**4. Discovery of small molecule modulators of PR function**

The identification of drug-like, potent and selective PR antagonists has been challenging. As well as being highly lipophilic, the ligand binding sites between homologous NHRs are highly conserved and PR has the highest sequence homology with GR, AR, MR and ER (Figure 1). Early classes of anti-progestagens were poorly selective, yet some, such as gestrinone, still found clinical utility in the treatment of endometriosis (Cornillie et al., 1986; Coutinho, 1982). Furthermore, the ligand binding domains of PR-A and PR-B are identical and yet several in vitro and in vivo lines of evidence suggest that the effects of progesterone on transcriptional activation and repression by PR-A and PR-B are different (Conneely et al., 2001; Tung et al., 2006). To date, however, there are no agonist or antagonist agents that have been characterised with selectivity for PR-A over PR-B or vice versa.

The development of selective and safe steroidal PRAs has been challenging, both due to reported hepatoxicity as well as potential dose-limiting anti-glucocorticoid effects, due to lack of selectivity (Robertson et al., 1999). More recently, an additional concern has emerged following histological evaluations of subjects dosed for more than 3 months on steroidal PRAs. The endometrium of these individuals undergoes a characteristic cystic histological change which may be difficult to distinguish from endometrial hyperplasia without specialist evaluation (Ioffe et al., 2009; Mutter et al., 2008; Williams et al., 2007).

Mifepristone/RU-486, the founding member of the steroidal class of PRAs, was originally produced by Roussel-Uclaf and licensed for use for medical abortion and as an emergency contraceptive. The in vivo pharmacokinetic/pharmacodynamic profile of RU-486 is challenging to model as RU-486 generates a large number of pharmacologically active metabolites (Heikinheimo et al., 1987). Since its identification, >100 related analogues, principally by modifying the C-11 and/or C-17 positions of the steroid ring, have been synthesized and have shown all degrees of anti-progestagenic activity. The pharmacological profile of these examples range from pure antagonists, such as RU-486 to those with mixed antagonist/agonist activity, such as J-867 (Table 1).

Progesterone Resistance and Targeting

N N O <sup>N</sup> <sup>S</sup>

> F N H O O

N

O

F N H O NC PR binding IC50=5

> <sup>H</sup> OH H H

<sup>O</sup> <sup>O</sup>

μM]

nM

CN PR binding Kd=4.4

μM

nM

nM

nM

nM

nM

nM

nM

PF-02413873

PRA-910

WAY-255348

Steroidal PRAs RU-486 / mifepristone <sup>N</sup>

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 163

Dose dependent inhibition in functionalis thickness & BrdU incorporation of follicular phase macaque endometrium; maximum effects at 10 mg/kg (p.o,

Dose dependent inhibition of the P4 induced rat decidual response (mean ED50 =0.3 mg/kg) with no evidence of agonism at 10 mg/kg. 5 mg/kg reduced BrdU incorporation in the E2/OVX macaque, but no effect on endometrial thickness c.f. control

Inhibition of P4-induced rat decidual response (ED50 = 0.3 mg/kg). Dose dependent inhibition of ovulation in the macaque. All the animals treated at 10 mg/kg had thinatrophied endometria

Dose dependent induction of menses in intact and E2/P4 artificially cycled macaque. Inhibition of ovulation and endometrial proliferation. ~75% reduction in peritoneal disease volume in a surgical model of endometriosis in the

macaque

(Howe et al., 2011)

(Zhang et al., 2002; 2007)

(Fensome et al., 2008)

(de Giorgio-Miller et al., 2008; Grow et al., 1996; Slayden & Brenner, 1994; Slayden et al., 2001; Wolf et al., 1989)

*b.i.d*)

PR binding Ki=2.6 nM; functional Ki=9.7 nM; pKB= 8.0 [Agonist format: ~25% activation at 10

AR binding IC50=2100 nM; functional Ki=1130 nM

MR functional Ki=307

nM; functional 14 nM [Agonist format: ~60- 70% activation at 0.1

AR binding IC50=1292

GR binding IC50=1756

MR binding IC50=2369

nM; functional IC50=5

ER binding IC50 >10000 nM

AR functional IC50=196 nM MR functional IC50=3700 nM >No significant activity at GR, ER

PR binding Ki=0.5 nM; T47D IC50=0.2 nM AR functional C50=20

GR binding Ki=1.4 nM; Functional IC50=3

MR functional IC50=3

Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

GR binding Ki=410 nM; functional Ki=2710 nM

Fig. 1. (a) X-ray structure of progesterone (cyan) bound to the PR ligand binding pocket (pink) (Williams & Sigler, 1998) and overlaid with the x-ray co-ordinates of GR, MR and AR (in green) to illustrate the high degree of structural similarity between homologous NHRs (b) Phylogentic tree illustrating the sequence similarity of homologous NHRs with PR.

More recently, several alternative non-steroidal chemical PRA scaffolds have been published. These might have advantages over the steroidal templates due to simpler synthetic route, and potential for greater selectivity and metabolic stability compared with steroidal templates (Dack et al., 2010; Fensome et al., 2008; Terefenko et al., 2005; Zhang et al., 2002),. With a few exceptions, these classes of agents tend to mimic the steroid A ring ketone with a cyanoaryl group isostere, as is also seen with tanaproget, the non-steroidal progestogen (Fensome et al., 2005).

