Mechanism of Inhibition of Estrogen Biosynthesis by Azole Fungicides

Abstract

Biosynthesis of estrogens from androgens is catalyzed by cytochrome P450 aromatase. Aromatase inhibition by the triazole compounds letrozole (LTZ) and anastrozole is a prevalent therapy for estrogen-dependent postmenopausal breast cancer. Azoles are widely used as agricultural fungicides and antimycotic drugs that target 14α-demethylase. Some were previously shown to inhibit aromatase, thereby raising the possibility of endocrine disruptive effects. However, mechanistic analysis of their inhibition has never been undertaken. We have evaluated the inhibitory effects of 3 common fungicides, bifonazole, imazalil, and flusilazole, in human aromatase purified from placenta and compared them with LTZ, the most potent inhibitor of aromatase. Bifonazole exhibits strong inhibitory effects with an IC50 of 270nM and Ki (Michaeles-Menten inhibition constant) of 68nM, compared with 10nM and 13nM, respectively, for LTZ. The IC50 and Ki are 1100nM and 278nM for imazilil and 3200nM and 547nM for flusilazole, respectively. Analyses of inhibition kinetics suggest that the modes of inhibition by azole fungicides are mixed or competitive, whereas LTZ inhibition could be noncompetitive or mixed. We interpret the inhibition mechanism in the context of the x-ray structure of aromatase-androstenedione complex. Structural data show that aromatase has 3 binding pockets in relation to the heme. The substrate-binding cavity at the heme-distal site closely compliments the structures of the natural substrate, androstenedione, and steroidal aromatase inhibitors. Because the structures of LTZ and the azole fungicides are entirely dissimilar to the androstenedione backbone, the azoles possibly inhibit by binding to a structurally rearranged active site, the 2 other catalytically important sites, or both, in agreement with the kinetics data.

In silico prediction of chemical reproductive toxicity using machine learning

“…Imidazole impedes the estrogen biosynthesis by the direct inhibition of aromatase enzyme activity and should thus be regarded as endocrine‐disrupting chemicals. In addition, it may initiate teratogenesis by inhibiting CYP26 enzymes, which plays a crucial role in maintaining proper gradients/concentrations of the morphogen retinoic acid in embryonic tissue (Egbuta, Lo, & Ghosh, ; Marotta & Tiboni, ). The third class of SA is 2‐fluoroethan‐1‐ol, which may be a potential alkylating agent toward DNA.…”

Recent Progress in the Discovery of Next Generation Inhibitors of Aromatase from the Structure–Function Perspective

“…We have reported the inhibition kinetics of LTZ and utilized statistical analyses to show that its mode of action could be either noncompetitive or mixed. 47 Furthermore, clinical data do not show superior efficacy of reversible AIs over irreversible AIs or of steroidal AIs over nonsteroidal AIs and vice versa. In the head-to-head trials evaluating the clinical efficacy of EXM and ANZ, 48 and LTZ and ANZ 49 in postmenopausal breast cancer, all were found to be equally effective.…”

“…Furthermore, recent inhibition kinetics data on LTZ and other azole compounds are indicative of mixed modes of inhibition, structural changes, and allostery. 47 Possibility of allosteric sites for nonsteroidal compounds has also been proposed from computational studies. 117 …”

“…151 In this model, the entrance to the active site access channel and the amphipathic F–G loop including the G′ helix sits on the membrane surface (Figure 7), while the hydrophobic loop of residues 462–471 between β 7 and β 8 touches the interior of the membrane. 47 The side group of compound 5 nearly extends to the “gatekeeper” residues Arg192 and Glu483 at the channel entrance at the membrane–protein interface. The juxtaposition of these catalytically important residues 6,14 at the end of a hydrophobic channel and lipid interface suggests that membrane integration has an important role not only in steroid passage and proton relay critical to aromatization 6 but in substrate and inhibitor selectivity as well.…”

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function

“…Studies on mice and rats demonstrate that fungicides cause testicular, bladder transitional cell and hepatocellular tumors. Fungicides that penetrate the soil and subsequently enter the aquatic environment cause reproductive and developmental side effects in fresh water fish [124,125]. …”