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R T Borchardt

Publications and source records attributed to R T Borchardt.

At least 19 recordsLinked to original sources
PubMed1999

Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeation.

PURPOSE: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of phenylpropionic acid-based cyclic prodrugs 1 and 2 of opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE, and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. RESULTS: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs 1 and 2 were slightly faster than in HBSS, pH 7.4. These accelerated rates of disappearance in 90% human plasma could be reduced to the rates observed in HBSS, pH 7.4, by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates of Caco-2 cells and rat liver, accelerated rates of disappearance of cyclic prodrugs 1 and 2 were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited stability similar to prodrug 1 when applied to the AP side of the Caco-2 cell monolayers. Prodrug 1 was 1680 fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with the phenylpropionic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In 90% human plasma but not in Caco-2 cell and rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon inhibition. However, the rate of this bioconversion appears to be extremely slow.

Animals↗
PubMed1999

Coumarinic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeability.

UNLABELLED: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of coumarinic acid-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of the cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. RESULTS: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded chemically to intermediates that further degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma and rat liver homogenate, the disappearance of cyclic prodrugs 1 and 2 was significantly faster than in HBSS, pH 7.4. The half-lives in 90% human plasma and in rat liver homogenate were substantially longer after pretreatment with paraoxon, a known inhibitor of serine-dependent esterases. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug 1 was 665-fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 31 fold more able to permeate a Caco-2 cell monolayer than was DADLE. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with the coumarinic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In various biological media, the opioid peptides were released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.

Animals↗
PubMed1999

Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers.

PURPOSE: To evaluate the chemical and enzymatic stability, as well as the cellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, were measured by HPLC in HBSS, pH = 7.4, and in various biological media (e.g., human plasma and Caco-2 cell and rat liver homogenates) having measurable esterase activity. The cellular permeation and metabolism characteristics of [Leu5]-enkephalin, DADLE and the cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HPLC. RESULTS: Cyclic prodrugs 1 and 2 degraded slowly but stoichiometrically to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% human plasma, the rates of disappearance of the cyclic prodrugs were significantly faster than in HBSS. The stabilities of the cyclic prodrugs 1 and 2 were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-dependent esterases, was included in the incubation mixtures. A similar stabilizing effect of paraoxon was not observed in 50% rat liver homogenates, but was observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs 1 and 2 exhibited significantly greater stability than [Leu5]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic prodrugs 1 and 2 should have exhibited high permeation across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permeability coefficients (P(App)) for cyclic prodrugs 1 and 2 across Caco-2 cell monolayers were significantly lower than the P(App) value determined for the metabolically stable opioid peptide DADLE. When the P(App) values for cyclic prodrugs 1 and 2 crossing Caco-2 cell monolayers in the BL-to-AP direction were determined, they were shown to be 36 and 52 times greater, respectively, than the AP-to-BL values. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with an acyloxyalkoxy promoiety, were shown to degrade in biological media (e.g., 90% human plasma) via an esterase-catalyzed pathway. The degradation of cyclic prodrug 1, which contained an ester formed with an L-amino acid, degraded more rapidly in esterase-containing media than did prodrug 2, which contained an ester formed with a D-amino acid. Cyclic prodrugs 1 and 2 showed very low AP-to-BL Caco-2 cell permeability, which did not correlate with their lipophilicities. These low AP-to-BL permeabilities result because of their substrate activity for apically polarized efflux systems.

Acylation↗
PubMed1999

Synthesis and evaluation of novel coumarin-based esterase-sensitive cyclic prodrugs of peptidomimetic RGD analogs with improved membrane permeability.

Earlier, we reported the development of a coumarin-based prodrug system that could be used for the preparation of cyclic prodrugs of opioid peptides. These cyclic prodrugs exhibited excellent membrane permeability characteristics. Therefore, it was of interest to determine the effects of this prodrug strategy on the membrane permeabilities of peptidomimetics which also have low membrane permeabilities. For this study, we have chosen two RGD (Arg-Gly-Asp) peptidomimetics, which have the potentials to be developed clinically as orally active antithrombotic agents. However, the clinical development of oral dosage forms of these RGD analogs has been hindered by their low intestinal mucosal permeability. Therefore, we have synthesized the corresponding coumarin-based cyclic prodrugs of these RGD peptidomimetics, which have the two most polar functional groups, a carboxyl and an amino group, masked as an ester and an amide, respectively. These cyclic prodrugs were shown to have higher membrane interaction potentials, as estimated by their partitioning between aqueous buffer and an immobilized artificial membrane, than the corresponding RGD analogs suggesting that they should exhibit good membrane permeation characteristics. Subsequently, in a separate study these cyclic prodrugs were shown to be 5 to 6-fold more able to permeate monolayers of Caco-2 cells, an in vitro cell culture model of the intestinal mucosa barrier, than the corresponding RGD peptidomimetics.

Caco-2 Cells↗
PubMed1998

Inactivation of S-adenosyl-L-homocysteine hydrolase and antiviral activity with 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosine analogues (4'-haloacetylene analogues derived from adenosine).

Treatment of a protected 9-(5, 6-dideoxy-beta-D-ribo-hex-5-ynofuranosyl)adenine derivative with silver nitrate and N-iodosuccinimide (NIS) and deprotection gave the 6'-iodo acetylenic nucleoside analogue 3c. Halogenation of 3-O-benzoyl-5,6-dideoxy-1, 2-O-isopropylidene-alpha-D-ribo-hex-5-enofuranose gave 6-halo acetylenic sugars that were converted to anomeric 1,2-di-O-acetyl derivatives and coupled with 6-N-benzoyladenine. These intermediates were deprotected to give the 6'-chloro 3a, 6'-bromo 3b, and 6'-iodo 3c acetylenic nucleoside analogues. Iodo compound 3c appears to inactivate S-adenosyl-L-homocysteine hydrolase by a type I ("cofactor depletion") mechanism since complete reduction of enzyme-bound NAD+ to NADH was observed and no release of adenine or iodide ion was detected. In contrast, incubation of the enzyme with the chloro 3a or bromo 3b analogues resulted in release of Cl- or Br- and Ade, as well as partial reduction of E-NAD+ to E-NADH. Compounds 3a, 3b, and 3c were inhibitory to replication of vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus, and reovirus-1 (3a < 3b < 3c, in order of increasing activity). The antiviral effects appear to correlate with type I mechanism-based inhibition of S-adenosyl-L-homocysteine hydrolase. Mechanistic considerations are discussed.

Adenosine↗
PubMed1998

Discovery of type II (covalent) inactivation of S-adenosyl-L-homocysteine hydrolase involving its "hydrolytic activity": synthesis and evaluation of dihalohomovinyl nucleoside analogues derived from adenosine.

Treatment of the 5'-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2',3'-O-isopropylideneadenosine (1) with the "(bromofluoromethylene)triphenylphosphorane" reagent and deprotection gave 9-(6-bromo-5, 6-dideoxy-6-fluoro-beta-d-ribo-hex-5-enofuranosyl)adenine (4). Parallel treatment with a "dibromomethylene Wittig reagent" and deprotection gave 9-(6,6-dibromo-5, 6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by successive bromination and dehydrobromination of the 6'-bromohomovinyl nucleoside 8. Bromination-dehydrobromination of the 5'-bromohomovinyl analogue 11 and deprotection gave (E)-9-(5, 6-dibromo-5,6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and 15 were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-l-homocysteine (AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could generate electrophilic acyl halide or alpha-halo ketone species that could undergo nucleophilic attack by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.

Adenosylhomocysteinase↗
PubMed1998

A novel mechanism-based inhibitor (6'-bromo-5', 6'-didehydro-6'-deoxy-6'-fluorohomoadenosine) that covalently modifies human placental S-adenosylhomocysteine hydrolase.

