly facilitates CYP3A4-associated fundamental and applied research studies.

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation, chemical profiling and biochemical assay.

Although herbal medicines (HMs) are widely used in the prevention and treatment of obesity and obesity-associated disorders, the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood. Recently, we assessed the inhibitory potentials of several HMs against human pancreatic lipase (hPL, a key therapeutic target for human obesity), among which the root-extract of Rhodiola crenulata (ERC) showed the most potent anti-hPL activity. In this study, we adopted an integrated strategy, involving bioactivity-guided fractionation techniques, chemical profiling, and biochemical assays, to identify the key anti-hPL constituents in ERC. Nine ERC fractions (retention time = 12.5-35 min), obtained using reverse-phase liquid chromatography, showed strong anti-hPL activity, while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS). Among the identified ERC constituents, 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) and catechin gallate (CG) showed the most potent anti-hPL activity, with pIC50 values of 7.59 ± 0.03 and 7.68 ± 0.23, respectively. Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner, with inhibition constant (K i) values of 0.012 and 0.082 µM, respectively. Collectively, our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC, as well as to elucidate their anti-hPL mechanisms. These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Discovery and characterization of amentoflavone as a naturally occurring inhibitor against the bile salt hydrolase produced by Lactobacillus salivarius.

Bile salt hydrolases (BSHs), a group of cysteine-hydrolases produced by gut microbes, play a crucial role in the hydrolysis of glycine- or taurine-conjugated bile acids and have been validated as key targets to modulate bile acid metabolism. This study aims to discover one or more efficacious inhibitors against a BSH produced by Lactobacillus salivarius (lsBSH) from natural products and to characterize the mechanism of the newly identified BSH inhibitor(s). Following screening of the inhibition potentials of more than 100 natural compounds against lsBSH, amentoflavone (AMF), a naturally occurring biflavone isolated from various medicinal plants, was discovered to be an efficacious BSH inhibitor (IC50 = 0.34 µM). Further investigation showed that AMF could strongly inhibit the lsBSH-catalyzed hydrolytic reaction in living gut microbes. Inhibition kinetic analyses demonstrated that AMF reversibly inhibited the lsBSH-catalyzed hydrolytic reaction in a mixed-inhibition manner, with an apparent Ki value of 0.65 µM. Fluorescence quenching assays suggested that AMF could quench the fluorescence of lsBSH via a static quenching procedure. Docking simulations suggested that AMF could be fitted into lsBSH at two distinct ligand-binding sites, mainly via hydrophobic interactions and hydrogen bonding, which explained well the mixed inhibition mode of this agent. Animal tests showed that the hydrolytic activities of BSHs in mice feces could be significantly blocked by AMF. In summary, this study reports that AMF is a strong, naturally occurring inhibitor of lsBSH, which offers a promising lead compound to develop novel agents for modulating bile acid metabolism in the host via targeting BSHs.

Discovery of triterpenoids as potent dual inhibitors of pancreatic lipase and human carboxylesterase 1.

Pancreatic lipase (PL) is a well-known key target for the prevention and treatment of obesity. Human carboxylesterase 1A (hCES1A) has become an important target for the treatment of hyperlipidaemia. Thus, the discovery of potent dual-target inhibitors based on PL and hCES1A hold great potential for the development of remedies for treating related metabolic diseases. In this study, a series of natural triterpenoids were collected and the inhibitory effects of these triterpenoids on PL and hCES1A were determined using fluorescence-based biochemical assays. It was found that oleanolic acid (OA) and ursolic acid (UA) have the excellent inhibitory effects against PL and hCES1A, and highly selectivity over hCES2A. Subsequently, a number of compounds based on the OA and UA skeletons were synthesised and evaluated. Structure-activity relationship (SAR) analysis of these compounds revealed that the acetyl group at the C-3 site of UA (compound 41) was very essential for both PL and hCES1A inhibition, with IC50 of 0.75 µM and 0.014 µM, respectively. In addition, compound 39 with 2-enol and 3-ketal moiety of OA also has strong inhibitory effects against both PL and hCES1A, with IC50 of 2.13 µM and 0.055 µM, respectively. Furthermore, compound 39 and 41 exhibited good selectivity over other human serine hydrolases including hCES2A, butyrylcholinesterase (BChE) and dipeptidyl peptidase IV (DPP-IV). Inhibitory kinetics and molecular docking studies demonstrated that both compounds 39 and 41 were effective mixed inhibitors of PL, while competitive inhibitors of hCES1A. Further investigations demonstrated that both compounds 39 and 41 could inhibit adipocyte adipogenesis induced by mouse preadipocytes. Collectively, we found two triterpenoid derivatives with strong inhibitory ability on both PL and hCES1A, which can be served as promising lead compounds for the development of more potent dual-target inhibitors targeting on PL and hCES1A.

