Lautaro D. Alvarez

Live Cell Imaging Unveils Multiple Domain Requirements for In Vivo Dimerization of the Glucocorticoid Receptor

The glucocorticoid receptors oligomerization state is revealed to not correlate with its activity; this challenges the current prevailing view that this state defines its transcriptional activity.

Synthesis and biological evaluation of novel substituted pyrrolo[1,2-a]quinoxaline derivatives as inhibitors of the human protein kinase CK2

Herein we describe the synthesis and properties of substituted phenylaminopyrrolo[1,2-a]quinoxaline-carboxylic acid derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 15 compounds was designed and synthesized using convenient and straightforward synthesis protocols. The compounds were tested for inhibition of human protein kinase CK2, which is a potential drug target for many diseases including inflammatory disorders and cancer. New inhibitors with IC50 in the micro- and sub-micromolar range were identified. The most promising compound, the 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid 1c inhibited human CK2 with an IC50 of 49 nM. Our findings indicate that pyrrolo[1,2-a]quinoxalines are a promising starting scaffold for further development and optimization of human protein kinase CK2 inhibitors.

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.

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.

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 (6 ns) 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.

Exploring the molecular basis of neurosteroid binding to the β3 homopentameric GABAA receptor.

Neurosteroids are the principal endogenous modulators of GABA(A) receptors (GABA(A)Rs), which are pentameric membrane-bound proteins that regulate the passage of chloride ions from the extracellular to the intracellular compartment. As consequence of their ability to modify inhibitory functions in the brain, neurosteroids have high physiological and clinical importance and may act as anesthetic, anticonvulsant and anxiolytic drugs. Despite their relevance, essential issues regarding neurosteroid action on GABA(A)Rs are still unsettled. In particular, residues taking part of the steroid recognition are not definitely identified. Taking as starting point the first reported crystal structure of a human GABAA receptor (a β3 homopentamer), we have explored through a combination of computational methods (a cavity-detection algorithm, docking and molecular dynamics simulations) the binding mode of two structurally different representative neurosteroids, pregnanolone and allopregnanolone. We have identified a neurosteroid binding site between the TM3 of one subunit and TM1 and TM4 of the adjacent subunit that is consistent with the set of experimental data reported for the action of neurosteroids on β3 homopentamers. These sites are able to properly accommodate both overall torsioned and flat steroidal structures and they specifically recognize the 3-OH group, explaining the requirement of a 3α-configuration for the activity. We believe that this work provides for first time convincing information about the molecular interaction between neurosteroids and a GABA(A)R. This information largely increases our understanding of this fundamental ligand-receptor system.

Synthesis and GABA A receptor activity of oxygen-bridged neurosteroid analogs

Three analogs of neuroactive steroids were prepared (4-6) in which 1,11- or 11,19-oxygen bridges give a constrained conformation. Their 3D structures were obtained by ab initio calculations and in the case of 3alpha-hydroxy-11,19-epoxypregn-4-ene-20-one (4), confirmed by X-ray analysis. Biological activity of the synthetic steroids was assayed in vitro using t-[(3)H]butylbicycloorthobenzoate as radiolabeled ligand for the GABA(A) receptor. The activity of compound 4 was similar to that of allopregnanolone (1). 1alpha,11alpha-Epoxypregnanolone (6) was more active than pregnanolone (2).

Biological activity and ligand binding mode to the progesterone receptor of A-homo analogues of progesterone

The biological activity of two seven-membered A-ring (A-homo) analogues of progesterone was evaluated by transactivation assays in Cos-1 cells and by determination of Bcl-x(L) expression levels in T47D cells. The results show that both compounds act as selective progesterone receptor (PR) agonists but lack mineralocorticoid receptor (MR) activity. Molecular modelling using semiempirical AM1 and ab initio HF/6-31G** calculations, showed that the A-ring of the A-homo steroids may adopt five different conformations, although only three correspond to low energy conformers. The low energy conformers of each analogue were introduced into the ligand binding pocket of the PR ligand binding domain (LBD) obtained from the PR LBD-progesterone crystal structure. The steroid binding mode was then analyzed using 10 ns of molecular dynamics (MD) simulation. The PR LBD-progesterone complex was also simulated as a control system. The MD results showed that both A-homo steroids have one conformer that may be properly recognized by the PR, in agreement with the observed progestagen activity. Moreover, the simulation revealed the importance of a water molecule in the formation of a hydrogen bonding network among specific receptor residues and the steroid A-ring carbonyl.