Most inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase function as substrates for the "3'-oxidative activity" of the enzyme and convert the enzyme from its active form (NAD+) to its inactive form (NADH) (Liu, S., Wolfe, M. S., and Borchardt, R. T. (1992) Antivir. Res. 19, 247-265). In this study, we describe the effects of a mechanism-based inhibitor, 6'-bromo-5', 6'-didehydro-6'-deoxy-6'-fluorohomoadenosine (BDDFHA), which functions as a substrate for the "6'-hydrolytic activity" of the enzyme with subsequent formation of a covalent linkage with the enzyme. Incubation of human placental AdoHcy hydrolase with BDDFHA results in a maximum inactivation of 83% with the remaining enzyme activity exhibiting one-third of the kcat value of the native enzyme. This partial inactivation is concomitant with the release of both Br- and F- ions and the formation of adenine (Ade). The enzyme can be covalently labeled with [8-3H]BDDFHA, resulting in a stoichiometry of 2 mol of BDDFHA/mol of the tetrameric enzyme. The 3H-labeled enzyme retains its original NAD+/NADH content. Tryptic digestion and subsequent protein sequencing of the [8-3H]BDDFHA-labeled enzyme revealed that Arg196 is the residue that is associated with the radiolabeled inhibitor. The partition ratio of the Ade formation (nonlethal event) to covalent acylation (lethal event) is approximately 1:1. From these experimental results, a possible mechanism by which BDDFHA inactivates AdoHcy hdyrolase is proposed: enzyme-mediated water addition at the C-6' position of BDDFHA followed by elimination of Br- ion results in the formation of homoAdo 6'-carboxyl fluoride (HACF). HACF then partitions in two ways: (a) attack by a proximal nucleophile (Arg196) to form an amide bond after expulsion of F- ion (lethal event) or (b) depurination to form Ade and hexose-derived 6-carboxyl fluoride (HDCF), which is further hydrolyzed to hexose-derived 6-carboxylic acid (HDCA) and F- ion (nonlethal event).

Adenosylhomocysteinase↗
PubMed1998

Kinetics of diketopiperazine formation using model peptides.

The intramolecular aminolysis of Phe-Pro-p-nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) was studied as a function of pH, temperature, buffer concentration, and buffer species using an HPLC assay that permits simultaneous analysis of the disappearance of the starting material and the appearance of degradation products. The degradation followed pseudo-first-order kinetics and showed significant dependence on pH. Phosphate (pH 5-8) and glycine (pH 9-10) buffers exhibit general base catalysis. The pH-rate profile suggested that the rate of Phe-Pro-DKP formation depends on the degree of ionization of the N-terminal amino group, with the unprotonated reactant being more reactive than the protonated form. The pKa value of 6.1, determined kinetically, and three microscopic rate constants were adequate to describe the shape of the pH-rate profile. In the pH range studied, Phe-Pro-DKP was the only product generated upon degradation of Phe-Pro-pNA. At pH values between 3 and 8, Phe-Pro-DKP was stable, while at pH less than 3 and greater than 8 it undergoes hydrolysis to the dipeptide, Phe-Pro-OH. Sequence inversion, a reaction normally associated with DKP formation, was not observed. The influence of primary sequence on the formation of DKP was also investigated using X-Pro-pNA analogues, where X = Gly, Ala, Val, Phe, beta-cyclohexylalanine, and Arg. Changing the amino acid preceding the proline residue had a significant effect on the rate of DKP formation at pH 7.0.

Amino Acid Sequence↗
PubMed1998

Chemical pathways of peptide degradation: IX. Metal-catalyzed oxidation of histidine in model peptides.

PURPOSE: To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides. METHODS: The degradation of AcAla-His-ValNH2 (Ala-peptide) and AcCysNH2-S-S-AcCys-His-VaNH2 (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O2) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry. RESULTS: The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 +/- 0.7% of the Cys-peptide and 18 +/- 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 +/- 3% of the Cys-peptide and 4 +/- 1% of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH2 and AcCysNH2-S-S-AcCys-2-oxo-His-ValNH2, respectively. CONCLUSIONS: Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.

Ascorbic Acid↗
PubMed1998

Transport characteristics of peptidomimetics. Effect of the pyrrolinone bioisostere on transport across Caco-2 cell monolayers.

PURPOSE: To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. METHODS: Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. RESULTS: In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (Papp) calculated for the BL-to-AP and AP-to-BL transport (P(BL-->AP)/P(AP-->BL)) varied between 1.7 and 36.2. When individual pairs were ompared, P(BL-->AP)/P(AP-BL) ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 microM cyclosporin A to the transport buffer reduced the P(BL-->AP)/P(AP-->BL) ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 microM cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. CONCLUSIONS: The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.

Biological Transport↗
PubMed1998

A comparison of the bioconversion rates and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs and methylester-based linear prodrugs of RGD peptidomimetics.

PURPOSE: To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (1a, 2a). METHODS: Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balances salt solution (HBSS), pH 7,4, at 37 degrees C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitro cell culture model of the intestinal mucosa. RESULTS. In HBSS, pH 7,4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics 1a and 2a, respectively (3a, t1/2 = 630+2-14 min; 3b, t1/2 = 301 +/-12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t1/2 > 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t1/2 < 5 min; 2b, t1/2 < 5 min; 3a, t1/2 < 91+/-1 min; 3b, 1/2 < 57+/-2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester prodrugs 1b and 2b underwent esterase bioconversion (>80%) to the RGD peptidomimetics 1a and 2a, respectively, In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics 1a and 2a. When similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics 1a and 12a, respectively. CONCLUSION: The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, then the methylester based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.

