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

Jiahai Zhou,Gordon L. Amidon,Kyung-Dall Lee,Zhaohui Xu,Longsheng Lai

doi:10.1074/jbc.m709530200

Jiahai Zhou, Gordon L. Amidon + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m709530200

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Abstract

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Highlights

VACVase catalyzes the hydrolytic activation of two clinically important antiviral nucleoside prodrugs, valacyclovir and valganciclovir (Fig. 1a), resulting in L-valine and their corresponding active drugs acyclovir and ganciclovir
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]
Crystal structures of the enzyme, two mutants, and a complex with a product analogue combined with biochemical data revealed the key elements in catalysis and substrate recognition

Summary

RESULTS

Substrate Specificity of VACVase—VACVase is synthesized in the cell as a precursor protein with a leader sequence of 20 residues [7, 25]. The enzyme efficiently hydrolyzed benzyl esters of L-Phe and L-Ala; its activity toward ␤-amino acid esters was 3 orders of magnitude lower than toward the corresponding ␣-amino acid esters (barely measurable). To gain further insight into the specificity of VACVase for the acyl moiety, a range of ␣-amino acid esters (mostly benzyl esters) were investigated (Table 2). Of the amino acid esters examined, the L-Pro ester was most efficiently hydrolyzed followed by the small amino acid esters, L-Gly and L-Ala esters. The hydrophobic and aromatic amino acid esters were efficiently hydrolyzed by the enzyme. The L-Asn ester is not a substrate for the enzyme, in contrast to the L-Ser ester, which was hydrolyzed very efficiently.

Substrate specificity of human VACVase

Specific activity

DISCUSSION

VACVases from various sources are needed to support these