**3.3.2 Additional examples**

The importance of the loop region between helix-11 and helix-12 has also been demonstrated by X-ray structure studies for ERα (Pike et al, 1999;Shiau et al, 1998), and mutagenesis studies on MR also support the conclusion that this region of the steroidreceptors is crucial for the agonism/antagonism balance (Fagart et al, 2005).

amongst this drug class. The role of this nitrogen is to form a salt-bridge to Asp351 in helix-3 of ERα (Asp303 in ERβ). The importance of this salt-bridge is that it requires Asp351 to adopt a new conformation and prevents it from undertaking is usual function of stabilizing the agonistic position of helix-12 by hydrogen-bonding to backbone residues in the helix. It also appears that the exact nature of the interaction between the basic amine and Asp351, including angle, distance and perhaps pKa can influence the biological effect of the ligands.

Due to the difficulty in crystallizing partial agonists in complex with steroid-receptors much of the evidence to support these mechanisms has to be inferred from other indirect sources. Some of the most valuable evidence comes from mutagenesis studies including those that indicate that the loop between helix-11 and helix-12 is a hotspot that is crucial to the

Mutation of **Ile747**, which sits in the middle of the helix-11 to helix-12 loop, to methionine results in GR having a reduced transactivation potential without affecting the binding of classic glucocorticoids (Vottero et al, 2002). Presumably, the increased size of the residue

**Tyr735** at the end of helix-11 is a surface residue whose role is poorly understood, but it has been shown that various mutations (W735F, W735V and W735S) result in a receptor with significant reduction in transactivation activity without affecting ligand binding (Ray et al,

**Thr739** is the last residue in helix-11 whose mutation to alanine has no effect on the binding of triamcinolone acetonide, but does result in a 16-fold reduction in transactivation (Lind et

In addition to these mutation studies, as discussed already, there is also overwhelming evidence across the family to support the hypothesis that **Asn564** is crucial for the agonistic activity of GR and related receptors (Bledsoe et al, 2005;Bledsoe, Stewart, and Pearce, 2004;Fagart et al, 1998;Hellal-Levy et al, 2000;Necela and Cidlowski, 2003;Rafestin-Oblin et al, 2002). The role of Asn564 (Asn705 in AR, Asn770 in MR) was previously discussed. Tyr735, Thr739 and Ile747, as shown in Fig 3, are all located at the end of helix-11 or in the following loop. Asn564 has an important role in stabilizing the loop. The studies associated with each of these residues indicate how sensitive this region to influencing the agonism/ antagonism balance and therefore the potential to modify its function by ligand design. The helix-11 to helix-12 loop in steroid-receptors is well suited to drug-design intervention as it forms around the 17β group of steroids and is therefore likely to be in close proximity

Bledsoe and colleagues recognized the importance of this region when solving the first GR-Dexamethasone structure (Bledsoe et al, 2002;Bledsoe, Stewart, and Pearce,2004) as did the group of Kauppi when solving GR complexed with Dexamethasone and RU486, including

The importance of the loop region between helix-11 and helix-12 has also been demonstrated by X-ray structure studies for ERα (Pike et al, 1999;Shiau et al, 1998), and mutagenesis studies on MR also support the conclusion that this region of the steroid-

receptors is crucial for the agonism/antagonism balance (Fagart et al, 2005).

**3.3 Modulate the end of helix-11 & the loop between helices-11 & 12** 

prevents the correct packing of the loop and therefore destabilizes helix-12.

**3.3.1 Case study: the glucocorticoid receptor** 

noting the flexibility of this loop (Kauppi et al, 2003).

agonism/antagonism balance in GR.

1999;Stevens et al, 2003).

al, 2000).

to most ligands.

**3.3.2 Additional examples** 

Fig. 3. The loop between helix-11 and helix-12 illustrating key residues believed to influence GR function
