Utilization of peptide phage display to investigate hotspots on IL-17A and what it means for drug discovery.

To date, IL-17A antibodies remain the only therapeutic approach to correct the abnormal activation of the IL-17A/IL-17R signaling complex. Why is it that despite the remarkable success of IL-17 antibodies, there is no small molecule antagonist of IL-17A in the clinic? Here we offer a unique approach to address this question. In order to understand the interaction of IL-17A with its receptor, we combined peptide discovery using phage display with HDX, crystallography, and functional assays to map and characterize hot regions that contribute to most of the energetics of the IL-17A/IL-17R interaction. These functional maps are proposed to serve as a guide to aid in the development of small molecules that bind to IL-17A and block its interaction with IL-17RA.

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.

Structural features underlying raloxifene's biophysical interaction with bone matrix.

Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure-activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.

Discovery of synthetic Leishmania inhibitors by screening of a 2-arylbenzothiophene library.

Tamoxifen has been shown to be active in vitro against Leishmania and effective in the treatment for leishmaniasis in murine models. Through the screening of a compound library of estrogen receptor modulator analogs, we identified the major characteristics required for antileishmanial activity. To overcome the difficulties presented by tamoxifen's propensity for E/Z isomerization, we used the 2-arylbenzothiophene compound BTP as a more stable alternative. Directed screening of a small compound library based on BTP led to active compounds against Leishmania. Subsequent structure-activity data for the synthetic 2-arylbenzothiophenes evaluated in this study indicate that optimal antileishmanial potency is dependent on the presence of two basic side chains. In addition, the primary structural features required for estrogen receptor binding, the phenols, are not required for inhibiting parasitic growth. Significantly, the most active antileishmanial benzothiophenes lack the pharmacophore for estrogen receptor activity and therefore address potential concerns about the undesirable effects of using selective estrogen receptor modulators in women and children with leishmaniasis. Three compounds selected from the screening have shown consistent activity against all species and stages of Leishmania in vitro although improvements in selectivity are needed. These compounds represent viable starting points for further optimization as antileishmanial agents.

Hydrophobic Interactions Improve Selectivity to ERα for Ben-zothiophene SERMs.

The discovery, pharmacology, and biophysical characterization of an ERα selective benzothiophene (BTPα) is described. BTPα (4) is a high affinity ligand with 140-fold greater selectivity for ERα (K(i)=0.25 nM) over ERbeta (K(i)=35 nM). In rodent models of estrogen action, BTPα blocks the effects of estrogen in the uterus but mimics the effects estrogen on bone. The basis of ERα selectivity for BTPα was evaluated by using protein crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry. HDX data supports that the n-butyl chain of BTPα stabilizes helix 7 in ERα relative to that of ERß which we propose leads to an enhancement of affinity to the alpha receptor sub-type.

Novel 3-aryl indoles as progesterone receptor antagonists for uterine fibroids.

We report the synthesis and characterization of novel 3-aryl indoles as potent and efficacious progesterone receptor (PR) antagonists with potential for the treatment of uterine fibroids. These compounds demonstrated excellent selectivity over other steroid nuclear hormone receptors such as the mineralocorticoid receptor (MR). They were prepared from 2-bromo-6-nitro indole in four to six steps using a Suzuki cross-coupling as the key step. Compound 8f was orally active in the complement 3 model of progesterone antagonism in the rat uterus and demonstrated partial antagonism in the McPhail model of progesterone activity.

Unique ligand binding patterns between estrogen receptor alpha and beta revealed by hydrogen-deuterium exchange.

Here we present the use of hydrogen-deuterium exchange (HDX) mass spectrometry in analyzing the estrogen receptor beta ligand binding domain (ERbeta LBD) in the absence and presence of a variety of chemical compounds with different binding modes and pharmacological properties. Previously, we reported the use of HDX as a method for predicting the tissue selectivity of ERalpha ligands. HDX profiles of ERalpha LBD in complex with ligand could differentiate compounds of the same chemotype. In contrast, similar analysis of ERbeta LBD showed correlation to the compound chemical structures but little correlation with compound tissue selectivity. The different HDX patterns observed for ERbeta LBD when compared to those for ERalpha LBD bound to the same chemical compounds serve as an indication that ERbeta LBD undergoes a different structural response to the same ligand when compared to ERalpha LBD. The conformational dynamics revealed by HDX for ERbeta LBD together with those for ERalpha LBD shed light on ER ligand interactions and offer new structural insights. The compound-specific perturbations in HDX kinetics observed for each of the two isoforms should aid the development of subtype-selective ER ligands.

Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method.

Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor-ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 5: Combined A- and C-ring structure-activity relationship studies.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe the synthesis of a late stage intermediate that allowed us to combine A-ring and C-ring modifications and carry out simultaneous SAR studies at both positions. Modification of both positions proved additive, maintaining affinity and improving ERbeta selectivity up to 83-fold. An X-ray cocrystal structure confirms the previously observed binding mode in ERbeta.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 4: functionalization of the benzopyran A-ring.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER receptor subtypes alpha and beta in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERalpha, thus improving ERbeta subtype selectivity. X-ray cocrystal structures with ERalpha and ERbeta are supportive of this approach to improve selectivity in this structural class.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 3: synthesis of cyclopentanone and cyclohexanone intermediates for C-ring modification.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe the syntheses of cyclopentanone and cyclohexanone intermediates for SAR studies of the C-ring on the benzopyran scaffold. Modification of the C-ring disrupts binding to ERalpha, thus improving ERbeta selectivity up to 100-fold. X-ray cocrystal structures confirm previously observed binding modes.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 2: structure-activity relationship studies on the benzopyran scaffold.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe structure-activity relationship studies that led to the discovery of bezopyran 5b. X-ray crystal structures of 5b and a non-selective analog 5c in ERalpha help explain the observed selectivity of the benzopyran platform.