Animals↗
PubMed1998

Structure determination of selenomethionyl S-adenosylhomocysteine hydrolase using data at a single wavelength.

S-Adenosylhomocysteine (AdoHcy) hydrolase regulates all adenosylmethionine-(AdoMet) dependent transmethylations by hydrolyzing the potent feedback inhibitor AdoHcy to homocysteine and adenosine. The crystallographic structure determination of a selenomethionyl-incorporated AdoHcy hydrolase inhibitor complex was accomplished using single wavelength anomalous diffraction data and the direct methods program, Snb v2.0, which produced the positions of all 30 crystallographically distinct selenium atoms. The mode of enzyme-cofactor binding is unique, requiring interactions from two protein monomers. An unusual dual role for a catalytic water molecule in the active site is revealed in the complex with the adenosine analog 2'-hydroxy, 3'-ketocyclopent-4'-enyladenine.

Adenosylhomocysteinase↗
PubMed1998

Structure-affinity relationships of Val-Val and Val-Val-Val stereoisomers with the apical oligopeptide transporter in human intestinal Caco-2 cells.

The objective of this study was to elucidate the structural features of the stereoisomers of Val-Val and Val-Val-Val that afford optimal binding affinity for the apical oligopeptide transporter in human intestinal Caco-2 cells. Three-dimensional conformations of cephalexin and Val stereoisomers were optimized using Chem-X molecular modeling software. Molecular features associated with the optimized conformations of the Val stereoisomers were analyzed to identify potential relationships with their binding affinities for the apical oligopeptide transporter. For Val-Val stereoisomers, the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C7), was found to have a linear relationship with their binding affinities at the 95% confidence level. For Val-Val-Val stereoisomers, three molecular features were found to have linear relationships with their binding affinities at the 95% confidence level. These features included: a) the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C11); b) the distance between the N-terminal amino group and the second peptide bond, d(N1-N9); and c) the molecular dipole moment. Principal component analysis on all molecular features of Val-Val-Val stereoisomers identified three components that accounted for 90% of the variance. A linear model built with these three components by multiple linear regression adequately described the binding affinities (r2 = 0.90). Results from the current study suggest that the distance between the N-terminal amino group and the C-terminal carboxyl group is important for interaction with the apical oligopeptide transporter in Caco-2 cells. In addition, the binding affinities of the Val-Val-Val stereoisomers appear to be influenced by additional factors, including the position of the second peptide bond and the molecular dipole moment.

ATP-Binding Cassette Transporters↗
PubMed1997

Pancreatic exocrine secretion is blocked by inhibitors of methylation.

A number of early experiments suggested a relationship between methyl group metabolism and the exocrine secretion of the pancreas. These included nutritional studies showing that ethionine, the ethyl analog of methionine which inhibits cellular methylation reactions, is a specific pancreatic toxin. Other studies indicated that protein carboxymethylation might be involved. We now show that in vivo ethionine inhibits amylase secretion from freshly isolated rat pancreatic acini, while in vitro ethionine inhibits amylase secretion from the AR42J pancreatic cell line. S-Adenosylhomocysteine (SAH) is a product inhibitor of all methyltransferase reactions involving S-adenosylmethionine (SAM), and treatments that elevate cellular levels of SAH such as inhibition of S-adenosylhomocysteine hydrolase and the in vitro addition of adenosine and homocysteine result in the inhibition of amylase secretion in both isolated pancreatic acini and AR42J cells. Measurement of SAM and SAH levels in AR42J cells shows that inhibition of secretion is more closely related to elevation of SAH levels than to a decrease in the SAM/SAH ratio. Small G-proteins are carboxymethylated on the C-terminal prenylated cysteine and inhibitors of membrane-associated prenylcysteine methyltransferase, N-acetylfarnesylcysteine, N-acetylgeranylgeranylcysteine, and farnesylthioacetic acid (FTA), block secretion in AR42J cells. N-Acetylgeranylcysteine is not an inhibitor of the methyltransferase and does not inhibit amylase secretion. FTA inhibits membrane-associated prenylcysteine methyltransferase from AR42J cells with a Ki in the 45-69 microm range. These results suggest that a methylation event is needed for pancreatic exocrine secretion which may be the reversible methylation of a G-protein involved in signal transduction or membrane trafficking.

Acetylcysteine↗
PubMed1997

The mechanism of inactivation of human placental S-adenosylhomocysteine hydrolase by (E)-4',5'-didehydro-5'-methoxyadenosine and adenosine 5'-carboxaldehyde oxime.

The mechanisms by which (E)-4',5'-didehydro-5'-methoxyadenosine (DMOA) and adenosine 5'-carboxaldehyde oxime (ACAO) inactivate S-adenosylhomocysteine (AdoHcy) hydrolase were elucidated in this study. Their inhibitory activities toward AdoHcy hydrolase were found to be time- and concentration-dependent, and DMOA and ACAO had K(i) and k2 values of 3.0 microM and 0.10 min(-1) and 0.67 microM and 0.16 min(-1), respectively. The inactivation of AdoHcy hydrolase by DMOA (and ACAO) occurs concomitantly with the reduction of the enzyme-bound NAD+ to NADH. The rates of enzyme inactivation correspond to the rates of NADH formation. Incubation of both DMOA and ACAO with the NAD+ form of AdoHcy hydrolase resulted in formation of 3'-ketoadenosine (3'-keto-Ado) 5'-carboxaldehyde and its 4'-epimer. Incubation of DMOA and ACAO with the apo form of the enzyme afforded adenosine (Ado) 5'-carboxaldehyde and its 4'-epimer. These results show that DMOA and ACAO are "proinhibitors" of the enzyme. They are first converted to the inhibitors (Ado 5'-carboxaldehyde and its 4'-epimer) in the active site of the enzyme; these inhibitors then inactivate the enzyme by a type I mechanism. The results from this study demonstrated that this is a common mechanism by which 4',5'-didehydroadenosine analogs, serving as substrates of both the 5'-hydrolytic activity and the 3'-oxidative activity of the enzyme, inactivate AdoHcy hydrolase. The results also provide further evidence supporting the hypothesis that AdoHcy hydrolase possesses a 5'-hydrolytic activity independent of the 3'-oxidation activity.

Adenosine↗
PubMed1997

Anticancer and antiviral effects and inactivation of S-adenosyl-L-homocysteine hydrolase with 5'-carboxaldehydes and oximes synthesized from adenosine and sugar-modified analogues.

Selectively protected adenine nucleosides were converted into 5'-carboxaldehyde analogues by Moffatt oxidation (dimethyl sulfoxide/dicyclohexylcarbodiimide/dichloroacetic acid) or with the Dess-Martin periodinane reagent. Hydrolysis of a 5'-fluoro-5'-S-methyl-5'-thio (alpha-fluoro thioether) arabinosyl derivative also gave the 5'-carboxaldehyde. Treatment of 5'-carboxaldehydes with hydroxylamine [or O-(methyl, ethyl, and benzyl)hydroxylamine] hydrochloride gave E/Z oximes. Treatment of purified oximes with aqueous trifluoroacetic acid and acetone effected trans-oximation to provide clean samples of 5'-carboxaldehydes. Adenosine (Ado)-5'-carboxaldehyde and its 4'-epimer are potent inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. They bind efficiently to the enzyme and undergo oxidation at C3' to give 3'-keto analogues with concomitant reduction of the NAD+ cofactor to give an inactive, tightly bound NADH-enzyme complex (type I cofactor-depletion inhibition). Potent type I inhibition was observed with 5'-carboxaldehydes that contain a ribo cis-2',3'-glycol. Their oxime derivatives are "proinhibitors" that undergo enzyme-catalyzed hydrolysis to release the inhibitors at the active site. The 2'-deoxy and 2'-epimeric (arabinosyl) analogues were much weaker inhibitors, and the 3'-deoxy compounds bind very weakly. Ado-5'-carboxaldehyde oxime had potent cytotoxicity in tumor cell lines and was toxic to normal human cells. Analogues had weaker cytotoxic and antiviral potencies, and the 3'-deoxy compounds were essentially devoid of cytotoxic and antiviral activity.

Adenosine↗
PubMed1997

Effect of limited proteolysis on the stability and enzymatic activity of human placental S-adenosylhomocysteine hydrolase.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface-exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, delta(delta Gu) [delta Gu(native)-delta Gu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface-exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X-ray crystallographic studies currently ongoing in our laboratories.

Adenosylhomocysteinase↗