| Valacyclovir is a valine ester prodrug of acyclovir, an effective anti-herpetic drug. The 3--5 fold increase in bioavailability of acyclovir after oral administration of valacyclovir is attributed to carrier-mediated intestinal absorption via hPEPT1, followed by the rapid and complete intracellular conversion to acyclovir. The human enzyme(s) responsible for the intracellular activation of valacyclovir to acyclovir; however, had not been identified. In this thesis, a human esterase that catalyzes the hydrolytic activation of valacyclovir was identified and its biochemical characteristics, substrate specificity, and three-dimensional structure were studied.; A human valacyclovir hydrolase purified from Caco-2 cells was identified as a novel serine hydrolase, biphenyl hydrolase-like (BPHL; gi: 4757862) protein, previously cloned from human breast carcinoma. Recombinant BPHL hydrolyzes valacyclovir with the specificity constant (kcat/Km) 420. The substrate specificity of BPHL was investigated with a series of amino acid ester prodrugs of anti-viral and anti-cancer nucleoside analogues. Results indicate that the substrate specificity of BPHL is largely determined by the structure, stereochemistry, and the site of esterification of amino acid promoiety, and is relatively less sensitive to the nucleoside parent drug. Irrespective of the parent drugs, BPHL prefers the hydrophobic amino acids to the charged amino acids and the 5'-esters to the 3' -esters and the 3',5'-diesters. BPHL is selective for L-isomers over D-isomers and more stereoselective for the valine isomers than the phenylalanine isomers. BPHL was predicted to have the alpha/beta hydrolase fold and a homology model of BPHL was developed, based on the crystal structure of 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase. The BPHL model suggests that BPHL has a hydrophobic acyl-binding pocket, charge-charge interaction sites for the alpha-amino group of the promoiety, a less constrained nucleoside binding site that opens toward the entrance of the substrate-binding pocket, and three free cysteines. Two free cysteines were confirmed by chemical labeling and mass spectrometry. In addition, residues M52, L53, D123, I158, G161, L179, I162, and L229 are identified as important residues for substrate specificity or catalysis.; In conclusion, BPHL, a human valacyclovirase and a novel prodrug activating enzyme was identified and has a great potential to serve as a prodrug design target for therapeutic nucleoside analogue prodrugs. |
