Relative inhibitory potency of molinate and metabolites with aldehyde dehydrogenase 2: implications for the mechanism of enzyme inhibition

Abstract

Molinate is a thiocarbamate herbicide used as a pre-emergent in rice patty fields. It has two predominant sulfoxidation metabolites, molinate sulfoxide and molinate sulfone. Previous work demonstrated an in vivo decrease in liver aldehyde dehydrogenase (ALDH) activity in rats treated with molinate and motor function deficits in dogs dosed chronically with this compound. ALDH is an enzyme important in the catabolism of many neurotransmitters, such as dopamine. Inhibition of this enzyme may lead to the accumulation of endogenous neurotoxic metabolites such as 3,4-dihydroxyphenylacetaldehyde, a dopamine metabolite, which may account for the observed neurotoxicity. In this study, the relative reactivity of molinate and both of its sulfoxidation metabolites toward ALDH was investigated, as well as the mechanism of inhibition. The ALDH activity was monitored in two different model systems, human recombinant ALDH (hALDH2) and mouse striatal synaptosomes. Molinate sulfone was found to be the most potent ALDH inhibitor, as compared to molinate and molinate sulfoxide. The reactivity of these three compounds was also assessed, using N-acetyl Cys, model peptides, and hALDH2. It was determined that molinate sulfone is capable of covalently modifying Cys residues, including catalytic Cys302 of ALDH, accounting for the observed enzyme inhibition.

Figure 1

Inhibition kinetics of hALDH2 by molinate sulfone. (A) Primary plot, representing the natural log transformation of percent activity calculated from treatment of hALDH2 with molinate sulfone (1–25µM). The slope of each curve gives k´ (s⁻¹). (B) Secondary plot, representing the linear correlation between k´ (s⁻¹) and [molinate sulfone], with the slope of this curve equal to k_i (µM⁻¹ s⁻¹).

Figure 2

Inhibition of aldehyde metabolism (i.e. DOPAL), represented by a decreased production of DOPAC, in mouse striatal synaptosomes (0.5mg/mL protein) pre-incubated with 0.1mM dopamine and treated with 50µM molinate (□), molinate sulfoxide (Δ), molinate sulfone (○), or control (◊). The values shown represent the mean ±SEM (n = 3, except for controls where n = 6), where peak area was converted to concentration using a standard curve.

Figure 3

Reactivity of molinate (□), molinate sulfoxide (Δ), and molinate sulfone (○), (0–100µM) with N-acetyl Cys (50µM), monitored using DTNB (500µM) to quantify free thiol concentration. The values shown represent the mean ±SEM (n = 3).

Figure 4

LC/MS and MS/MS analysis of the model peptide, ANP(1–11) (MH⁺ at m/z 1225.4, SLRSSCFGGR) (50µM), incubated with molinate sulfone (0µM (panels A–C) or 500µM (panels D–F)). (A) ANP(1–11) peptide HPLC chromatogram, RT 4.57min. (B) Mass spectrum at 4.57min with peaks 613.4 and 409.2 m/z corresponding to the doubly and triply charged, unmodified peptide. (C) Deconvoluted MS/MS of unmodified peptide (408.95 m/z). (D) HPLC chromatogram of ANP(1–11) peptide modified by molinate sulfone, RT 7.37min. (E) Mass spectrum at 7.37min with peaks at 676.4, 451.3, and 338.7 m/z corresponding to the doubly, triply, and quadruply charged peptide modified by molinate sulfone. (F) Deconvoluted MS/MS of modified peptide (450.8 m/z). *, charged loss of adduct (126.1 m/z).

Figure 5

Sequence of the thiocarbamate-modified ANP(1–11) peptide with y and b fragment ions identified from MS/MS analysis. C*, modified Cys residue.

Scheme 1

Metabolism of Molinate

Scheme 2

Reactivity of Molinate Sulfone with Cys Containing Peptide, ANP(1–11)