Some 3-(4-aminophenyl)pyrrolidine-2,5-diones as all-trans-retinoic acid metabolising enzyme inhibitors (RAMBAs)

A series of (+/-)-3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3-position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC50 = 98.8 microM, ketoconazole, 22.15 microM) showed that it was not stereoselective in its inhibition. (+/-)-(1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC50 = 20.9 microM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC50 = 211.6 microM respectively; ketoconazole, 38.8% and 85.95 microM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (+/-)-(1) was a weak inhibitor (c. 53% at 200 microM) whereas ketoconazole showed high potency (c. 65% at 0.625 microM and 0.25 microM respectively). The nature of the induced target enzyme is discussed.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Retinoic acid stimulates peptide leukotriene-syntheses in rat basophilic leukemia-1 (RBL-1) cells

Overnight incubation of rat basophilic leukemia-1 (RBL-1) cells with retinoic acid enhanced calcium ionophore-stimulated syntheses of LTC4 by more than 28-times (from 5.91 +/- 0.31 to 168.31 +/- 22.66 ng/2.10(6) cells) nd LTD4 by more than 7-times (from 5.27 +/- 0.12 to 39.38 +/- 14.89 ng/2.10(6) cells). The stimulatory action first appeared after a 10 h incubation with retinoic acid and was completely abolished by concomitant presence of low concentration cycloheximide (0.5 micrograms/ml) in the medium. However, LTB4 synthesis was dose-dependently inhibited by incubation with retinoic acid. Reduced form of glutathione significantly increased the synthesis of LTC4, but showed no action on the syntheses of LTD4 or LTB4. These results indicate a new enzyme synthesis of LTC4 synthetase.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.     

Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3.

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg.kg(-1)), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD(50) value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild-type (92-95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.     

Expression of cellular retinoic acid binding protein (CRABP) in Escherichia coli. Characterization and evidence that holo-CRABP is a substrate in retinoic acid metabolism.

Cellular retinoic acid binding protein (CRABP) has been expressed efficiently in Escherichia coli from the cDNA of bovine adrenal CRABP and characterized, especially with respect to affinity for endogenous retinoids and a role for it in retinoic acid metabolism. The purified E. coli-expressed CRABP was similar to authentic mammalian CRABP in molecular weight (approximately 14,700), isoelectric point (4.76), absorbance maxima (apo-CRABP, 280 nm; holo-CRABP, 350 and 280 nm with the ratio A350/A280 = 1.8), and in fluorescence excitation (350 nm) and emission spectra (475 nm). The equilibrium dissociation constant, Kd, of E. coli-derived CRABP and all-trans-retinoic acid was 10 +/- 1 nM (mean +/- S.D., n = 4) by retinoid fluorescence and 7 +/- 1 nM (mean +/- S.D., n = 3) by quenching of protein fluorescence, but neither retinol nor retinal bound in concentrations as high as 7 microM. All-trans-cyclohexyl ring derivatives of retinoic acid (3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, 18-hydroxy-) had affinities similar to that of all-trans-retinoic acid, whereas 13-cis-retinoic acid and 4-oxo-13-cis-retinoic acid had approximately 25-fold lower affinity. Holo-CRABP was a substrate for retinoic acid catabolism in rat testes microsomes by three criteria: 1) the rate of retinoic acid metabolism with CRABP in excess of retinoic acid exceeded the rate supported by the free retinoic acid; 2) increasing the apo-CRABP did not decrease the rate as predicted if free retinoic acid were the only substrate; and 3) holo-CRABP had a lower Michaelis constant (1.8 nM) for retinoic acid elimination than did free retinoic acid (49 nM). These data indicate a direct role for CRABP in retinoic acid metabolism and suggest a mechanism for discriminating metabolically between all-trans- and 13-cis-retinoids.     

The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin.

