**3.2.2 Additional examples**

The residue equivalent to AR residue Asn705 in MR is Asn770. Extensive X-ray, SAR and mutation studies have been conducted on Asn770 which demonstrate clearly the existence of a ligand-mediated hydrogen bonding network which is critical for the activation of MR in a similar fashion to the one described for AR (Bledsoe et al, 2005;Hellal-Levy et al, 2000).

Agonistic steroidal ligands for GR and MR are typified by 11β-hydroxyl groups which hydrogen bond to Asn564 in GR and Asn770 in MR respectively. Despite the similarity between MR and PR, the endogenous PR agonist progesterone behaves as an antagonist of PR. This appears to at least in part be due to a lack of an 11β-hydroxyl group on progesterone. It is interesting how the lack of the hydroxyl group doesn't disturb the agonistic activity of PR but does MR.

Another important example of disrupting the function of stabilizing interactions can be seen in the estrogen receptors. In addition to their role in sterically precluding helix-12, SERM side-chains also contain an important basic amine function which is almost ubiquitous

Drug Design Approaches to Manipulate the Agonist-Antagonist Equilibrium in Steroid Receptors 227

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

Methods for antagonizing or reducing the agonism of steroid receptors that do not involve direct steric clashes with the receptor are often referred to as "passive antagonism". This term was coined by the group of Geoffrey Greene to explain their observations when studying the binding of tetrahydrochyrsene (THC) and its interactions with ERα and ERβ

THC is an ERα agonist and an ERβ antagonist. The group of Greene was able to conclude, after generating X-ray structures of both complexes that THC stabilizes ERα in its agonist conformation but ERβ is in an antagonist conformation. This difference on its own is of significant interest, but the study also demonstrated that the reason for ERβ not adopting an agonist conformation was due to missing stabilizing interactions between the receptor and the ligand. They observe that in ERβ residues Leu476 and Met479 are not positioned correctly by the ligand to form interactions with relevant residues in helix-12 to stabilize its agonist conformation. The result is a failure of THC to stabilize the agonist conformation of helix-12 and therefore a shift in the agonist-antagonist equilibrium. The fact that THC has such differing effects on two such similar receptors illustrates the challenge when following

It is generally accepted that steroid-receptor activation is facilitated by interactions between helix-3 and helix-5. The correct positioning of the basic component of the charge clamp (Lys579 in GR and Lys785 in MR) and the formation of the hydrophobic pocket in which co-

**3.4 Reduce the stabilizing interactions between the ligand and helix-12** 

**3.4.1 Case study: the estrogen receptors alpha & beta** 

this or any of the five described approaches in drug-design.

**3.5.1 Case study: the mineralocorticoid receptor** 

**3.5 Straighten helix-3, and/or disrupt interactions between helices-3 & 5** 

GR function

(Shiau et al, 2002).

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.