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation,chemical profiling and biochemical assay / 药物分析学报

Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5-35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 μM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Discovery and Characterization of the Biflavones From Ginkgo biloba as Highly Specific and Potent Inhibitors Against Human Carboxylesterase 2.

Human carboxylesterase 2 (CES2), one of the most abundant hydrolases distributed in the small intestine, has been validated as a key therapeutic target to ameliorate the intestinal toxicity caused by irinotecan. This study aims to discover efficacious CES2 inhibitors from natural products and to characterize the inhibition potentials and inhibitory mechanisms of the newly identified CES2 inhibitors. Following high-throughput screening and evaluation of the inhibition potency of more than 100 natural products against CES2, it was found that the biflavones isolated from Ginkgo biloba displayed extremely potent CES2 inhibition activities and high specificity over CES1 (>1000-fold). Further investigation showed that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed hydrolysis of various substrates, including the CES2 substrate-drug irinotecan. Notably, the inhibition potentials of four biflavones against CES2 were more potent than that of loperamide, a marketed anti-diarrhea agent used for alleviating irinotecan-induced intestinal toxicity. Inhibition kinetic analyses demonstrated that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed fluorescein diacetate hydrolysis via a reversible and mixed inhibition manner, with K i values of less than 100 nM. Ensemble docking and molecular dynamics revealed that these biflavones could tightly and stably bind on the catalytic cavity of CES2 via hydrogen bonding and π-π stacking interactions, while the interactions with CES1 were awfully poor. Collectively, this study reports that the biflavones isolated from Ginkgo biloba are potent and highly specific CES2 inhibitors, which offers several promising lead compounds for developing novel anti-diarrhea agent to alleviate irinotecan-induced diarrhea.

Design, synthesis and biological evaluation of novel chalcone-like compounds as potent and reversible pancreatic lipase inhibitors.

Pancreatic lipase (PL), a crucial enzyme responsible for hydrolysis of dietary lipids, has been validated as a key therapeutic target to prevent and treat obesity-associated metabolic disorders. Herein, we report the design, synthesis and biological evaluation of a series of chalcone-like compounds as potent and reversible PL inhibitors. Following two rounds of structural modifications at both A and B rings of a chalcone-like skeleton, structure-PL inhibition relationships of the chalcone-like compounds were studied, while the key substituents that would be beneficial for PL inhibition were revealed. Among all tested chalcone-like compounds, compound B13 (a novel chalcone-like compound bearing two long carbon chains) displayed the most potent PL inhibition activity, with an IC50 value of 0.33 µM. Inhibition kinetic analyses demonstrated that B13 could potently inhibit PL-mediated 4-MUO hydrolysis in a mixed inhibition manner, with the Ki value of 0.12 µM. Molecular docking simulations suggested that B13 could tightly bind on PL at both the catalytic site and a non-catalytic site that was located on the surface of PL, which was consistent with the mixed inhibition mode of this agent. In addition, B13 displayed excellent stability in artificial gastrointestinal fluids and good metabolic stability in human liver preparations. Collectively, our findings suggested that chalcone-like compounds were good choices for design and development of orally administrated PL inhibitors, while B13 could be served as a promising lead compound to develop novel anti-obesity agents via targeting on PL.

Design, synthesis and biological evaluation of indanone-chalcone hybrids as potent and selective hCES2A inhibitors.

Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intestine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some important hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanone-chalcone hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition assays demonstrated that most indanone-chalcone hybrids displayed strong to moderate hCES2A inhibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of a hydroxyl at the C4' site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A inhibition. Particularly, B7 (an N-alkylated 1-indanone-chalcone hybrid) exhibited the most potent inhibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated Ki value of 0.068 µM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed good metabolic stability. Collectively, our findings show that indanone-chalcone hybrids are good choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.

Rapalogues as hCES2A Inhibitors: In Vitro and In Silico Investigations.

BACKGROUND AND OBJECTIVE: Rapamycin and its semi-synthetic analogues (rapalogues) are frequently used in combination with other prescribed medications in clinical settings. Although the inhibitory effects of rapalogues on cytochrome P450 enzymes (CYPs) have been well examined, the inhibition potentials of rapalogues on human esterases have not been investigated. Herein, the inhibition potentials and inhibitory mechanisms of six marketed rapalogues on human esterases are investigated. METHODS: The inhibitory effects of six marketed rapalogues (rapamycin, zotarolimus, temsirolimus, everolimus, pimecrolimus and tacrolimus) on three major esterases, including human carboxylesterases 1 (hCES1A), human carboxylesterases 2 (hCES2A) and butyrylcholinesterase (BuChE), were assayed using isozyme-specific substrates. Inhibition kinetic analyses and docking simulations were performed to investigate the inhibitory mechanisms of the rapalogues with strong hCES2A inhibition potency. RESULTS: Zotarolimus and pimecrolimus displayed strong inhibition of human hCES2A but these agents did not inhibit hCES1A or BuChE. Further investigation demonstrated that zotarolimus could strongly inhibit intracellular hCES2A in living HepG2 cells, with an estimated IC50 value of 4.09 µM. Inhibition kinetic analyses revealed that zotarolimus inhibited hCES2A-catalyzed fluorescein diacetate hydrolysis in a mixed manner, with the Ki value of 1.61 µM. Docking simulations showed that zotarolimus could tightly bind on hCES2A at two district ligand-binding sites, consistent with its mixed inhibition mode. CONCLUSION: Our findings demonstrate that several marketed rapalogues are potent and specific hCES2A inhibitors, and these agents can serve as leading compounds for the development of more efficacious hCES2A inhibitors to modulate the pharmacokinetic profiles and toxicity of hCES2A-substrate drugs (such as the anticancer agent irinotecan).

Discovery of natural alkaloids as potent and selective inhibitors against human carboxylesterase 2.

Human Carboxylesterase 2A (hCES2A), one of the most important serine hydrolases, plays crucial roles in the hydrolysis and the metabolic activation of a wide range of esters and amides. Increasing evidence has indicated that potent inhibition on intestinal hCES2A may reduce the excessive accumulation of SN-38 (the hydrolytic metabolite of irinotecan with potent cytotoxicity) in the intestinal tract and thereby alleviate the intestinal toxicity triggered by irinotecan. In this study, more than sixty natural alkaloids have been collected and their inhibitory effects against hCES2A are assayed using a fluorescence-based biochemical assay. Following preliminary screening, seventeen alkaloids are found with strong to moderate hCES2A inhibition activity. Primary structure-activity relationships (SAR) analysis of natural isoquinoline alkaloids reveal that the benzo-1,3-dioxole group and the aromatic pyridine structure are beneficial for hCES2A inhibition. Further investigations demonstrate that a steroidal alkaloid reserpine exhibits strong hCES2A inhibition activity (IC50 = 0.94 µM) and high selectivity over other human serine hydrolases including hCES1A, dipeptidyl peptidase IV (DPP-IV), butyrylcholinesterase (BChE) and thrombin. Inhibition kinetic analyses demonstrated that reserpine acts as a non-competitive inhibitor against hCES2A-mediated FD hydrolysis. Molecular docking simulations demonstrated that the potent inhibition of hCES2A by reserpine could partially be attributed to its strong σ-π and S-π interactions between reserpine and hCES2A. Collectively, our findings suggest that reserpine is a potent and highly selective inhibitor of hCES2A, which can be served as a promising lead compound for the development of more efficacious and selective alkaloids-type hCES2A inhibitors for biomedical applications.

Pentacyclic triterpenoid acids in Styrax as potent and highly specific inhibitors against human carboxylesterase 1A.

Human carboxylesterase 1A1 (hCES1A) is a promising target for the treatment of hyperlipidemia and obesity-associated metabolic diseases. To date, the highly specific and efficacious hCES1A inhibitors are rarely reported. This study aims to find potent and highly specific hCES1A inhibitors from herbs, and to investigate their inhibitory mechanisms. Following large-scale screening of herbal products, Styrax was found to have the most potent hCES1A inhibition activity. After that, a practical bioactivity-guided fractionation coupling with a chemical profiling strategy was used to identify the fractions from Styrax with strong hCES1A inhibition activity and the major constituents in these bioactive fractions were characterized by LC-TOF-MS/MS. The results demonstrated that seven pentacyclic triterpenoid acids (PTAs) in two bioactive fractions from Styrax potently inhibit hCES1A, with IC50 values ranging from 41 nM to 478 nM. Among all the identified PTAs, epibetulinic acid showed the most potent inhibition activity and excellent specificity towards hCES1A. Both inhibition kinetic analyses and in silico analysis suggested that epibetulinic acid potently inhibited hCES1A in a mixed inhibition manner. Collectively, our findings demonstrate that some PTAs in Styrax are potent and highly specific inhibitors of hCES1A and these constituents can be used as promising lead compounds for the development of more efficacious hCES1A inhibitors.

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism.

BACKGROUND: Styrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions. PURPOSE: This study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered. STUDY DESIGN: The inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated. METHODS: The inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis. RESULTS: In vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6ß-hydroxylation. CONCLUSION: Styrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.

Pancreatic Lipase Inhibitory Cyclohexapeptides from the Marine Sponge-Derived Fungus Aspergillus sp. 151304.

Three new cyclohexapeptides, petrosamides A-C (1-3), were isolated from the sponge-derived fungus Aspergillus sp. 151304. Their structures were elucidated by detailed 1D and 2D spectroscopic analyses, and the absolute configurations of the amino acid residues were determined by the advanced Marfey's method. These peptides displayed significant and dose-dependent pancreatic lipase (PL) inhibitory activities, with IC50 values of 7.6 ± 1.5, 1.8 ± 0.3, and 0.5 ± 0.1 µM, respectively. Further inhibition kinetics analyses showed that compound 3 inhibited PL in a noncompetitive manner, while molecular dynamics simulation revealed that it could bind to PL at the entrance of the catalytic pocket.

Reversible and Irreversible Inhibition of Cytochrome P450 Enzymes by Methylophiopogonanone A.

Methylophiopogonanone A (MOA), an abundant homoisoflavonoid bearing a methylenedioxyphenyl moiety, is one of the major constituents in the Chinese herb Ophiopogon japonicas This work aims to assess the inhibitory potentials of MOA against cytochrome P450 enzymes and to decipher the molecular mechanisms for P450 inhibition by MOA. The results showed that MOA concentration-dependently inhibited CYP1A, 2C8, 2C9, 2C19, and 3A in human liver microsomes (HLMs) in a reversible way, with IC50 values varying from 1.06 to 3.43 µM. By contrast, MOA time-, concentration-, and NADPH-dependently inhibited CYP2D6 and CYP2E1, along with KI and kinact values of 207 µM and 0.07 minute-1 for CYP2D6, as well as 20.9 µM and 0.03 minutes-1 for CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in an HLM incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate. Additionally, the potential risks of herb-drug interactions triggered by MOA or MOA-related products were also predicted. Collectively, our findings verify that MOA is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. SIGNIFICANCE STATEMENT: Methylophiopogonanone A (MOA), an abundant homoisoflavonoid isolated from the Chinese herb Ophiopogon japonicas, is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in a human liver microsome incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate.