Three-Dimensional Model of the Human Aromatase Enzyme and Density Functional Parameterization of the Iron- Containing Protoporphyrin IX for a Molecular Dynamics Study of Heme-Cysteinato Cytochromes Angelo Danilo Favia, 1 Andrea Cavalli, 2 * Matteo Masetti, 2 Angelo Carotti, 1 Maurizio Recanatini 2 1 Department of Medicinal Chemistry, University of Bari, Via E. Orabona 4, I-70124 Bari, Italy 2 Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy ABSTRACT Mammalian cytochromes P450 (CYP) are enzymes of great biological and pharmaco- toxicological relevance. Due to their membrane- bound nature, the structural characterization of these proteins is extremely difficult, and therefore computational techniques, such as comparative modeling, may help obtaining reliable structures of members of this family. An important feature of CYP is the presence of an iron-containing porphyrin group at the enzyme active site. This calls for quan- tum chemical calculations to derive charges and parameters suitable for classical force field-based investigations of this proteins family. In this report, we first carried out density functional theory (DFT) computations to derive suitable charges for the Fe 2 -containing heme group of P450 enzymes. Then, by means of the homology modeling technique, and taking advantage of the recently published crystal structure of the human CYP2C9, we built a new model of the human aromatase (CYP19) enzyme. Furthermore, to study the thermal stability of the new model as well as to test the suitability of the new DFT-based heme parameters, molecular dynam- ics (MD) simulations were carried out on both CYP2C9 and CYP19. Finally, the last few ns of aromatase MD trajectories were investigated follow- ing the essential dynamics protocol that allowed the detection of some correlated motions among some protein domains. Proteins 2006;62:1074 –1087. © 2006 Wiley-Liss, Inc. Key words: mammalian cytochromes P450 (CYP); DFT parameterization; heme-cystein- ato cytochromes INTRODUCTION Mammalian cytochromes P450 (CYP) are involved in the biosynthesis of several important hormones, mainly of a steroidal nature and in the metabolism of more than 90% of the drugs in current clinical use. 1 In addition, they participate in both toxification and detoxification pro- cesses of many xenobiotics. The primary sequences of CYP are extremely diverse with levels of identity as low as 16%, 2 whereas the overall folding, comprising 12 helices and loops denoted as A-L (Fig. 1), has remained unaltered through the evolution. Furthermore, CYP share a common prosthetic group constituted by an iron-containing proto- porphyrin IX, which is linked to the protein through both a covalent bond between the metal and the sulphur atom of a proximal cysteine, and a network of hydrogen bonds among its propionate groups and the side chains of posi- tively charged amino acids. The heme group is located between helices I and L, while helices B, C, F, and G contribute to the substrate recognition. Although the key steps of the catalytic mechanism seem to be similar among all isoforms, CYP show a surprising degree of selectivity versus their own specific substrates. 3 The cytochrome P450 19 (CYP19; EC 1.14.14.1), commonly called aromatase (AR), metabolizes a wide variety of important substrates in many species of bacteria, plants, and animals. In the human body, AR catalyzes the conver- sion of androgens into estrogens, through the aromatiza- tion of the A ring of substrates like testosterone and androstenedione. 4 In the anti-cancer therapy, AR is an important pharmacological target because of its critical role in the progression of post-menopausal breast cancer. 5 Actually, the reduction of the levels of circulating estro- gens in women with the disease has been demonstrated to be clinically effective, 6,7 and AR inhibitors (ARIs) such as anastrozole, vorozole, letrozole, and fadrozole are widely used, even as the first-line drugs in the therapy of breast cancer. 8,9 However, some important side effects associated with the prolonged clinical use of ARIs 10,11 called for the search of new, potent, more selective, and less toxic CYP19 inhibitors. In the era of the so-called “molecular targeted therapy,” there is a strong need to develop reliable and validated 3D models of P450 enzymes of therapeutic interest, enabling the application of the structure-based design (SBD) of potent and selective inhibitors. Unfortunately, the SBD of P450 inhibitors has been strongly hampered by the ab- Grant sponsor: MIUR (Ministero dell’Istruzione dell’Universita `e della Ricerca, Rome, Italy). *Correspondence to: Andrea Cavalli, Department of Pharmaceuti- cal Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy. E-mail: andrea.cavalli@unibo.it Received 13 May 2005; Accepted 5 October 2005 Published online 4 January 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.20829 PROTEINS: Structure, Function, and Bioinformatics 62:1074 –1087 (2006) © 2006 WILEY-LISS, INC.