The kinetics and mechanisms of the reactions of a number of pyrogallol-based ligands with iron(III) have been investigated in aqueous solution at 25 degrees C and ionic strength 0.5 M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are generally consistent with a mechanism in which the 1:1 complex that is formed initially when the metal reacts with the ligand subsequently decays through an electron transfer reaction. There was also some evidence for the formation of a 1:2 ligand-to-metal complex at higher pH values. The kinetics of complex formation were investigated with either the ligand or metal in pseudo-first-order excess. Rate constants for k(1) of 2.83(+/-0.09)x10(3), 1.75(+/-0.045)x10(3) and 3300(+/-200) M(-1) s(-1) and k(-1) of 20(+/-6.0), 35(+/-13) and 25+/-7.6 M(-1) s(-1) have been evaluated for the reaction of Fe(OH)(2+) with gallic acid, gallic acid methyl ester and catechin, respectively. The stability constant of each [Fe(L)](+) complex has been calculated from the kinetic data. The iron(III) assisted decomposition of the initial iron(III) complex formed was investigated. Analysis of the kinetic data yielded both the equilibrium constants for protonation of the iron(III) complexes initially formed together with the rate constants for the intramolecular electron transfers for gallic acid and gallic acid methyl ester. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time-dependent spectra.     

Retinoic acid is a modulator of thyroid hormone activation of Ca2+-ATPase in the human erythrocyte membrane

The thyroid hormones thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) stimulate plasma membrane Ca2+-ATPase (EC 3.6.1.3) activity in human erythrocytes by a mechanism independent of the cell nucleus. The current studies were conducted to determine the effect of retinoic acid on the extranuclear activation by T4 and T3 of Ca2+-ATPase in the human red cell. The retinoid inhibited basal and T4-stimulatable activity of that enzyme in a dose-dependent manner. At the highest tested concentration (10(-6) M), retinoic acid inhibited basal enzyme activity by 25% and T4-stimulated activity by 72%. A concentration as low as 5 x 10(-10) M retinoic acid shifted the dose-response curve of both T4 and T3 so that the concentration of each associated with maximal enzyme stimulation was 10(-9) M instead of 10(-10) M. Retinoic acid displaced [125I]T4 binding to red cell membranes as effectively as unlabeled T4. Retinol failed to influence either basal or T4-stimulated enzyme activity or to displace T4 binding. These results indicate that retinoic acid can partially block the T4 and T3 stimulation of Ca2+-ATPase in human red cell membranes and suggest a physiologic role for the retinoid as a modulator of this peripheral action of thyroid hormone. They suggest that the red cell membrane is an important site of action for this active retinoid.     

Homozygous deletion of the CRABPI gene in AB1 embryonic stem cells results in increased CRABPII gene expression and decreased intracellular retinoic acid concentration

The cellular retinoic acid (RA) binding proteins I and II (CRABPI and CRABPII), intracellular proteins which bind retinoic acid with high affinity, are involved in the actions of RA, though their exact roles are not fully understood. We have generated several genetically engineered AB1 cell lines in which both alleles of the CRABPI gene have been deleted by homologous recombination. We have used these CRABPI knockout cell lines to examine the consequences of functional loss of CRABPI on RA-induced gene expression and RA metabolism in the murine embryonic stem cell line, AB1, which undergoes differentiation in response to RA. Complete lack of CRABPI results in decreased intracellular [3H]RA concentrations under conditions in which external concentrations of [3H]RA are low (1-10nM) and in an altered distribution of [3H] polar metabolites of [3H]RA in the cell and in the medium. Fewer [3H] polar metabolites are retained within the CRABPI(-/-) cells compared to the wild-type cells. These data suggest that CRABPI functions to regulate the intracellular concentrations of retinoic acid and to maintain high levels of oxidized retinoic acid metabolites such as 4-oxoretinoic acid within cells.     

Metabolism of deuterium-labeled jasmonic acid and OPC 8:0 in the potato plant (Solanum tuberosum L.)

