Inhibition of enzyme activity at high substrate concentrations, so-called "substrate inhibition," is commonly observed and has been recognized in drug metabolism reactions since the last decade. ...Although the importance of such "atypical" kinetics in vivo remains poorly understood, a substrate with substrate inhibition kinetics has been shown to unconventionally alter the metabolism of other substrates. In recent years, it is becoming increasingly evident that the mechanisms for substrate inhibition are highly complex, which are possibly contributed by multiple (at least two) binding sites within the enzyme protein, the formation of a ternary dead-end enzyme complex, and/or the ligand-induced changes in enzyme conformation. This review primarily discusses the mechanisms for substrate inhibition displayed by the important drug-metabolizing enzymes, such as cytochrome p450s, UDP-glucuronyltransferases, and sulfotransferases. Kinetic modeling of substrate inhibition in the absence or presence of a modifier is another central issue in this review because of its importance in the determination of kinetic parameters and in vitro/in vivo predictions.
The roles of Rev-erbα and circadian clock in colonic inflammation remain unclarified. Here we show colon clock genes (including Rev-erbα) are dysregulated in mice with DSS-induced colitis. In turn, ...disruption of the circadian clock exacerbates experimental colitis. Rev-erbα-deficient mice are more sensitive to DSS-induced colitis, supporting a critical role of Rev-erbα in disease development. Further, Rev-erbα ablation causes activation of Nlrp3 inflammasome in mice. Cell-based experiments reveal Rev-erbα inactivates Nlrp3 inflammasome mainly at the priming stage. Rev-erbα directly represses Nlrp3 transcription through specific binding to the promoter region. Additionally, Rev-erbα represses p65 transcription and indirectly repressed Nlrp3 via the NF-κB pathway. Interestingly, Rev-erbα activation in wild-type mice by SR9009 attenuates DSS-induced colitis, whereas the protective effects are lost in Nlrp3
and Rev-erbα
mice. Taken together, Rev-erbα regulates experimental colitis through its repressive action on the NF-κB/Nlrp3 axis. Targeting Rev-erbα may represent a promising approach for prevention and management of colitis.
Glutathione transferases (GSTs) are important detoxifying enzymes that catalyze the conjugation of electrophilic substrates to glutathione. In recent years, GSTs have been of great interest in ...pharmacology and drug development because of their involvement in many important biological processes such as steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. This review describes crystal structures for cytosolic GSTs and correlates active-site features with enzyme functions (e.g., steroid synthesis, tyrosine degradation, and dehydroascorbate reduction) and substrate selectivity. Use of these crystal structures for the design of specific inhibitors for several GST enzymes is also discussed.
Dependence of drug metabolism on dosing time has long been recognized. However, only recently are the underlying mechanisms for circadian drug metabolism being clarified. Diurnal rhythmicity in ...expression of drug-metabolizing enzymes is believed to be a key factor determining circadian metabolism. Supporting the notion that biological rhythms are generated and maintained by the circadian clock, a number of diurnal enzymes are under the control of the circadian clock. In general, circadian clock genes generate and regulate diurnal rhythmicity in drug-metabolizing enzymes via transcriptional actions on one or two of three
-elements (i.e., E-box, D-box, and Rev-erb response element or RAR-related orphan receptor response element). Additionally, cycling or clock-controlled nuclear receptors such as hepatocyte nuclear factor 4
and peroxisome proliferator-activated receptor
are contributors to diurnal enzyme expression. These newly discovered mechanisms for each of the rhythmic enzymes are reviewed in this article. We also discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics. Our discussion is also extended to two diurnal transporters (P-glycoprotein and multidrug resistance-associated protein 2) that have an important role in drug absorption. Although the experimental evidence is lacking in metabolism-based chronoefficacy, circadian genes (e.g.,
) as drug targets are shown to account for diurnal variability in drug efficacy. SIGNIFICANCE STATEMENT: Significant progress has been made in understanding the molecular mechanisms for generation of diurnal rhythmicity in drug-metabolizing enzymes. In this article, we review the newly discovered mechanisms for each of the rhythmic enzymes and discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics.
The role of intestine clock in energy homeostasis remains elusive. Here we show that mice with Bmal1 specifically deleted in the intestine (Bmal1
mice) have a normal phenotype on a chow diet. ...However, on a high-fat diet (HFD), Bmal1
mice are protected against development of obesity and related abnormalities such as hyperlipidemia and fatty livers. These metabolic phenotypes are attributed to impaired lipid resynthesis in the intestine and reduced fat secretion. Consistently, wild-type mice fed a HFD during nighttime (with a lower BMAL1 expression) show alleviated obesity compared to mice fed ad libitum. Mechanistic studies uncover that BMAL1 transactivates the Dgat2 gene (encoding the triacylglycerol synthesis enzyme DGAT2) via direct binding to an E-box in the promoter, thereby promoting dietary fat absorption. Supporting these findings, intestinal deficiency of Rev-erbα, a known BMAL1 repressor, enhances dietary fat absorption and exacerbates HFD-induced obesity and comorbidities. Moreover, small-molecule targeting of REV-ERBα/BMAL1 by SR9009 ameliorates HFD-induced obesity in mice. Altogether, intestine clock functions as an accelerator in dietary fat absorption and targeting intestinal BMAL1 may be a promising approach for management of metabolic diseases induced by excess fat intake.
