stampidine and phosphoramidic-acid

Stampidine and other halogen substituted stavudine phosphoramidates can be activated by lipase-mediated hydrolysis. The target site for the lipase appears to be the methyl ester group of the L-alanine side chain. Accordingly, the D-amino acid substituted isomers {Rp or Sp}are resistant to lipase-mediated hydrolysis and exhibit substantially less anti-HIV activity. Molecular modeling results indicate that the L-amino acid configured isomers {Rp or Sp} are preferred in the lipase binding pocket.

Topics: Amides; Chromatography, High Pressure Liquid; Dideoxynucleotides; Humans; Hydrolysis; In Vitro Techniques; Lipase; Magnetic Resonance Spectroscopy; Models, Molecular; Phosphoric Acids; Reverse Transcriptase Inhibitors; Spectrophotometry, Ultraviolet; Stavudine; Stereoisomerism; T-Lymphocytes; Thymidine Monophosphate

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.

Topics: Amides; Animals; Cathepsin B; Cattle; Dideoxynucleotides; Endopeptidase K; Hydrolysis; Kinetics; Molecular Structure; Peptide Hydrolases; Phosphoric Acids; Stavudine; Thymidine Monophosphate

Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.

Topics: Amides; Biotransformation; Dideoxynucleotides; Magnetic Resonance Spectroscopy; Peptide Hydrolases; Phosphoric Acids; Prodrugs; Spectrophotometry, Ultraviolet; Stavudine; Stereoisomerism; Thymidine Monophosphate

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.

Topics: Amides; Animals; Anti-HIV Agents; Cell Line, Tumor; Dideoxynucleotides; Enzyme Inhibitors; Esterases; Humans; Kinetics; Lipase; Lymphocytes; Microbial Sensitivity Tests; Molecular Conformation; Peptide Hydrolases; Phenol; Phosphoric Acids; Stavudine; Stereoisomerism; Structure-Activity Relationship; Thymidine Monophosphate; Zidovudine

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.

Topics: Amides; Antiviral Agents; Candida; Chromatography, High Pressure Liquid; Dideoxynucleotides; Esterases; Hydrolysis; Inhibitory Concentration 50; Lipase; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Phosphoric Acids; Protein Conformation; Stavudine; Stereoisomerism; Thymidine Monophosphate