This section describes examples of usage of the methods that were introduced above. Molecular dynamics simulations have proven useful for drug design purposes on many different targets.88-103 Here, we will focus on the ligand-binding domain of the estrogen receptor. The estrogen receptor is a member of the nuclear hormone receptors and plays a key role in the growth, development, and maintenance of a diverse range of tissues. It consists of an N-terminal DNA-binding domain, a ligand-binding domain, and a C-terminal activation domain. Upon binding of an agonist to the ligand-binding domain a conformational change in the estrogen receptor takes place, allowing it to homodimerize and subsequently to translocate to the nucleus. Here, the DNA-binding domain directly interacts with response elements on the DNA, thereby activating or repressing transcription.104 Apart from the physiological effects, the estrogen receptor is involved in a range of diseases such as breast cancer, osteoporosis, endometrial cancer, and prostate hypertrophy.105
Naturally occurring estrogens are steroid hormones, such as the endogenous ligand 17b-estradiol. Apart from this endogenous ligand, the estrogen receptor is known to show affinity for a wide range of structurally diverse compounds, including synthetic estrogens, phytoestrogens, pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs).106-110 PAHs are products of incomplete combustion of fossil fuels, wood, and other organic matter and as such are ubiquitous in the environment. Oxidative biotransformation by cytochrome P450 introduces hydroxyl groups, which results in structures that can mimic estradiol.111-113 Everyday foodstuffs are another source of estrogenlike compounds. Some foods are known to contain a wide range of phytoestrogens, such as the isoflavonoids genistein and daidzain that show estrogenic activity in vitro and in vivo. The most important human sources of these isoflavonoids are soybeans and soybean products.114 Although the production and use of polychlorinated biphenyls was banned in the late 1970s, they have still been observed in all kinds of tissues and species.115-117 Again, one of the metabolic pathways of the PCBs involves the hydroxylation of (one of the) aromatic rings,118 after which the affinity for the estrogen receptor is altered.119
Several crystal structures of the estrogen receptor ligand-binding domain in complex with various agonists and antagonists are available from which it becomes clear that the ligand occupies a deeply buried, largely hydrophobic cavity within the ligand-binding domain. In most structures there is no clear path or channel connecting this cavity with the exterior of the protein, in contrast to typical protein-ligand complexes. In addition, the structure of an antagonist bound to the estrogen receptor ligand-binding domain shows that a bulky side chain moiety of the antagonist serves to prevent the formation of the active tertiary structure of helices 11 and 12, known to be crucial for activity.120,121
The overall topology of the estrogen receptor ligand-binding domain, the wide structural diversity of known agonists, its physiological relevance, and the availability of crystal structures of the target make the estrogen receptor an ideal candidate to apply and develop computational methods for the prediction of binding affinities, to be used for drug design purposes. The following sections will describe some of our experiences of molecular dynamics simulations and free energy calculations of the estrogen receptor ligand-binding domain, the prediction and structural interpretation of binding affinities, and the design of a new ligand binding to the estrogen receptor.72'101'122'123
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