Direct spectroscopic evidence for binding of anastrozole to the iron heme of human aromatase. Peering into the mechanism of aromatase inhibition

Abstract

Aromatase (CYP19A1), is a microsomal cytochrome P450 catalysing the conversion of androgens to estrogens. Non-steroidal inhibitors, such as anastrozole, are important drugs in breast cancer therapy. Using hyperfine sublevel correlation (HYSCORE) spectroscopy we provide the first experimental evidence of the binding of anastrozole to the iron heme of human aromatase.

Effect of sildenafil on human aromatase activity: From in vitro structural analysis to catalysis and inhibition in cells

“…Since the spectral features of rArom-sildenafil complex are similar to those previously observed for rArombound to anastrozole [34,36], the possibilities to have or a direct N-Fe bond formation (as in the case of anastrozole) either a hydrogen bond via a water molecule was investigated by EPR spectroscopy.…”

“…This change is also evidenced in the difference spectra with a decrease of the absorbance at 412 nm and a concomitant increase at 434 nm (inset Figure 1A). This spectroscopic behaviour is usually ascribed to the so-called type II ligands of cytochromes P450 [30,32,33] and it was previously observed also for rArom in complex with the known inhibitor anastrozole [34,36]. In contrast, rArom in complex with the substrate androstenedione and the steroidal inhibitor exemestane give rise to type I spectra, consistent with the displacement of the sixth water ligand of the heme iron [36].…”

“…The results are compared to those previously obtained on ligand-free aromatase and on the protein in complex with the known inhibitor anastrozole [34]. Figure 2A shows the X-band CW EPR spectra recorded for the different samples.…”

“…The spectra of all three samples are dominated by cross peaks in the (-,+) quadrant due to the typical interactions of the unpaired electron localized on the Fe(III) centers with the nitrogen nuclei of the porphyrin ring ( Figure 2B (a), (b) and (c)) [34,39,40]. Proton ridges are also present in all spectra, which are not assigned due to the large number of potential contributors.…”

“…This is spectroscopically detectable as a shift of the maximum absorbance peak from 417-419 nm to 390-394 nm [31]. On the other hand, type II ligands are nitrogen-containing compounds that displace the water molecule while directly coordinating to the heme iron, or alternatively they form a hydrogen bond with the water molecule present as the sixth ligand [32][33][34]. In the case of type II ligands, the heme iron is hexa-coordinated and the maximum absorbance peak shift from 418 nm to 422-426 nm [35].…”