Adriana S. Veleiro

Antiproliferative activity of withanolides against human breast cancer cell lines.

The in vitro antiproliferative activity of a series of 22 naturally occurring withanolides was examined against the T-47D, MCF7, MCF7/BUS, MDA-MB-231, and SK-Br-3 human solid tumor breast cancer cell lines. The most active compound showed GI(50) values in the range 0.16-0.71 muM. The aromatic withanolide 19 exhibited specific activity for the estrogen-receptor-positive cell lines (T-47D, MCF7, and MCF7/BUS). Overall, the results demonstrated the relevance of the substitution pattern on the A and B rings on the resultant antiproliferative activity.

Structure-Activity Relationships of Neuroactive Steroids Acting on the GABAA Receptor

The term neuroactive steroid (NAS) refers to steroids which, independent of their origin, are capable of modifying neural activities. These steroids positively or negatively modulate the function of members of the ligand-gated ion channel superfamily. Those with positive allosteric actions on the gamma-amino butyric acid type A receptor (GABAA receptor) have been shown to be potent anticonvulsants, anxiolytics, and antistress agents and to possess sedative, hypnotic, and anesthetic activities. New types of neuroactive steroids have been widely sought and structural modifications of the naturally occurring metabolites allopregnanolone, pregnanolone and allotetrahydrodeoxycorticosterone, have been examined in the light of the vast family of GABA receptor subtypes within the brain. Here we review the structure-activity relationship (SAR) of neuroactive steroid analogues obtained by modification of the steroid nucleus, including substitutions at the A, B, C, and D rings and the side chain, with emphasis on the different pharmacophores proposed.

A ring-D aromatic withanolide from Salpichroa origanifolia

Abstract From Salpichroa origanifolia plants, a new withanolide (20 S ,22 R ,24 S ,25 S ,26 R )-5,6α: 22,26: 24,25-triepoxy-26-hydroxy-17(13 → 18) abeo -5α-ergosta-2,13,15,17-tetraen-1-one was isolated and characterized by spectroscopic and chemical methods.

Insights on Glucocorticoid Receptor Activity Modulation through the Binding of Rigid Steroids

Background The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptors ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior.

Chemistry and bioactivity of withanolides from south american Solanaceae

Since the isolation of withaferin A in 1965 over 300 withanolides have been described, largely from genera belonging to the Solanaceae. Although until the mid eighties most of the withanolides appeared to adhere to the basic structure of withaferin A, nowadays a considerable number of withanolides and withanolide related compounds are known, which present modified skeletons, aromatic rings, additional rings, etc., posing challenging problems of structure elucidation. Many of these structures coexist in the plants with “normal” withanolides and thus allow us to infer the biogenetic relationships among them and the transformations they suffer in the plant. As a further bonus, several species show marked seasonal and geographical variations in the amount and type of withanolides present, thus adding to the structural diversity. The latter is particularly noteworthy in (but not restricted to) species of the Jaborosa and Salpichroa genera. In recent years these genera, both native to South America, have rendered several novel withanolide types. Exodeconus, Dunalia, Deprea and Vassobia are other southamerican genera where unusual structures have also been found. Recently, some of the withanolides isolated from these plants have shown interesting biological activities as cancer chemopreventive agents (inductors of quinone reductase), as feeding deterrants for several insects, and displaying selective phytotoxicity towards monocotiledoneous and dicotiledoneous species. Trypanocidal and leishmanicidal activities have also been reported.

Structure of the Glucocorticoid Receptor, a Flexible Protein That Can Adapt to Different Ligands

Glucocorticoids (GCs) are a class of endogenous steroid hormones that regulate a variety of cell-, tissue-, and organ-specific biological functions including metabolism, growth, development behavior, and apoptosis. Clinically, GCs represent one of the most commonly prescribed drugs worldwide for the treatment of asthma, dermatitis, rheumatoid arthritis, and autoimmune diseases, due to their potent immunosuppressive and anti-inflammatory activities. They are also used for the treatment of leukemias, lymphomas, and myelomas because of their role in the induction of apoptosis. Despite the great benefits of GCs, their curative use is often limited by severe side effects such as osteoporosis, diabetes, hypertension, and obesity. The development of novel steroids that can dissociate the therapeutic effects from the undesired adverse effects is therefore highly desirable but such development will require a profound understanding of how GCs act at the molecular level. GCs mediate their effects by binding to the intracellular glucocorticoid receptor (GR), which functions as a ligand-activated nuclear transcriptional regulator. The GR belongs to the nuclear receptor (NR) superfamily, which includes receptors for the mineralocorticoids (MR), estrogens (ER), progestins (PR), and androgens (AR), as well as receptors for peroxisome proliferators (PPARs), vitamin D (VDR), and thyroid hormone (TR). Phylogenetic analysis shows that GR, MR, PR, and AR form a subfamily. As the structures of GR, PR, and AR are closely related, there is considerable overlap in ligand specificity and action. The GR and the MR descend from duplication of a single ancient gene, the ancestral corticoid receptor (AncCR), that can be traced back 450 million years in the vertebrate lineage. The GR is activated by the adrenal steroid cortisol and, even though the MR is also activated by cortisol, the latter receptor is considerably more sensitive to the endogenous metabolites aldosterone and deoxycorticosterone (DOC; Figure 1). Using X-ray crystallography, Thornton and colleagues determined the structures of the resurrected AncCR ligand binding domain (LBD) in complex with aldosterone, DOC, and cortisol, and identified the specific set of historical mutations that show the evolution of the hormone specificity of the GR from an MR-like ancestor. Although GR was the first steroid receptor to be cloned, the three-dimensional structure of the GR LBD proved elusive due to the difficulty in obtaining a purified receptor. After several crystal structures of other NR LBDs had been described, including those of the PR, AR, and ER, the first crystal structure of the GR LBD was reported by Bledsoe et al. in 2002 as a complex with the agonist dexamethasone (1; Figure 2) and a co-activator motif derived from the cofactor TIF2 (transcriptional intermediary factor 2). A few years later the crystal structure of the MR was reported completing the set of steroid hormone receptors. Several recent reviews have covered different aspects of GR function and structure and the mechanism of action of GC; herein we summarize recent findings on the GR LBD structure with emphasis on receptor– ligand and receptor–cofactor interactions.

Synthesis and GABAA receptor activity of a 6,19-Oxido analogue of pregnanolone

3 alpha-Hydroxy-6,19-oxidopregn-4-ene-20-one (4) was prepared in seven steps from pregnanolone acetate. At 0.1 microM concentration 4 significantly increased GABA induced (36)Cl(-) influx in hamster cerebral cortex synaptoneurosomes while at 20 mg/kg it decreased the percentage of hamsters showing seizures induced by 3-mercaptopropionic acid.

New lead compounds in the search for pure antiglucocorticoids and the dissociation of antiglucocorticoid effects.

Antiglucocorticoids that act as antagonists at the glucocorticoid receptor (GR) level may be used to block or modulate the undesirable effects of glucocorticoid excess (from endogenous or exogenous origin). RU486 developed in the early 80s, is an antiglucocorticoid but also a potent antiprogestin and abortifacient, nevertheless it still remains as the only GR antagonist drug in the market. Further on, in view of the variety of physiological processes in which glucocorticoids are involved, selective antiglucocorticoids that can block only some of these processes (eventually with tissue specificity) would be highly desirable. The bridged pregnane 21-hydroxy-6,19-epoxyprogesterone, was developed as an alternative lead being an antagonist of the GR with no affinity for mineralocorticoid and progesterone receptors. Antagonistic activity was evidenced by partial blocking of dexamethasone induction of tyrosine aminotransferase (TAT) and thymocyte apoptosis. Replacement of the oxygen bridge by a sulfur bridge gave a less bent, more flexible molecule. 21-Hydroxy-6,19-epithioprogesterone exhibited improved antiapoptotic activity on thymocytes but was not effective blocking TAT induction. This selectivity was improved further by oxidation to the sulfone. The sulfone but not the reduced compound also reverted the dexamethasone-mediated inhibition of NFkappaB activity in HeLa cells. Blocking of the apoptotic effect of TNFalpha by dexamethasone in the L929 cell line (mouse fibroblasts), was only reverted partially by the sulfone which exhibited a mild agonistic/antagonistic activity in this assay. None of these compounds showed antiprogestin activity. Similar overall molecular shapes but more lipophylic and with higher metabolic stability were obtained by introduction of a methylene bridge (6,19-methanoprogesterone) or by a direct bond between C-6 and C-19 (6,19-cycloprogesterone and its 21-hydroxy derivative). The latter highly bent steroids showed affinity for the GR. Recently we performed molecular dynamics simulations of GR-ligand complexes to investigate the molecular basis of the passive antagonism exhibited by 21-hydroxy-6,19-epoxyprogesterone. On the basis of our findings, we proposed that the passive antagonist mode of action of this antiglucocorticoid analog resides, at least in part, in the incapacity of GR-21-hydroxy-6,19-epoxyprogesterone complex to dimerize, making the complex unable to activate gene transcription.

Exploring the molecular basis of action of the passive antiglucocorticoid 21-hydroxy-6,19-epoxyprogesterone.

21-Hydroxy-6,19-epoxyprogesterone (21OH-6,19OP) is a selective antiglucocorticoid that lacks the bulky substituent at C-11 found in active antagonists of the glucocorticoid receptor (GR). Ligand-free GR ligand-binding domain (LBD) and GR LBD complexed with 21OH-6,19OP or the agonist dexamethasone were simulated during 6 ns using molecular dynamics. Results suggest that the time fluctuation and average position adopted by the H1-H3 loop affect the ability of GR LBD-21OH-6,19OP complex to homodimerize, a necessary step in transcriptome assembly. A nuclear localization and a transactivation experiment showed that, although 21OH-6,19OP activates the translocation of the GR, the nuclear complex is unable to induce the transcription of a reporter driven by a promoter, that requires binding to a GR homodimer to be activated. These findings support the hypothesis that the passive antagonist mode of action of 21OH-6,19OP resides, at least in part, in the incapacity of the GR-21OH-6,19OP complex to dimerize.

Hemisuccinate of 21-hydroxy-6,19-epoxyprogesterone: a tissue-specific modulator of the glucocorticoid receptor.

The introduction of a hemisuccinate group at the 21‐position of the passive antiglucocorticoid 21OH‐6,19OP leads to a compound (21HS‐6,19OP) with a notable activity profile toward the glucocorticoid receptor (GR). In contrast to the parent steroid, 21HS‐6,19OP behaves as a pure agonist of GR activity in direct transactivation assays. However, the apoptotic effects of 21HS‐6,19OP show that the effect depends on cell type: while 21HS‐6,19OP is a pure agonist in L929 mouse fibroblasts, in thymocytes 21HS‐6,19OP had significant antiglucocorticoid activity. This tissue‐specific activity makes 21HS‐6,19OP a novel selective GR modulator. To investigate the molecular basis of action of 21HS‐6,19OP, we carried out molecular dynamics simulations (6u2005ns) of the GR ligand binding domain (LBD) complexed with 21HS‐6,19OP. Our results indicate that the hemisuccinate moiety may play a key role in stabilizing the active conformation of the receptor dimerization interface, reverting the changes observed with the antagonist 21OH‐6,19OP. Other changes in regions of the GR related to cofactor recruitment (possibly tissue‐specific), could explain this particular activity profile.