Rescue of expression and signaling of human luteinizing hormone G protein-coupled receptor mutants with an allosterically binding small-molecule agonist.

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace (125)I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

Potent Dual Inhibitors of Steroid Sulfatase and 17ß-Hydroxysteroid Dehydrogenase Type 1 with a Suitable Pharmacokinetic Profile for In Vivo Proof-of-Principle Studies in an Endometriosis Mouse Model.

Treating estrogen-dependent diseases like endometriosis with drugs suppressing local estrogen activation may be superior to existing endocrine therapies. Steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) are key enzymes of local estrogen activation. We describe the rational design, synthesis, and biological profilation of furan-based compounds as a novel class of dual STS/17ß-HSD1 inhibitors (DSHIs). In T47D cells, compound 5 showed irreversible inhibition of STS and potent, reversible inhibition of 17ß-HSD1. It was selective over 17ß-HSD2 and displayed high metabolic stabilities in human and mouse liver S9 fractions. No effect on cell viability was detected up to 31 µM (HEK293) and 23 µM (HepG2), respectively, and there was no activation of the aryl hydrocarbon receptor (AhR) up to 3.16 µM. Single daily application to mice revealed steady-state plasma levels high enough to make this compound eligible for an in vivo proof-of-principle study in a mouse endometriosis model.

17ß-Hydroxysteroid Dehydrogenase Type 1 Inhibition: A Potential Treatment Option for Non-Small Cell Lung Cancer.

In the face of the clinical challenge posed by non-small cell lung cancer (NSCLC), the present need for new therapeutic approaches is genuine. Up to now, no proof existed that 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a viable target for treating this disease. Synthesis of a rationally designed library of 2,5-disubstituted furan derivatives followed by biological screening led to the discovery of 17ß-HSD1 inhibitor 1, capable of fully inhibiting human NSCLC Calu-1 cell proliferation. Its pharmacological profile renders it eligible for further in vivo studies. The very high selectivity of 1 over 17ß-HSD2 was investigated, revealing a rational approach for the design of selective inhibitors. 17ß-HSD1 and 1 hold promise in fighting NSCLC.

Oligoproline helices as structurally defined scaffolds for oligomeric G protein-coupled receptor ligands.

Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the monomeric counterparts.

Molecular mechanisms of posaconazole- and itraconazole-induced pseudohyperaldosteronism and assessment of other systemically used azole antifungals.

Recent reports described cases of severe hypertension and hypokalemia accompanied by low renin and aldosterone levels during antifungal therapy with posaconazole and itraconazole. These conditions represent characteristics of secondary endocrine hypertension caused by mineralocorticoid excess. Different mechanisms can cause mineralocorticoid excess, including inhibition of the adrenal steroidogenic enzymes CYP17A1 and CYP11B1, inhibition of the peripheral cortisol oxidizing enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) or direct activation of the mineralocorticoid receptor (MR). Compared to previous experiments revealing a threefold more potent inhibition of 11ß-HSD2 by itraconazole than with posaconazole, the current study found sevenfold stronger CYP11B1 inhibition by posaconazole over itraconazole. Both compounds most potently inhibited CYP11B2. The major pharmacologically active itraconazole metabolite hydroxyitraconazole (OHI) resembled the effects of itraconazole but was considerably less active. Molecular modeling calculations assessed the binding of posaconazole, itraconazole and OHI to 11ß-HSD2 and the relevant CYP enzymes, and predicted important interactions not formed by the other systemically used azole antifungals, thus providing an initial explanation for the observed inhibitory activities. Together with available clinical observations, the presented data suggest that itraconazole primarily causes pseudohyperaldosteronism through cortisol-induced MR activation due to 11ß-HSD2 inhibition, and posaconazole by CYP11B1 inhibition and accumulation of the mineralocorticoids 11-deoxycorticosterone and 11-deoxycortisol because of hypothalamus-pituitary-adrenal axis (HPA) feedback activation. Therapeutic drug monitoring and introduction of upper plasma target levels may help preventing the occurrence of drug-induced hypertension and hypokalemia. Furthermore, the systemically used azole antifungals voriconazole, isavuconazole and fluconazole did not affect any of the mineralocorticoid excess targets, offering alternative therapeutic options.

Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells.

Profiling of anabolic androgenic steroids and selective androgen receptor modulators for interference with adrenal steroidogenesis.

Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treatment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain unknown. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spectrometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone increased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell-free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3ß-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxymetholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, suggesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic information. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.

