Exploiting allostery as a strategy to target human aromatase with new generation inhibitors

Human aromatase (HA) is the member of the cytochrome P450 superfamily that catalyses the formation of the characteristic phenolic A-ring of estrogens from androgenic substrates. Estrogens, beyond their role in reproductive functions, are involved in the development and growth of several hormone-dependent disorders, in particular mammary and endometrial carcinomas. Since more than 70% of breast cancers are positive for estrogen receptor (ER), the reduction of estrogen concentrations through HA inhibition is one of most important therapeutic strategies against this cancer type. Recently, computational simulations show the presence of possible allosteric sites for aromatase activity inhibition, in close proximity to the active site. On this basis, new generation non-competitive inhibitors with high anti-cancer activity and targeting the putative allosteric sites were developed. Moreover, the role of the redox partner protein as allosteric modulator in cytochromes P450 is currently emerging as a possible mechanism of activity control. Thus, in this project, a recombinant form of human aromatase is used i) to investigate the presence of possible allosteric binding sites for the new inhibitors designed in silico and for some emerging pollutants and ii) to study the role of the redox partner in promoting substrate binding. The binding of new generation aromatase inhibitors on the recombinant form of aromatase is investigated by visible spectroscopy, by monitoring the Soret peak position that usually shifts upon binding of substrate and competitive inhibitors. The results show that they do not induce any transition of the Soret peak. However, in their presence, the binding of the substrate androstenedione is affected with a shift of the dissociation constant (KD) toward higher values. Thus, the inhibitors bind to an allosteric site, affecting substrate binding. The same results were obtained when testing some emerging pollutants such as glyphosate and neonicotinoid pesticides (thiacloprids, imidacloprid) that resulted to inhibit aromatase activity with U-shaped inhibition curves. Finally, substrate binding curves were built in the absence and presence of aromatase redox partner, cytochrome P450 reductase (CPR). The results show a decrease in the dissociation constant (KD) by three folds when the CPR is present, suggesting that it may stabilize the open conformation of the enzyme ready to bind the substrate, as reported for other bacterial cytochromes P450. The data show the presence of allosteric sites on HA, where small molecules as well as the redox partner bind and inhibit or promote substrate binding, respectively. The identification of the allosteric sites can provide new insights into the mechanism of action of this important enzyme and allow the design of new allosteric inhibitors.