Steroidal PRAs have been characterised as facilitating PR dimerisation and nuclear translocation, but induce a conformation of the DNA-bound complex which recruits corepressors to directly shutdown transcriptional PRE activity and other transcriptional promoters by trans-repression. The pharmacological profile of the non-steroidal compounds appears to be subtly different from steroidal examples (Howe et al., 2011; Zhang et al., 2007). In vitro pharmacological profiling of PRA-910 and PF-02413873 has indicated that at low concentrations the compounds inhibit the expression of progesterone-reporter genes, but at high concentrations, they induce agonism. For PF-02413873, the inhibition of progesteronereporter gene expression appears to be due to blocking PR nuclear translocation and then at high concentrations PF-02413873 facilitates it, recruits co-activators and induces gene transcription.


Table 1. Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

Fig. 1. (a) X-ray structure of progesterone (cyan) bound to the PR ligand binding pocket (pink) (Williams & Sigler, 1998) and overlaid with the x-ray co-ordinates of GR, MR and AR (in green) to illustrate the high degree of structural similarity between homologous NHRs (b) Phylogentic tree illustrating the sequence similarity of homologous NHRs with PR.

More recently, several alternative non-steroidal chemical PRA scaffolds have been published. These might have advantages over the steroidal templates due to simpler synthetic route, and potential for greater selectivity and metabolic stability compared with steroidal templates (Dack et al., 2010; Fensome et al., 2008; Terefenko et al., 2005; Zhang et al., 2002),. With a few exceptions, these classes of agents tend to mimic the steroid A ring ketone with a cyanoaryl group isostere, as is also seen with tanaproget, the non-steroidal

Steroidal PRAs have been characterised as facilitating PR dimerisation and nuclear translocation, but induce a conformation of the DNA-bound complex which recruits corepressors to directly shutdown transcriptional PRE activity and other transcriptional promoters by trans-repression. The pharmacological profile of the non-steroidal compounds appears to be subtly different from steroidal examples (Howe et al., 2011; Zhang et al., 2007). In vitro pharmacological profiling of PRA-910 and PF-02413873 has indicated that at low concentrations the compounds inhibit the expression of progesterone-reporter genes, but at high concentrations, they induce agonism. For PF-02413873, the inhibition of progesteronereporter gene expression appears to be due to blocking PR nuclear translocation and then at high concentrations PF-02413873 facilitates it, recruits co-activators and induces gene

Compound Structure Pharmacology data In vivo observations Reference

Dose dependent inhibition of arborisation of the immature rabbit and luteal phase endometrium of the intact macaque

PR binding Ki=76 nM; functional IC50=40.3

Table 1. Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486

>3000x functional selectivity over GR,

was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

**a b**

progestogen (Fensome et al., 2005).

transcription.

Non-steroidal PRAs PF-02367982

N

N N O

HN O

nM

AR, MR

**GR AR MR PR** 

**ERR2B2 ERR2B1** 

**ER**β

**ER**α

**ERR1**

**ERR3** 

(de Giorgio-Miller et al., 2008)


Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

Progesterone Resistance and Targeting

H H H

ZK-137316 PR relative binding

OH

O

N

ZK-98299 / Onapristone

**5.1 Rodent** 

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 165

Inhibition of arborisation of the immature rabbit. Reduction in cell proliferation of cells in ectopic lesion in a rat endometriosis model. Inhibition of ovulation and endometrial growth (Elger et al., 2000; Gopalkrishn an et al., 2003; Ishwad et al., 1993; Stoeckeman n et al., 1995)

(Borman et al., 2003; Slayden et al., 1998; 2001; Slayden & Brenner, 2003; Stoeckeman n et al., 1995; Zelinski-Wooten et al., 1998)

in monkeys

Inhibition of arborisation of the immature rabbit. Reduction in cell proliferation of cells in ectopic lesion in a rat endometriosis model. Inhibition of ovulation and endometrial growth

in macaques.

OH PR relative binding affinity equivalent to

affinity equivalent to

Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

The study of the effects of PRAs in pre-clinical models has shed light on the site of and mechanism of action of PRAs in normal reproductive physiology and disease. The classical models for quantifying PRA activity are the modified assay in juvenile rabbits according to McPhail (McPhail, 1933), the induction of luteolysis in the guinea pig (Elger et al., 2000) and the inhibition of decidualisation in the rat. For the purposes of this review, I have focussed on the pre-clinical models which have been used to support a role for PR in the pathogenesis and treatment of endometriosis, principally focussing on studies in the rodent and macaque. Many data have been acceded from studies in the normal animal, but several experimental models of endometriosis have been also developed in normal as well as immune compromised rodents and non-human primates (D'Hooghe et al., 2009; Grummer, 2006; Laschke & Menger, 2007). In these models, cystic ectopic endometrial lesions develop following the transplantation of syngeneic or human uterine endometrial tissue under the control of ovarian estradiol. Measuring temporal changes in the size of these lesions and their proliferative capacity as well other aspects of the disease presentation is a powerful

Mouse knockout studies have elegantly described differences in the function of PR-A and PR-B. Both PR null mutation (PRKO) and selective disruption of the PR-A isoform (PRAKO)

RU-486

RU-486

**5. Pre-clinical effects of PR antagonists in pre-clinical models** 

pre-clinical yardstick for testing the efficacy of experimental drugs.


Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM


Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM
