**3.2 Disrupt the function of direct stabilizing interactions 3.2.1 Case study: the androgen receptor**

224 Steroids – Basic Science

Introducing bulky groups onto PR modulating non-steroidal scaffolds has also been demonstrated to result in partial agonists on a number of occasions (Jones et al,

The existence of a clash between antagonists and helix-12 was first demonstrated for ER by studies comparing the X-ray structures of Estradiol to Raloxifene (Brzozowski et al, 1997) and Diethylstilbestrol to Tamoxifen (Shiau et al, 1998). Numerous reviews of these two studies have been published (Hubbard et al, 2000;Kong, Pike, and Hubbard, 2003;Mueller-Fahrnow and Egner, 1999;Pike et al, 2000;Pike, Brzozowski, and Hubbard, 2000) as have many further studies on the X-ray structures of SERMs, full antagonists and full agonists bound to the ERs (Blizzard et al, 2005;Dykstra et al, 2007;Heldring et al, 2007;Kim et al,

Fig. 1. Binding of PR agonist Norethindrone (orange) from X-ray structure compared to PR antagonist Mifepristone (green) demonstrating clash between antagonists and Met909 in

The same helix-12 clash has also been demonstrated for AR (Cantin et al, 2007) and GR (Schoch et al, 2010) in recent X-ray structure determination studies. It was also suggested for GR by a mutagenesis study (Hillmann et al, 2002) that showed that mutating Leu753 (equivalent to Met909 in PR) to a phenylalanine results in a receptor defective in transactivation. We can conclude that the reason for this loss of activation is that an increase in the size of the residue at this position prevents helix-12 from adopting its agonistic

2005;Kallander et al, 2010;Thompson et al, 2009;Washburn et al, 2009).

2004;Renaud et al, 2003;Renaud et al, 2005;Tan et al, 2005;Vajdos et al, 2007).

**3.1.2 Additional examples** 

helix-12.

conformation due to a clash with the ligand.

The binding of testosterone and dihydrotestosterone to AR demonstrate the existence of crucial receptor stabilizing interactions mediated by agonistic ligands. As we will discuss later, the loop between helix-11 and helix-12 is a key region for mediating partial agonism. As shown in figure 2, AR is stabilized by a ligand mediated hydrogen-bond network from Thr877 in helix-11 to the 17β-OH group in the endogenous steroidal agonists to Asn705 in helix-3 and finally to the backbone of Asp890 in the loop itself (Matias et al, 2000).

Hydroxyflutamide is the active metabolite of the androgen receptor antagonist flutamide. Its antagonism appears to be a result of its inability to complete the entire network of stabilizing hydrogen-bonds (Bohl et al, 2005) also shown in figure 2. The result is that Thr877 is left buried in a predominately hydrophobic pocket, destabilizing the receptor and shifting the agonist-antagonist equilibrium.

Fig. 2. Left shows the X-ray structure of DHT bound to AR including full hydrogen-bond network. Right shows a model of hydroxyflutamide bound to AR based on the X-ray structure of hydroxyflutamide bound to an AR-T877A mutant.