Effects of 17ß-HSD2 inhibition in bones on osteoporosis based on an animal rat model.

Hormone replacement therapy is a viable option to protect bone from postmenopausal osteoporosis. Systemically elevated estrogen levels, however, are disadvantageous because of the risk of harmful side effects in other organs. The rationale of the study presented here is to target a key enzyme in estradiol (E2) and testosterone (T) metabolism to increase E2 levels in an organ-specific manner, thereby avoiding the disadvantages of systemically increased E2 levels. The 17ß-hydroxysteroid dehydrogenase (17ß-HSD2), which is e.g. expressed in bone, catalyzes the oxidation of E2 and T into estrone (E1) and androstenedione. We postulate that inhibiting 17ß-HSD2 should lead to elevated E2 and T levels in organs expressing the enzyme. Therefore, we can use the benefits of E2 directly, or those of T following aromatization into E2, in the bone without affecting systemic levels. We tested for the first time, the novel and potent 17ß-HSD2 inhibitor, compound 24 (C24), to explore the therapeutic potential of a 17ß-HSD2 inhibition in an ovariectomy (ovx)-induced rat model of bone loss. We tested the inhibitor alone and, together with low dose estrogen supplementation to model estrogen levels in the postmenopausal situation. Female mature Wistar-Hannover rats were treated for 8 weeks with doses of 2, 10, 50 mg C24 per kg body weight per day alone or in the presence of estradiol benzoate (E2B) supplementation to alleviate ovx-induced bone loss. Ovx placebo and sham operated animals served as negative and positive controls. The experiment was evaluated regarding aspects of efficacy and safety: Bone was analyzed to evaluate bone protective effects, and uterus for potential, unwanted E2-mediated side effects. We observed a good bioavailability of C24 as very high plasma concentrations were measured, up to a group mean of 15,412 nM for the ovx C24-high group. Histomorphometrical analyses and in vivo &ex vivo µCT revealed significant bone protective effects for the lowest inhibitor concentration used. Irrespective of the plasma concentration, no proliferative effects in the uterus could be observed. These results support our approach of intracellular targeting key enzymes of E2 and T metabolism to increase E2 and T levels in an organ specific manner.

Development of potential preclinical candidates with promising in vitro ADME profile for the inhibition of type 1 and type 2 17ß-Hydroxysteroid dehydrogenases: Design, synthesis, and biological evaluation.

Estrogens are the major female sex steroid hormones, estradiol (E2) being the most potent form in humans. Disturbing the balance between E2 and its weakly active oxidized form estrone (E1) leads to diverse types of estrogen-dependent diseases such as endometriosis or osteoporosis. 17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the biosynthesis of E2 by reduction of E1 while the type 2 enzyme catalyzes the reverse reaction. Thus, 17ß-HSD1 and 17ß-HSD2 are attractive targets for treatment of estrogen-dependent diseases. Recently, we reported the first proof-of-principle study of a 17ß-HSD2 inhibitor in a bone fracture mouse model, using subcutaneous administration. In the present study, our aim was to improve the in vitro ADME profile of the most potent 17ß-HSD1 and 17ß-HSD2 inhibitors described so far. The optimized compounds show strong and selective inhibition of both the human enzymes and their murine orthologs. In addition, they display good metabolic stability in human liver microsomes (S9 fraction), low in vitro cytotoxicity as well as better aqueous solubility and physicochemical properties compared to the lead compounds. These achievements make the compounds eligible for testing in preclinical in vivo animal model studies on the effects of inhibition of 17ß-HSD1 and 17ß-HSD2.

Targeted Endocrine Therapy: Design, Synthesis, and Proof-of-Principle of 17ß-Hydroxysteroid Dehydrogenase Type 2 Inhibitors in Bone Fracture Healing.

Current therapies of steroid hormone-dependent diseases predominantly alter steroid hormone concentrations (or their actions) in plasma, in target and nontarget tissues alike, rather than in target organs only. Targeted therapy through the inhibition of steroidogenic enzymes may pose an attractive alternative with much less side effects. Here, we describe the design of a nanomolar potent 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) inhibitor (compound 15) and successful targeted intracrine therapy in a mouse bone fracture model. Blockade of 17ß-HSD2 in bone is thought to increase intracellular estradiol (E2) and testosterone (T), which thereby inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts, respectively. Administration of compound 15 in the mouse fracture model strongly increases the mechanical stability of the healing fractured bone because of a larger periosteal callus with newly formed bone without changing the plasma E2 and T concentrations. Steroidogenic 17ß-HSD2 inhibition thus enables targeted intracrine therapy.

