Comparative Study

Authors: Hygor M R de Souza, Jéssica S Guedes, Rosana H C N Freitas...

Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.

Topics: Animals; Carboxylesterase; Dose-Response Relationship, Drug; Enzyme Inhibitors; Esters; Hydrolysis; Male; Microsomes, Liver; Models, Molecular; Molecular Structure; Prodrugs; Rats; Rats, Wistar; Structure-Activity Relationship

PubMed: 35156494
DOI: 10.1080/14756366.2022.2027933

Authors: Navid J Ayon, William G Gutheil

The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library. Comparative screening of the un-metabolized (UM) and pre-metabolized (PM) libraries allows identification of intrinsically active agents from the UM library screen and of agents with active metabolites from the PM library screen. To further enhance this screening effort, it was combined with a -/+ resistant to antibiotic screen (-/+ cefoxitin; Cef). This allows the identification of agents that can act synergistically with the resistant to antibiotic. This approach revealed five compounds with substantially improved activity after metabolism and four compounds with substantial synergistic activity with cefoxitin. Capecitabine in particular only had significant antibacterial activity after metabolism. Its metabolites were isolated, identified, and characterized for spectrum of activity along with several other anticancer drugs with anti-MRSA activity. Floxuridine, gemcitabine, novobiocin, and rifaximin were identified as having substantial synergy with cefoxitin from the -/+Cef screens. Checkerboard assays verified synergy for these agents. Floxuridine demonstrated a particularly high degree of synergy with cefoxitin (FIC = 0.14). This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and strategies for countering antibacterial agent resistance.

Topics: Anti-Bacterial Agents; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Microsomes, Liver

PubMed: 31675203
DOI: 10.1021/acschembio.9b00745

Review

Present Status and Future Trends of Natural-Derived Compounds Targeting T Helper (Th) 17 and Microsomal Prostaglandin E Synthase-1 (mPGES-1) as Alternative Therapies for Autoimmune and Inflammatory-Based Diseases.

Authors: Anella Saviano, Federica Raucci, Gian Marco Casillo...

Several natural-based compounds and products are reported to possess anti-inflammatory and immunomodulatory activity both in vitro and in vivo. The primary target for these activities is the inhibition of eicosanoid-generating enzymes, including phospholipase A2, cyclooxygenases (COXs), and lipoxygenases, leading to reduced prostanoids and leukotrienes. Other mechanisms include modulation of protein kinases and activation of transcriptases. However, only a limited number of studies and reviews highlight the potential modulation of the coupling enzymatic pathway COX-2/mPGES-1 and Th17/Treg circulating cells. Here, we provide a brief overview of natural products/compounds, currently included in the Italian list of botanicals and the BELFRIT, in different fields of interest such as inflammation and immunity. In this context, we focus our opinion on novel therapeutic targets such as COX-2/mPGES-1 coupling enzymes and Th17/Treg circulating repertoire. This paper is dedicated to the scientific career of Professor Nicola Mascolo for his profound dedication to the study of natural compounds.

Topics: Anti-Inflammatory Agents; Autoimmune Diseases; Biological Products; Complementary Therapies; Cyclooxygenase 1; Cyclooxygenase 2; Humans; Inflammation; Microsomes; Th17 Cells

PubMed: 33353211
DOI: 10.3390/molecules25246016

Review

Authors: Ruth E Gimeno, Jingsong Cao

Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Ethylmaleimide; Glycerol-3-Phosphate O-Acyltransferase; Humans; Isoenzymes; Microsomes

PubMed: 18658143
DOI: 10.1194/jlr.R800013-JLR200

Authors: Daniel Scotcher, Sarah Billington, Jay Brown...

In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver ( = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex ( = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney-specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo.

Topics: Animals; Cytochrome P-450 Enzyme System; Cytosol; Dogs; Female; Glucose-6-Phosphatase; Humans; Kidney Cortex; Male; Microsomes; Species Specificity

PubMed: 28270564
DOI: 10.1124/dmd.117.075242

Review

Authors: J H Capdevila, R C Harris, J R Falck...

