Potent inhibitors of equine steroid isomerase EcaGST A3-3

Abstract

Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 μM concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Δ5-androstene-3,17-dione.

Fig 1. The steroid double-bond isomerization reactions catalyzed by GST A3-3 with the substrates Δ⁵-androstene-3,17-dione and Δ⁵-pregnene-3,20-dione.

Fig 2. The most potent inhibitors (≥50% inhibition at 10 μM) from the US drug library screened with EcaGST A3-3.

Enzymatic activity was measured with 1 mM GSH and 1mM CDNB with and without 10 μM inhibitor in sodium phosphate buffer at pH 6.5 and 30°C.

Fig 3. Dose-response curves of potent inhibitors of EcaGST A3-3 tested with the alternative substrates CDNB and Δ⁵-AD.

Data points are means and standard deviations (SD) of triplicate measurements. The Hill coefficients represented by mean ± SD are for Δ⁵-AD inhibition: n_anthralin = 1.9±0.19, n_{sennoside A} = 2.1±0.29, n_{tannic acid} = 2.1±0.63, n_{ethacrynic acid} = 1.0, and for CDNB inhibition: n_anthralin = 2.0±0.43, n_{sennoside A} = 2.2±0.41, n_{tannic acid} = 2.2±0.23, n_{ethacrynic acid} = 2.5±0.21. The curve fitting for ethacrynic acid with androstenedione was not improved by using the Hill coefficient as a parameter and was therefore pre-set = 1.0 and presented no SD.

Fig 4

Substrate-saturation curves (A) and double reciprocal plot (B) of the most potent inhibitor anthralin with EcaGST A3-3 at varied concentrations of Δ⁵-AD. Data points are represented as means and standard deviations (SD) of triplicate measurements. The reaction was followed spectrophotometrically for 1 min at 1 mM GSH in the absence (blue) and presence (red) of 0.09 μM anthralin.