Structure-activity relationships of SERMs optimized for uterine antagonism and ovarian safety.

Structure-activity relationship studies are described, which led to the discovery of novel selective estrogen receptor modulators (SERMs) for the potential treatment of uterine fibroids. The SAR studies focused on limiting brain exposure and were guided by computational properties. Compounds with limited impact on the HPO axis were selected using serum estrogen levels as a biomarker for ovarian stimulation.

Benzopyrans are selective estrogen receptor beta agonists with novel activity in models of benign prostatic hyperplasia.

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.

A selective estrogen receptor modulator for the treatment of hot flushes.

A selective estrogen receptor modulator (SERM) for the potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol, LSN2120310, potently binds ERalpha and ERbeta and is an antagonist in MCF-7 breast adenocarcinoma and Ishikawa uterine cancer cell lines. The compound is a potent estrogen antagonist in the rat uterus. In ovariectomized rats, the compound lowers cholesterol, maintains bone mineral density, and is efficacious in a morphine dependent rat model of hot flush efficacy.

A selective estrogen receptor modulator designed for the treatment of uterine leiomyoma with unique tissue specificity for uterus and ovaries in rats.

The design of a novel selective estrogen receptor modulator (SERM) for the potential treatment of uterine leiomyoma is described. 16 (LY2066948-HCl) binds with high affinity to estrogen receptors alpha and beta (ERalpha and ERbeta, respectively) and is a potent uterine antagonist with minimal effects on the ovaries as determined by serum biomarkers and histologic evaluation.

A new selective estrogen receptor modulator with potent uterine antagonist activity, agonist activity in bone, and minimal ovarian stimulation.

The use of selective estrogen receptor modulators for the treatment of estrogen-dependent diseases in premenopausal women has been hindered by undesirable ovarian stimulation and associated risks of ovarian cysts. We have identified a selective estrogen receptor modulator compound (LY2066948) that is a strong estrogen antagonist in the uterus yet has minimal effects on the ovaries of rats. LY2066948 binds with high affinity to both estrogen receptors and has potent estrogen antagonist activity in human uterine and breast cancer cells. Oral administration of LY2066948 to immature rats blocked uterine weight gain induced by ethynyl estradiol with an ED50 of 0.07 mg/kg. Studies in mature rats demonstrated that LY2066948 decreases uterine weight by 51% after 35 d treatment, confirming potent uterine antagonist activity over several estrous cycles. This strong uterine response contrasted with the minimal effects on the ovaries: serum estradiol levels remained within the normal range, whereas histologic evaluation showed granulosa cell hyperplasia in few of the rats. Bone studies demonstrated that LY2066948 prevented ovariectomy-induced bone loss and treatment of ovary-intact rats caused no bone loss, confirming estrogen receptor agonist skeletal effects. Collectively, these data show that LY2066948 exhibits a tissue-specific profile consistent with strong antagonist activity in the uterus, agonist activity in bone, and minimal effects in the ovaries.

Synthesis and biological evaluation of an orally active ghrelin agonist that stimulates food consumption and adiposity in rats.

2-(2-Amino-2-methyl-propionylamino)-5-phenyl-pentanoic acid [1-[1-(4-methoxy-phenyl)-1-methyl-2-oxo-2-pyrrolidin-1-yl-ethyl]-1H-imidazol-4-yl]-amide (LY444711, 6) is an orally active ghrelin agonist that binds with high affinity to and is a potent activator of the growth hormone secretagogue receptor 1a (GHS-R1a) receptor. In rat models of feeding behavior and pharmacology, 6 creates a positive energy balance and induces adiposity by stimulating food consumption and sparing fat utilization. As an orally active ghrelin agonist, 6 represents a new pharmacological tool to investigate the orexigenic role of ghrelin in regulating energy homeostasis.

Estrogen receptor modulators: relationships of ligand structure, receptor affinity and functional activity.

The estrogen receptor is a regulator of a wide range of physiological functions, including the female reproductive system, in addition to bone, cardiovascular and CNS function. ER ligands have been approved for the treatment of menopausal symptoms, breast cancer and osteoporosis, however the search continues for new modulators of ER function with improved properties. Progress in medicinal chemistry programs has resulted in the identification of structurally diverse molecules with unique biological properties. Recent advances in the design and synthesis of these non-steroidal and steroidal estrogen receptor ligands is reviewed. The relationship between the structural features of the ligand and receptor function is also discussed.

Tetrahydroquinoline-based selective estrogen receptor modulators (SERMs).

A new series of estrogen receptor ligands based on a 6-hydroxy-tetrahydroquinoline scaffold is described, in addition to their binding affinity and functional activity in MCF-7 cells. Several 1,2-disubstituted tetrahydroquinolines bearing a basic side chain were shown to be high affinity ligands and antagonists in the MCF-7 proliferation assay. Compounds lacking the basic side chain were agonists in the MCF-7 assay.