The demonstration of a role for microsomal P450 in the metabolism of endogenous pools of arachidonic acid established this enzyme system as a member of the arachidonic acid cascade and characterized a new an important metabolic function for this enzyme system. Studies from several laboratories documenting the powerful biological activities of the P450-derived eicosanoids have suggested important roles for the P450 arachidonic acid monooxygenase in renal and vascular physiology, and in the pathophysiology of experimental hypertension. These studies provide significant evidence to indicate that in addition to its recognized traditional toxicological and pharmacological roles, microsomal P450s also play important physiological roles in the control of tissue and body homeostasis.

Topics: Animals; Cytochrome P-450 Enzyme System; Eicosanoids; Homeostasis; Humans; Microsomes; Microsomes, Liver; NADP; Oxidation-Reduction

PubMed: 12088278
DOI: 10.1007/s00018-002-8466-y

Authors: Adam Kawiński, Magdalena Miklaszewska, Szymon Stelter...

BACKGROUND

Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.

RESULTS

During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.

CONCLUSIONS

In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Topics: Caryophyllales; Esters; Germination; Hydrolysis; Lipase; Lipids; Microsomes; Orlistat; Plant Proteins; Seeds; Substrate Specificity; Temperature; Triglycerides; Waxes

PubMed: 33468064
DOI: 10.1186/s12870-020-02823-4

Review

Authors: Annaleise V Sampey, Seetha Monrad, Leslie J Crofford...

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG)H2 to PGE2. Proinflammatory stimuli markedly increase levels of mPGES-1 expression both in vivo and in vitro. mPGES-1 knockout studies and animal models of inflammatory arthritis also provide a strong basis for the contribution of mPGES-1 in the increased local production of PGE2 observed in inflammatory arthritis. The focus of this article is to review some recent advances in our understanding of mechanisms specific to the regulation of inducible mPGES-1 in inflammatory arthritis.

Topics: Animals; Dinoprostone; Enzyme Induction; Humans; Intramolecular Oxidoreductases; Microsomes; Prostaglandin-E Synthases

PubMed: 15899061
DOI: 10.1186/ar1748

Authors: Saki Iwamori, Emiko Sato, Daisuke Saigusa...

Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.

Topics: Animals; Bilirubin; Biliverdine; Calibration; Chromatography, High Pressure Liquid; Heme Oxygenase (Decyclizing); Mice; Mice, Inbred C57BL; Microsomes; Tandem Mass Spectrometry

PubMed: 26224716
DOI: 10.1152/ajprenal.00210.2015

Comparative Study

Authors: K T Chung, G E Fulk, A W Andrews...

Seventeen commonly used dyes and 16 of their metabolites or derivatives were tested in the Salmonella-mammalian microsome mutagenicity test. Mutagens active with and without added Aroclor-induced rat liver microsome preparations (S9) were 3-aminopyrene, lithol red, methylene blue (USP), methyl yellow, neutral red, and phenol red. Those mutagenic only with S9 activation were 4-aminopyrazolone, 2,4-dimethylaniline, N,N-dimethyl-p-phenylenediamine, methyl red, and 4-phenyl-azo-1-naphthylamine. Orange II was mutagenic only without added S9. Nonmutagenic azo dyes were allura red, amaranth, ponceau R, ponceau SX, sunset yellow, and tartrazine. Miscellaneous dyes not mutagenic were methyl green, methyl violet 2B, and nigrosin. Metabolites of the azo dyes that were not mutagenic were 1-amino-2-naphthol hydrochloride, aniline, anthranilic acid, cresidine salt, pyrazolone T,R-amino salt (1-amino-2-naphthol-3,6-disulfonic disodium salt), R-salt, Schaeffer's salt (2-naphthol-6-sulfonic acid, sodium salt), sodium naphthionate, sulfanilamide, and sulfanilic acid. 4-Amino-1-naphthalenesulfonic acid sodium salt was also not mutagenic. Fusobacterium sp. 2 could reductively cleave methyl yellow to N,N-dimethyl-p-phenylenediamine which was then activated to a mutagen.

Topics: Coloring Agents; Microsomes, Liver; Mutagenicity Tests; Mutagens; Salmonella; Salmonella typhimurium

PubMed: 7039509
DOI: 10.1128/aem.42.4.641-648.1981