The Antiviral Drug Acyclovir Is a Slow-Binding Inhibitor of d-Amino Acid Oxidase

Abstract

d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

Identification of Novel d-Aspartate Oxidase Inhibitors by in Silico Screening and Their Functional and Structural Characterization in Vitro

“…Escherichia coli BL21­(DE3)­pLysS cells were transformed with expression plasmids and cultured at 37 °C with shaking in Luria–Bertani medium containing ampicillin (100 μg/mL). Crude extracts were prepared from cells transformed with pRSET-His-hDDO, pRSET-His-rDDO, pRSET-His-mDASPO, and pRSET-His-hDAO, as described previously. ,, …”

“…Crude extracts were prepared from cells transformed with pRSET-His-hDDO, pRSET-His-rDDO, pRSET-His-mDASPO, and pRSET-His-hDAO, as described previously. 38,57,59 All recombinant proteins were purified by affinity chromatography using a chelating column. Specifically, crude extracts were applied to a His GraviTrap column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 10 mM imidazole.…”

Identification of an l-serine/l-threonine dehydratase with glutamate racemase activity in mammals

“…Escherichia coli BL21(DE3)pLysS (Invitrogen) or Rosetta(DE3)pLysS cells (Merck KGaA, Darmstadt, Germany) were transformed with expression plasmids and cultured at 37°C with shaking in Luria-Bertani (LB) medium containing 100 g/mL ampicillin and 34 g/mL chloramphenicol. Crude extracts were prepared from cells transformed with pRSET-His-hDAO, as described previously [45]. Cells transformed with pRSET-His-rSDHL were grown to an absorbance at 620 nm of 0.5 and incubated for 30 min at a reduced temperature (30°C).…”

Quantitative in silico analysis of selective enzyme reaction of mammalian D-amino acid oxidase and acidic D-amino acid oxidase mutants

“…Previously, we performed conformation analysis of HDAO complex with several active site-directed inhibitors [28,29], but this analysis did not quantitatively demonstrate the enzyme reactivity (inhibitory activity). On the other hand, the reactivity can be quantitatively described by electron localization of key atoms as used in organic synthesis chemistry.…”