Evidence for substrate-dependent inhibition profiles for human liver aldehyde oxidase

Abstract

The goal of this study was to provide a reasonable assessment of how probe substrate selection may impact the results of in vitro aldehyde oxidase (AO) inhibition experiments. Here, we used a previously studied set of seven known AO inhibitors to probe the inhibition profile of a pharmacologically relevant substrate N-[(2-dimethylamino)ethyl]acridine-4-carboxamide (DACA). DACA oxidation in human liver cytosol was characterized with a measured V(max) of 2.3 ± 0.08 nmol product · min(-1) · mg(-1) and a K(m) of 6.3 ± 0.8 µM. The K(ii) and K(is) values describing the inhibition of DACA oxidation by the panel of seven inhibitors were tabulated and compared with previous findings with phthalazine as the substrate. In every case, the inhibition profile shifted to a much less uncompetitive mode of inhibition for DACA relative to phthalazine. With the exception of one inhibitor, raloxifene, this change in inhibition profile seems to be a result of a decrease in the uncompetitive mode of inhibition (an affected K(ii) value), whereas the competitive mode (K(is)) seems to be relatively consistent between substrates. Raloxifene was found to inhibit competitively when using DACA as a probe, and a previous report showed that raloxifene inhibited uncompetitively with other substrates. The relevance of these data to the mechanistic understanding of aldehyde oxidase inhibition and potential implications on drug-drug interactions is discussed. Overall, it appears that the choice in substrate may be critical when conducting mechanistic inhibition or in vitro drug-drug interactions prediction studies with AO.

Scheme 1.

DACA oxidation reaction catalyzed by AO in HLC.

Fig. 1.

Michaelis-Menten curve fit and solved kinetic parameters for the oxidation of DACA in HLC. Each point represents a mean of at least seven determinations. Solved parameters are from the best global fit ± S.E.M.

Fig. 2.

Structures of AO inhibitors used in K_i studies.

Fig. 3.

L-B plots comparing the inhibition of the AO-catalyzed oxidation of two different substrates in HLC: phthalazine (left) and DACA (right). Inset graphs are subsequent replots of slope and/or y-intercept of the L-B plots. The top two panels show inhibition by estradiol, center panels show inhibition by clozapine, and bottom panels show inhibition by chlorpromazine. Each point represents the mean ± S.E.M. of a single assay vial analyzed in triplicate by LC-MS/MS.

Fig. 4.

L-B plot of the inhibition of AO-catalyzed DACA oxidation by raloxifene. Inset graph is a subsequent replot of L-B slopes versus inhibitor concentration. Each point represents the mean ± S.E.M. of a single assay vial analyzed in triplicate by LC-MS/MS.