A Novel Nucleoside Prodrug-Activating Enzyme:  Substrate Specificity of Biphenyl Hydrolase-like Protein

Abstract

Biphenyl hydrolase-like protein (BPHL, NCBI accession number NP_004323) is a novel human serine hydrolase recently identified as a human valacyclovirase, catalyzing the hydrolytic activation of the antiviral prodrugs valacyclovir and valganciclovir. The substrate specificity of BPHL was investigated with a series of amino acid ester prodrugs of the therapeutic nucleoside analogues: acyclovir, zidovudine, floxuridine, 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl) benzimidazole, and gemcitabine. The hydrolysis of typical esterase and aminopeptidase substrates by BPHL was also investigated. The results indicate that the substrate specificity of BPHL is largely determined by the amino acid acyl promoiety, and is less sensitive to the nucleoside parent drugs. For all nucleoside parent drugs, BPHL preferred the hydrophobic amino acids valine, phenylalanine, and proline over the charged amino acids lysine and aspartic acid. The position and monoester or diester form of the prodrug were also important, with BPHL exhibiting higher affinity for the 5'-esters than for the 3'-esters and the 3',5'-diesters irrespective of amino acid type. Further, the presence of the 3'-amino acid ester considerably reduced the hydrolysis rate of the 5'-amino acid ester functionality. BPHL exhibited stereoselectivity with an L/D specificity ratio of 32 for 5'-valyl floxuridine and 1.5 for 5'-phenylalanyl floxuridine. The substrate specificity suggests that the substrate-binding pocket of BPHL has a hydrophobic acyl binding site which can accommodate the positively charged alpha-amino group, while having an alcohol leaving group binding site that can accommodate nucleoside analogues with a relatively generous spatial allowance. In conclusion, BPHL catalyzes the hydrolytic activation of amino acid esters of a broad range of therapeutic nucleoside analogues in addition to valacyclovir and valganciclovir and has considerable potential for utilization as an activation target for design of antiviral and anticancer nucleoside analogue prodrugs.

Targeted delivery to PEPT1-overexpressing cells: Acidic, basic, and secondary floxuridine amino acid ester prodrugs

“…A free lysyl aamino group could render these esters more prone to potential aminopeptidase activity as observed with other lysyl prodrugs (30). Biphenyl hydrolase-like protein seems to prefer hydrophobic amino acids and is less active against lysyl and aspartyl ester prodrugs (19). Aspartyl esters were significantly more stable in Caco-2 homogenates than lysyl and prolyl ester prodrugs, consistent with previous enzymatic stability data for Asp-acyclovir prodrugs (31).…”

“…Individual prodrug-converting enzymes expressed in Caco-2 cells, such as biphenyl hydrolase-like protein, have been reported to hydrolyze the amino acid ester prodrugs valacyclovir and valganciclovir (18). In Caco-2 cell homogenates, the prolyl prodrugs were most rapidly converted to floxuridine potentially due to hydrolysis by biphenyl hydrolase-like protein or by other hydrolase enzymes (19). Lysyl prodrugs were also rapidly hydrolyzed; however, because they are not good substrates for biphenyl hydrolase-like protein, other enzymes may be responsible (19).…”

“…In Caco-2 cell homogenates, the prolyl prodrugs were most rapidly converted to floxuridine potentially due to hydrolysis by biphenyl hydrolase-like protein or by other hydrolase enzymes (19). Lysyl prodrugs were also rapidly hydrolyzed; however, because they are not good substrates for biphenyl hydrolase-like protein, other enzymes may be responsible (19). A free lysyl aamino group could render these esters more prone to potential aminopeptidase activity as observed with other lysyl prodrugs (30).…”

“…Because the enzymatic activity toward these prodrugs could be influenced by the structure of the promoiety and drug molecule, a variety of prodrugs need to be investigated. Recently, the influence of stereochemistry and site of esterification on the activation rate of nucleoside analogue prodrugs was reported (6,19). Beyond these studies, there are very limited structureactivation studies evaluating the influence of the promoiety on the prodrug activation rate.…”

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

“…After absorption via intestinal peptide transporters (3,14,15), both prodrugs are found to be rapidly activated (10 -12), with about 50% of the absorbed dose converted to acyclovir before reaching the portal circulation (16). In addition to activating valacyclovir and valganciclovir, VACVase has been shown to hydrolyze the prodrugs of a broad range of antiviral and anticancer nucleoside analogues such as zidovudine, floxuridine, and gemcitabine (7,17).VACVase contains the consensus sequence motif Sm-X-Nu-X-Sm-Sm (Nu ϭ nucleophile, Sm ϭ small residue) of the ␣/␤ hydrolase-fold superfamily (18) as well as a putative catalytic triad, Ser-122-His-255-Asp-227 (Fig. 1b).…”