**3.5 Straighten helix-3, and/or disrupt interactions between helices-3 & 5 3.5.1 Case study: the mineralocorticoid receptor**

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-

Drug Design Approaches to Manipulate the Agonist-Antagonist Equilibrium in Steroid Receptors 229

The binding of Dexamethasone to the Glucocorticoid Receptor (GR) is shown to illustrate the five major routes for reducing agonist efficacy in steroid receptors via the destabilisation of the binding of co-activating proteins. Co-activating proteins bind in a hydrophobic pocket on the surface of the ligand-binding domain (LBD) stabilised by a charge-clamp formed by residues Lys579 in helix-3 and Glu755 in helix-12. **[1]** Direct clashes between ligands and helix-12 prevent Glu755 from adopting its necessary position and thus prevent the formation of the charge clamp. It has been shown for some receptors that clashes with helix-12 result in the helix adopting a new orientation actually precluding the binding of coactivators by binding in the required hydrophobic pocket. **[2]** It is probably therefore not a surprise that the positioning of helix-12 can be influenced by the residues that directly precede it. The loop before helix-12 influences its position and is clearly a hotspot that can influence degree of agonism by modifying the ligand. **[3]** Other interactions also help stabilise helix-12 in its agonist position. For example, in GR, there is a hydrogen-bond network from the ligand to Asn564 in helix-3 to Glu748 in the loop before helix-12. Disruption of this network, by perhaps removing the hydrogen-bonding function in the ligand, can influence the stabilisation of helix-12. **[4]** In a number of nuclear receptors Helix-12 also makes direct hydrophobic interactions to the ligand. Loss of these interactions, by changing the properties of the ligand, can decrease the stabilisation of helix-12 and therefore alter the agonistic capability of the complex. **[5]** Finally, the first four approaches are directly or indirectly related to ensuring Glu755, as half of the charge-clamp, is correctly positioned. The second residue in the charge-clamp, Lys579, should not be overlooked. Lys579 is part of helix-3 which itself bends midway along its length. This bend is crucial for ensuring that Lys579 is in the correct position to form the chargeclamp. The bend in helix-3 is partly as a result of its interaction with helix-5. For GR this is largely mediated by a hydrogen-bond network between Gln570 in helix-3, the ligand and Arg611 in helix-5. Disrupting this network by modifying the ligand may influence the distortion in helix-3 and therefore the correct formation of the charge-clamp and therefore

In addition to exploring the development of partial agonists, structure-based approaches continue to play an important role in the identification of new ligands via virtual screening approaches and other compound optimization tasks. An important lesson in this regard has been our change in understanding the dynamic nature of the steroid-receptor binding pocket. We have seen examples of extensive induced fits for amongst others the glucocorticoid receptor which is able to bind ligands beyond the conventional confines of its binding pocket whilst remaining in an agonistic conformation (Biggadike et al, 2009;Madauss et al, 2008;Suino-Powell et al, 2008). The pocket, behind the crucial helix-3 and helix-5 binding residues, Gln570 and Arg611, is normally water filled. It has already been demonstrated to be a viable ligand-binding region with the potential to improve ligand potency. An interesting note regarding the exploration of the pocket is that GSK report difficulty in combining the use of this pocket with the maintenance of partial agonism (Biggadike et al, 2009). PR has been shown to adapt to steroids baring bulky 17α groups (Madauss et al, 2004) and Trp741 in AR adapts to different ligands, adopting a new position

**4.1 Binding mode of DEX to GR illustrates each of the five design approaches** 

co-activator binding.

**5. Other structure-based design considerations** 

to open an additional channel in the receptor (Bohl et al, 2005).

activators bind is dependent on a bend forming in the middle of helix-3. That bend in helix-3 is induced by a ligand mediated hydrogen bond to helix-5 via the 3-keto group of steroidal ligands. It was initially believed that the importance of the classic interactions between the 3-keto group of steroids and the Glutamine (Glutamate in ERα and ERβ) and Arginine residues in steroid hormones was purely to ensure potent binding of the steroids, but the work of Bledsoe (Bledsoe et al, 2005) and Huyet (Huyet et al, 2007) have demonstrated that is also has a role in the agonism-antagonism balance. Huyet *et al* demonstrated that mutation of either Gln776 or Arg817 in MR to alanine results in previously ligand-mediated agonistic responses being lost.

Bledsoe *et al* have further demonstrated the importance of this bend in helix-3 by characterizing the S810L mutation in MR. This mutation has the effect of stabilizing the agonist conformation of MR, rendering some antagonistic ligands to have an increased agonistic response. Their analysis shows that the role of the S810L mutation is to increase the hydrophobic stabilization between helix-3 and helix-5.
