Enzymatic hydrolysis of stampidine and other stavudine phosphoramidates in the presence of mammalian proteases

Mammalian proteases have not been implicated in the metabolism of any nucleoside phosphoramidate prodrug. The results presented herein provide unprecedented and conclusive experimental evidence that mammalian proteases are capable of hydrolyzing stavudine phosphoramidates. Specifically, cathepsin B and Proteinase K are able to metabolize stampidine and other phosphoramidate derivatives of stavudine. Additionally, cathepsin B exhibits chiral selectivity at the phosphorus center. The elucidation of the metabolic pathways leading to activation of stampidine may provide the basis for pharmacologic interventions aimed at modulating the metabolism and thereby improving the therapeutic window of stampidine as an anti-HIV agent.     

Lipase-mediated stereoselective hydrolysis of stampidine and other phosphoramidate derivatives of stavudine

Enzymatic hydrolysis of stampidine and other aryl phosphate derivatives of stavudine were investigated using the Candida Antarctica Type B lipase. Modeling studies and comparison of the hydrolysis rate constants revealed a chiral preference of the lipase active site for the putative S-stereoisomer. The in vitro anti-HIV activity of these compounds correlated with their susceptibility to lipase- (but not esterase-) mediated hydrolysis. We propose that stampidine undergoes rapid enzymatic hydrolysis in the presence of lipase according to the following biochemical pathway: During the first step, hydrolysis of the ester group results in the formation of carboxylic acid. Subsequent step involves an intramolecular cyclization at the phosphorous center with simultaneous elimination of the phenoxy group to form a cyclic intermediate. In the presence of water, this intermediate is converted into the active metabolite Ala-d4T-MP. We postulate that the lipase hydrolyzes the methyl ester group of the l-alanine side chain to form the cyclic intermediate in a stereoselective fashion. This hypothesis was supported by experimental data showing that chloroethyl substituted derivatives of stampidine, which possess a chloroethyl linker unit instead of a methyl ester side chain, were resistant to lipase-mediated hydrolysis, which excludes the possibility of a direct hydrolysis of stampidine at the phosphorous center. Thus, our model implies that the lipase-mediated formation of the cyclic intermediate is a key step in metabolism of stampidine and relies on the initial configuration of the stereoisomers.     

Synthesis and biological evaluation of LNA phosphoramidates

The synthesis of LNA phosphoramidates is presented. The LNA phosphoramidates were evaluated for their ability to inhibit cell proliferation of the human prostate cancer cell line 15PC3. A number of the LNA phosphoramidates showed cell proliferation inhibition determined by the MTS assay.     

Inhibitory effect of lysophosphatidylcholine on pancreatic lipase-mediated hydrolysis in lipid emulsion

In the lipid metabolism pathway, dietary lipid emulsified with bile salts and phospholipids is mainly digested by pancreatic lipase into free fatty acids and monoacylglycerols. In order to study substrate recognition mechanism of a pancreatic lipase, we investigated its catalytic property toward the lipid emulsion prepared with long- or intermediate-chain acylglycerols and several physiological surfactants. When lysophosphatidylcholine (LysoPC), rather than bile salts or phospholipid, was incorporated into the lipid emulsion, it caused an increase in the Km(app) and a decrease in the Vmax(app) values in the interactions between the lipase and triacylglycerol (triolein or tricaprin). This indicated that LysoPC inhibited hydrolysis by decreasing both the substrate affinities and the catalytic activity of this lipase. Interestingly, further addition of taurodeoxycholic acid sodium salts or phospholipid completely restored the inhibitory effect of LysoPC on hydrolysis by lipase. On the other hand, the change in these kinetic values between the lipase and two 1-monoacylglycerols (1-monocaprin and 1-monoolein) were not particularly large when LysoPC was added. Particle size analysis of the lipid emulsion composed of LysoPC and triacylglycerols showed that most of the particles were less than 200 nm in size, which was smaller than the particle size in the triacylglycerol emulsions containing bile salts or phospholipid. The composition of the emulsion would affect its surface characteristics and thus contribute to changing lipase activity.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Stereochemical reaction mechanism formulations for enzyme-catalyzed pyrophosphate hydrolysis,ATP hydrolysis,and ATP synthesis

Fundamental concepts pertaining to the stereochemistry paths of polar additionelimination (nucleophilic substitution) reactions at phosphate phosphorus centers are reviewed and employed to analyze ¹⁸O exchange reactions catalyzed by inorganic pyrophosphatase and mitochondrial ATP synthetase. The analysis suggests reasonable choices for the stereochemistry path of the ¹⁸O exchanges. This, in turn, permits reasonable choices for the stereochemistry paths of hydrolysis of pyrophosphate catalyzed by pyrophosphatase and of hydrolysis and synthesis of ATP catalyzed by ATP synthetase.     

Apolipoproteins regulate the kinetics of endothelial lipase-mediated hydrolysis of phospholipids in reconstituted high-density lipoproteins

Endothelial lipase (EL) is a newly identified member of the triglyceride lipase gene family that hydrolyzes high-density lipoprotein (HDL) phospholipids. This study investigates the ability of the major apolipoproteins of rHDL to regulate the kinetics of EL-mediated phospholipid hydrolysis in well-characterized, homogeneous preparations of spherical rHDL. The rHDL contained either apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid composition and contained cholesteryl esters (CE) as their sole core lipid. Phospholipid hydrolysis was quantitated as the mass of nonesterified fatty acids (NEFA) released from the rHDL during incubation with EL. The V(max) of phospholipid hydrolysis for (A-I/A-II)rHDL [391.9 +/- 12.9 nmol of NEFA formed (mL of EL)(-1) h(-1)] was greater than (A-I)rHDL [152.8 +/- 4.7 nmol of NEFA formed (mL of EL)(-1) h(-1)]. The energy of activation (E(a)) for the hydrolysis reactions was calculated to be 52.1 and 34.8 kJ mol(-1) for (A-I)rHDL and (A-I/A-II)rHDL, respectively. Minimal phospholipid hydrolysis was observed for the (A-II)rHDL. Kinetic analysis showed that EL has a higher affinity for the phospholipids in (A-I)rHDL [K(m)(app) = 0.10 +/- 0.01 mM] than in (A-I/A-II)rHDL [K(m)(app) = 0.27 +/- 0.03 mM]. Furthermore, (A-I)rHDL is a competitive inhibitor of the EL-mediated phospholipid hydrolysis of (A-I/A-II)rHDL. These results establish that apolipoproteins are major determinants of the kinetics of EL-mediated phospholipid hydrolysis in rHDL.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.     

Enzymatic hydrolysis and biological activity of oligonucleotides containing 5-substituted pyrimidine bases.

Two series of alternating ODNs containing 5-n.alkyl-, alkenyl- and alkynyl-dU and -dC units have been prepared in order to study the kinetics of their hydrolysis by SV PDE and human serum, respectively. Both in (r5dUpdA)10 and (r5dCpdG)6 series the rate of hydrolysis decreased with increasing length of side-chain. Replacement of thymidines by 5-hexynyl-dU in different antisense oligomers resulted in considerably higher biological activity relative to that of the thymidine-containing counterparts.     

Stereochemical enhancement of specificity in biological recognition systems]

Specificity of the reaction of equilibrium association as a model of recognition process has been analysed. It has been shown that subdividing the centres of recognition into two uncoupled groups may lead to the effect of stereochemical enhancement of specificity, i. e. an increase of the fraction of the correct complex. If the second group of centers is unspecific, then a compensation of mistakes on a specific group of centers by unspecific ones takes place. Stereochemical enhancement of specificity requires the presence in the recognition protein molecule of a mechanism of switching on - off interaction on a group of centers. The most universal method of such switching on - off is a conformational transition in the protein molecule and (or) the "substrate".     

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.     

Antitumor activity and other biological properties of two phenotypes isolated from BCG

Summary Two phenotypes isolated from the BCG strain of Mycobacterium tuberculosis were found to differ from each other and from the parental strain by their chemical and antigenic compositions and by some of their biological properties. When used as immunotherapeutic agents in admixture with cancerous cells, one of these phenotypes enhanced whereas the other inhibited tumor growth. As immunoprophylactic agents, both phenotypes exerted, but at different degrees, antitumor protection in mice.     

Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analogue of the inhibitor pepstatin have been refined recently at 1.8-A resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide nitrogen is protonated, either from the solvent after nitrogen inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.     

The influence of lime and biological activity on sediment pH,redox and phosphorous dynamics

The addition of powdered limestone to intact sediment cores from oligotrophic, acid Lake Hovvatn caused pH to increase, redox potential (E₇) to drop, and permitted net precipitation of phosphorous (P) from the water column. Significant pH increase was found to a sediment depth of 6 cm and a maximum increase in pH from 4.9 to 6.5 was found at a depth of 0.5 cm when dosed with 36 g m^–2 of lime. Such pH increase creates important changes in sediment equilibrium chemistry and enhances habitat suitability. In the case of Hovvatn, however, sediments would consume only 5 kg of the 91 tons of applied limestone. Superficial sediments remained oxidized, but below 0.5 cm, E₇ in limed sediment declined significantly more than in unlimed sediments, with a maximum difference of 102 mV versus –66 mV at a depth of 6 cm in unlimed and limed cores, respectively. Abiotic reactions account for 82 ± 54% of this reduction and the remainder is due to the oxidation of organic matter by bacteria. Precipitation of CaSO₄, reduction of the sediments by organic compounds at elevated pH and inhibition of the downward diffusion of O₂ by the limestone powder are potential abiotic mechanisms which could drive E₇ down. Enhanced P release was not found at lowered E₇, and supernatent TP concentrations dropped from 11.7 to 4.4 µg P l^–1. More P was swept from solution in cores which recieved larger lime doses. The presence of chironomids caused sediment pH to increase by as much as 1.2 pH units, presumably due to NH₄ release, reduced sediment E₇ by as much as 171 mV and facilitated TP release during the first 17 d of core incubation. Field measurements of vertical distributions of sediment pH and E₇ before and after the liming of Hovvtn corroborated laboratory findings.     

Stereochemical aspects of peptide hydrolysis catalyzed by serine proteases of the chymotrypsin type

The steric course of peptide hydrolysis catalyzed by serine proteases has been studied on the basis of the available, extensive structural data and taking into account the stereoelectronic theory of Deslongchamps (Heterocycles, 7, 1271 (1977)). These studies allowed elucidation of the structure of intermediates, in particular of the tetrahedral intermediate, and of the main structural events taking place during catalysis. They reveal a difficulty inherent in the generally accepted mechanism of peptide hydrolysis: protonation of the leaving nitrogen in the configuration arising from nucleophilic attack of Ser-195 on the carbonyl carbon cannot take place internally from His-57. Two alternative mechanisms are discussed which are compatible with all implications of the stereoelectronic theory. The main features of the more probable mechanism are: (i) a conformational change allowing the imidazole ring of His-57 to occupy two distinct positions; in one position a proton is abstracted from O^γ of Ser-195, and in the other this proton is donated to the leaving nitrogen; (ii) a configurational change (inversion) of the pyramidal leaving nitrogen reorienting the lone-pair orbital developed during nucleophilic attack; in one orientation CO bond breaking, and in the other CN bond breaking, is allowed. This inversion process confers on the nitrogen the property of a switch controlling the breakdown of the tetrahedral intermediate.