To design potentially more effective therapies, we need to further understand the mechanisms underlying epilepsy. Here, we uncover the role of Rev-erbα in circadian regulation of epileptic seizures. ...We first show up-regulation of REV-ERBα/Rev-erbα in brain tissues from patients with epilepsy and a mouse model. Ablation or pharmacological modulation of Rev-erbα in mice decreases the susceptibility to acute and chronic seizures, and abolishes diurnal rhythmicity in seizure severity, whereas activation of Rev-erbα increases the animal susceptibility. Rev-erbα ablation or antagonism also leads to prolonged spontaneous inhibitory postsynaptic currents and elevated frequency in the mouse hippocampus, indicating enhanced GABAergic signaling. We also identify the transporters Slc6a1 and Slc6a11 as regulators of Rev-erbα-mediated clearance of GABA. Mechanistically, Rev-erbα promotes the expressions of Slc6a1 and Slc6a11 through transcriptional repression of E4bp4. Our findings propose Rev-erbα as a regulator of synaptic function at the crosstalk between pathways regulating the circadian clock and epilepsy.
Sulfotransferase 1a1 (Sult1a1) is a phase II enzyme that contributes extensively to metabolism and detoxification of various drugs and chemicals. Here we aimed to investigate a potential role of the ...clock protein Bmal1 (brain and muscle Arnt-like protein-1) in circadian regulation of Sult1a1 in mice. The regulatory effects of Bmal1 on Sult1a1 were assessed both in vivo (using Bmal1- deficient mice) and in vitro (using both normal and serum-shocked Hepa-1c1c7 cells). The relative mRNA and protein levels of Sult1a1 in the cells or mouse livers were measured by RT-qPCR and Western blotting, respectively. Sulfation activities of two Sult1a1 substrates (i.e.,
-nitrophenol and galangin) were determined using mouse liver S9 fractions. Transcriptional regulation of Sult1a1 by Bmal1 was investigated using luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays. We first showed that hepatic Sult1a1 was rhythmically expressed at both mRNA and protein levels (higher expressions during the night than the daytime). Consistently, the liver sulfation activities toward two Sult1a1 substrates were circadian time dependent with a higher activity at ZT14 than at ZT2. Furthermore, deletion of
in mice blunted the circadian rhythmicity of hepatic Sult1a1 (with reduced expression levels). Likewise, Bmal1 positively regulated Sult1a1 expression in conventionally cultured Hepa-1c1c7 cells, and
knockdown blunted expression rhythmicity of Sult1a1 in serum-shocked Hepa-1c1c7 cells. A combination of promoter analysis, EMSA and ChIP assays revealed that Bmal1 stimulated Sult1a1 transcription through its specific binding to the-571- to -554-bp region (an E-box element) in the promoter. In conclusion, Bmal1 activated the transcription of Sult1a1 and controlled circadian expression and activity of the enzyme.
Diabetes mellitus (DM) remains a great challenge in treatment due to pathological complexity. It has been proven that phytomedicines and natural medicines have prominent antidiabetic effects. This ...work aimed to develop selenium-layered nanoparticles (SeNPs) for oral delivery of mulberry leaf and
extracts (MPE), a group of phytomedicines with significant hypoglycemic activities, to achieve a synergic antidiabetic effect. MPE-loaded SeNPs (MPE-SeNPs) were prepared through a solvent diffusion/
reduction technique and characterized by particle size,
potential, morphology, entrapment efficiency (EE) and drug loading (DL). The resulting MPE-SeNPs were 120 nm around in particle size with EE of 89.38% for rutin and 90.59% for puerarin, two marker components in MPE. MPE-SeNPs exhibited a slow drug release and good physiological stability in the simulated digestive fluid. After oral administration, MPE-SeNPs produced significant hypoglycemic effects both in the normal and diabetic rats.
intestinal imaging and cellular examinations demonstrated that MPE-SeNPs were provided with outstanding intestinal permeability and transepithelial transport aptness. It was also revealed that MPE-SeNPs could alleviate the oxidative stress, improve the pancreatic function, and promote the glucose utilization by adipocytes. Our study provides new insight into the use of integrative nanomedicine containing phytomedicines and selenium for DM treatment.
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