Design, Synthesis, and Biological Characterization of Orally Active 17ß-Hydroxysteroid Dehydrogenase Type 2 Inhibitors Targeting the Prevention of Osteoporosis.

Osteoporosis is predominantly treated with drugs that inhibit further bone resorption due to estrogen deficiency. Yet, osteoporosis drugs that not only inhibit bone resorption but also stimulate bone formation, such as potentially inhibitors of 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2), may be more efficacious in the treatment of osteoporosis. Blockade of 17ß-HSD2 is thought to increase intracellular estradiol and testosterone in bone, thereby inhibiting bone resorption by osteoclasts and stimulating bone formation by osteoblasts, respectively. We here describe the design, synthesis, and biological characterization of a novel bicyclic-substituted hydroxyphenylmethanone 17ß-HSD2 inhibitor (compound 24). Compound 24 is a nanomolar potent inhibitor of human 17ß-HSD2 (IC50 of 6.1 nM) and rodent 17ß-HSD2 with low in vitro cellular toxicity, devoid of detectable estrogen receptor α affinity, displays high aqueous solubility and in vitro metabolic stability, and has an excellent oral pharmacokinetic profile for testing in a rat osteoporosis model. Administration of 24 in a rat osteoporosis model demonstrates its bone-sparing efficacy.

beta-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1.

beta-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of beta-galactosidase to measure receptor-beta-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter beta-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed beta-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter cell line at the level of beta-arrestin recruitment, G(i/o) protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of beta-arrestin recruitment was largely unaffected upon blockade of G(i/o) protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of beta-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully G(i/o) protein regulated. The data indicate that the beta-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of "biased ligands" (i.e., compounds that differ in G-protein coupling and beta-arrestin-mediated cellular effects).

G protein-independent cell-based assays for drug discovery on seven-transmembrane receptors.

Conventional cell-based assays for seven-transmembrane receptors, also known as G protein-coupled receptors, rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins. New assay methods have become available that are not based on G protein activation, but that apply the molecular mechanism underlying the attenuation of G protein signaling mediated by beta-arrestin. beta-arrestin is a cytoplasmic protein that targets receptors to clathrin-coated endocytotic vesicles for degradation or recycling. This process has been visualized and quantified in high-content imaging assays using receptor- or beta-arrestin-chimeras with green fluorescent protein. Other assay methods use bioluminescence resonance energy transfer, enzyme fragment complementation, or a protease-activated transcriptional reporter gene, to measure receptor-beta-arrestin proximity. beta-arrestin recruitment assays have been applied successfully for receptors coupling to Galpha(q), Galpha(s) and Galpha(i) proteins, thus providing a generic assay platform for drug discovery on G protein-coupled receptors. The best understood signal transduction pathway elicited by the seven-transmembrane Frizzled receptors does not involve G proteins. The activation of Frizzleds by their cognate ligands of the Wnt family recruits the phosphoprotein dishevelled. Dishevelled regulates a protein complex involved in the destruction of beta-catenin. Activation of Frizzled blocks degradation of beta-catenin, which translocates to the nucleus to activate transcription of Wnt-responsive genes. The cytoplasm-to-nuclear translocation of beta-catenin forms the basis of several high-content assays to measure Wnt/Frizzled signal transduction. Interestingly, Frizzled receptors have recently been shown to internalize and to recruit beta-arrestin. This suggests that beta-arrestin recruitment assays may be applied for drug discovery on seven-transmembrane receptors beyond G protein-coupled receptors.

A signaling-selective, nanomolar potent allosteric low molecular weight agonist for the human luteinizing hormone receptor.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) activate the LH receptor/cyclic AMP (cAMP) signaling pathway to induce ovulation. As an alternative to parenterally administered hCG to treat anovulatory infertility, orally active low molecular weight (LMW) LHR agonists have been developed at Organon. In this paper, we present the mechanism of action of a prototypic, nanomolar potent and almost full LHR agonist, Org 43553. Org 43553 interacts with the endodomain of the LHR, whereas LH acts via the N-terminal exodomain. LH stimulates the cAMP pathway with an EC50 of 35 pM, but this stimulation is not antagonized by simultaneous incubation with Org 43553. At nanomolar concentrations, LH also stimulates phospholipase C (PLC), but Org 43553 is hardly able to do so. In contrast, Org 43553 inhibits LH-induced PLC (IC50 approximately 10 nM). While Org 43553 stimulates dissociation of [125I]hCG from the LHR and reduces [125I]hCG binding, LH reduces specific [3H]Org 43553 binding. We conclude that Org 43553 is a signaling-selective, allosteric LHR agonist. We hypothesize that Org 43553 and LH induce a similar LHR conformation necessary for activating adenylyl cyclase, which initiates most, if not all, physiological responses of LH.