The metabolism of deuterium-labeled (+/-)-jasmonicacid and 3-oxo-2-[(Z)pent-2'-enyl]-cyclopentan-1- octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z) pent-2'-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (+/-)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z)-2'-pentenyl]cyclopentan-1-octanoic acid to 5' and 4'-O-glucopyranosyloxyjasmoic acids was strongly suggested.     

Retinoic acid-induced modulation of rat liver transglutaminase and total polyamines in vivo.

The effect of a single intraperitoneal injection of retinoic acid on liver transglutaminase (EC 2.3.2.13) activity and total putrescine, spermidine and spermine was studied. The results demonstrate that: (1) transglutaminase activity is increased over control values as early as 4-6 h after treatment, reaching a maximum (2-fold increase) at 12 h and returning to control values at 36 h; (2) the retinoic acid-induced form of enzyme is the soluble tissue transglutaminase; (3) actinomycin D treatment does not completely inhibit the early (6 h) increase of activity, while suppressing that at 12 h; (4) the immunoassay of the soluble transglutaminase shows that, 6 h after treatment, there is no increase in the protein, whereas at 12 and 24 h a significant increase is observed; (5) putrescine, but not spermidine and spermine, increases (5-7-fold) 6 and 18 h after the retinoic acid treatment. The possibility also that the expression of soluble transglutaminase is modulated in vivo by retinoic acid and the relationship to polyamine levels are discussed.     

A biologically active metabolite of retinoic acid from rat liver

1. Four major radioactive fractions have been isolated from the livers of vitamin A-deficient rats given [6,7-(14)C(2)]retinoic acid. 2. At least one of these was more potent than retinoic acid and approximately equal to retinol in the growth assay for vitamin A activity. 3. The biologically active material was chromatographically distinct from retinoic acid, retinol and retinal. 4. Alkaline hydrolysis of this material yielded an acidic compound containing all the radioactivity. 5. The methyl ester of the acidic product was unlike the methyl ester of retinoic acid in its chromatographic behaviour. 6. It is suggested that this metabolite may represent the active form of retinol in its growth-supporting role.     

Retinoic acid induced suicidal erythrocyte death.

Vitamin A and retinoic acid have previously been shown to confer some protection against a severe course of malaria by fostering the phagocytosis of parasitized erythrocytes. Phagocytosis of erythrocytes is stimulated by phosphatidylserine exposure at the cell surface. The present study has thus been performed to explore the effect of retinoic acid and the specific retinoic acid receptor (RAR) agonist 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB) on erythrocyte annexin V binding, which reflects phosphatidylserine exposure at the cell surface. A 24 hours exposure to either, retinoic acid (3 microM) or TTNPB (3 microM), indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter reflecting cell shrinkage. According to Fluo3 fluorescence, exposure to either, retinoic acid (10 microM, 24 hours) or TTNPB (10 microM, 6 hours), significantly increased cytosolic Ca(2+)-activity, a known trigger of phosphatidylserine exposure. Infection of erythrocytes with Plasmodium falciparum increased phosphatidylserine exposure, an effect increased in the presence of TTNPB. In conclusion, retinoid acid and TTNPB trigger phosphatididylserine exposure and cell shrinkage of erythrocytes, typical features of suicidal erythrocyte death or eryptosis. The eryptosis could participate in the accelerated clearance of parasitized erythrocytes from circulating blood following treatment with retinoids.     

Synthesis and antifungal properties of alpha-methoxy and alpha-hydroxyl substituted 4-thiatetradecanoic acids

4-thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC=4.8-12.7 mM). It has been demonstrated that alpha-methoxylation efficiently blocks beta-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by alpha-substitution. The unprecedented (+/-)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (+/-)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (+/-)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC=0.8-1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (+/-)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC=0.17 mM) than the alpha-methoxylated analog, but not as effective against A. niger (MIC=5.5 mM). The enhanced fungitoxicity of the (+/-)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked beta-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells.     

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.     

Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor

Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.