Accelerated skin wound healing by selective 11ß-Hydroxylase (CYP11B1) inhibitors.

Previous studies have shown that inhibition of cortisol biosynthesis in skin leads to accelerated wound healing. Here, pyridylmethyl pyridine type 11ß-hydroxylase (CYP11B1) inhibitors were optimized for topical application to avoid systemic side effects. The resulting very potent, non-toxic CYP11B1 inhibitor 14 (IC50 = 0.8 nM) exhibited good selectivity over 11ß-HSD1, CYP17A1 and CYP19A1. The compound showed high stability toward human plasma (t1/2= > 150 min, as a substitute for wound fluid) and low stability toward HLS9 (t1/2 = 19 min) for rapid metabolic clearance after absorption. Compound 14 was able to accelerate wound healing in human skin.

Highly Potent 17ß-HSD2 Inhibitors with a Promising Pharmacokinetic Profile for Targeted Osteoporosis Therapy.

Intracellular elevation of E2 levels in bone by inhibition of 17ß hydroxysteroid dehydrogenase type 2 (17ß-HSD2) without affecting systemic E2 levels is an attractive approach for a targeted therapy against osteoporosis, a disease which is characterized by loss of bone mineral density. Previously identified inhibitor A shows high potency on human and mouse 17ß-HSD2, but poor pharmacokinetic properties when applied perorally in mice. A combinatorial chemistry approach was utilized to synthesize truncated derivatives of A, leading to highly potent compounds with activities in the low nanomolar to picomolar range. Compound 33, comparable to A in terms of inhibitor potency against both human and mouse enzymes, displays high in vitro metabolic stability in human and mouse liver S9 fraction as well as low toxicity and moderate hepatic CYP inhibition. Thus, compound 33 showed a highly improved peroral pharmacokinetic profile in comparison to A, making 33 a promising candidate for further development.

Cryopreserved cells facilitate cell-based drug discovery.

Advances in detection technologies have enabled an increased use of cell-based functional assays in early drug discovery, in particular for G protein-coupled receptors. Screening assays that use live cells are less prone to generate false positives than assays using lysed cell samples. The use of cryopreserved cells instead of cells that are continuously maintained in culture decreases day-to-day variation, removes passage effects and improves the consistency of cell-based assay results. Cryopreservation techniques uncouple cell culturing from drug-screening activities and allow the use of cells as reagents, just like enzymes in biochemical assays.

Regulation and functional roles of sphingosine kinases.

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

Lead Optimization Generates CYP11B1 Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmacological Profile for the Treatment of Cushing's Disease.

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11ß-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC50= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC50 = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC50 = 2 µM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a selective 17ß-HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study.

Current endocrine therapeutics for the estrogen-dependent disease endometriosis often lead to considerable side-effects as they act by reducing estrogen action systemically. A more recent approach takes advantage of the fact that the weak estrogen estrone (E1) which is abundant in the plasma, is activated in the target cell to the highly estrogenic estradiol (E2) by 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). 17ß-HSD1 is overexpressed in endometriosis and thus a promising target for the treatment of this disease, with the prospect of less target-associated side-effects. Potent inhibitors from the class of bicyclic substituted hydroxyphenylmethanones with sulfonamide moiety recently described by us suffered from high molecular weight and low selectivity over 17ßHSD2, the physiological adversary of 17ß-HSD1. We describe the structural optimizations leading to the discovery of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-yl)(2,6-difluoro-3-hydroxyphenyl)methanone 20, which displayed a sub-nanomolar IC50 towards 17ß-HSD1 as well as high selectivity over the type 2 enzyme, the estrogen receptors α and ß and a range of hepatic CYP enzymes. The compound did neither show cellular toxicity, nor PXR activation nor mutagenicity in the AMES II assay. Additional favourable pharmacokinetic properties (rat) make 20 a suitable candidate for proof-of-principle studies using xenotransplanted immunodeficient rats.