The transportosome system as a model for the retrotransport of soluble proteins.

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

Roles of GR Isoforms and Hsp90-binding Immunophilins in the Modulation of Glucocorticoid Biological Responses.

Glucocorticoid steroids play cardinal roles during the life span of an individual, modulating almost all aspects of the physiology, including the metabolism of carbohydrates, lipids and amino acids, as well as the immune response, neurological biology, stress adaptation, apoptosis, cell division, cell fate, inflammatory responses, etc. Glucocorticoids exert their biological effects by activation of the glucocorticoid receptor (GR), a bona fide ligand-activated transcription factor belonging to the nuclear receptor superfamily. The GR is expressed in virtually all cells of the human body showing isoformic versions and also transcription variants. GR forms oligomeric heterocomplexes that include the 90-kDa heat-shock protein (Hsp90) as an essential hub of the chaperone oligomer. The nature of chaperones associated with this heterocomplex is responsible for the modulation of the subcellular localization of the GR and its biological actions in a given tissue or cell type. In this sense, the discovery that immunophilins containing tetratricopeptide repeats (TPR) domains are responsible for the GR cytoplasmic transport mechanism and the nuclear retention half-time of the receptor opened new trends in our understanding of its complex mechanism of action. Because the properties of GR ligands influence these protein-protein interactions, specific steroid•receptor complexes may confer the GR different features providing new therapeutic opportunities to manage the disease. In this article, we analyze multiple aspects of the GR mechanism of action, some properties of the GR isoforms, and the latest findings revealing the roles of Hsp90-binding immunophilins to manage the glucocorticoid biological response.

Corticosteroid receptors as a model for the Hsp90•immunophilin-based transport machinery.

Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.

Differential regulation of the glucocorticoid receptor nucleocytoplasmic shuttling by TPR-domain proteins.

A dimer of the heat-shock protein of 90-kDa (Hsp90) represents the critical core of the chaperone complex associated to the glucocorticoid receptor (GR) oligomer. The C-terminal end of the Hsp90 dimer shapes a functional acceptor site for co-chaperones carrying tetratricopeptide repeat (TPR) domains, where they bind in a mutually exclusive and competitive manner. They impact on the biological properties of the GR•Hsp90 complex and are major players of the GR transport machinery. Recently, we showed that the overexpression of a chimeric TPR peptide influences the subcellular distribution of GR. In this study, the functional role of endogenous proteins carrying TPR or TPR-like sequences on GR subcellular distribution was characterized. It is demonstrated that, contrarily to the positive influence of FKBP52 on GR nuclear accumulation, FKBP51 and 14-3-3 impaired this property. While SGT1α showed no significant effect, the overexpression of the Ser/Thr phosphatase PP5 resulted in a nearly equal nuclear-cytoplasmic redistribution of GR rather than its typical cytoplasmic localization in the absence of steroid. This observation led to analyse the influence of the phosphorylation status of GR, which resulted not linked to its nucleo-cytoplasmic shuttling mechanism. Nonetheless, it was evidenced that both PP5 and FKBP52 are related to the anchorage of the GR to nucleoskeleton structures. The influence of these TPR domain proteins on the steroid-dependent transcriptional activity of GR was also characterized. It is postulated that the pleiotropic actions of the GR in different cell types may be the consequence of the relative abundance of different TPR-domain interacting co-chaperones.

Role of Mitochondrial Heat-shock Proteins and Immunophilins in Neuro Degenerative Diseases.

Pathophysiologic conditions of neurodegenerative diseases are unquestionably related to protein misfolding. The accumulation of misfolded proteins into relatively ordered structures such as fibrillar intracellular and extracellular amyloids results in tissue lesions that lead to neuronal loss and brain damage. In these pathologies, the occurrence of protein aggregates suggests certain inefficient or insufficient cellular responses of those molecular chaperones that should properly assist the folding of the client proteins. In this regard, most experimental models for neurodegenerative diseases have demonstrated that the overexpression of molecular chaperones provides effective neuroprotection. A subset of these molecular chaperones corresponds to a group of proteins that exhibit peptidylprolyl isomerase enzymatic activity, the immunophilins. Most of the family members of the latter group were first described as being responsible for the immunosuppressive response or they were reported as members of the chaperone complex associated with HSP90 in steroid receptor oligomers. In this article, we review some aspects of the liaison between molecular chaperones and neurodegenerative diseases, in particular heat-shock proteins and immunophilins with demonstrated influence on the proper function of mitochondria. This article is intended to address a field that represents a yet critical unmet clinical need for the development of neuroprotective molecules focused on potentially novel molecular targets.

Nucleocytoplasmic shuttling of the glucocorticoid receptor is influenced by tetratricopeptide repeat-containing proteins.

It has been demonstrated that tetratricopeptide-repeat (TPR) domain proteins regulate the subcellular localization of glucocorticoid receptor (GR). This study analyses the influence of the TPR domain of high molecular weight immunophilins in the retrograde transport and nuclear retention of GR. Overexpression of the TPR peptide prevented efficient nuclear accumulation of the GR by disrupting the formation of complexes with the dynein-associated immunophilin FKBP52 (also known as FKBP4), the adaptor transporter importin-ß1 (KPNB1), the nuclear pore-associated glycoprotein Nup62 and nuclear matrix-associated structures. We also show that nuclear import of GR was impaired, whereas GR nuclear export was enhanced. Interestingly, the CRM1 (exportin-1) inhibitor leptomycin-B abolished the effects of TPR peptide overexpression, although the drug did not inhibit GR nuclear export itself. This indicates the existence of a TPR-domain-dependent mechanism for the export of nuclear proteins. The expression balance of those TPR domain proteins bound to the GR-Hsp90 complex may determine the subcellular localization and nucleocytoplasmic properties of the receptor, and thereby its pleiotropic biological properties in different tissues and cell types.

Rational Identification of Hsp90 Inhibitors as Anticancer Lead Molecules by Structure Based Drug Designing Approach.

BACKGROUND: Heat shock protein 90 (Hsp90) is an encouraging anticancer target for the development of clinically significant molecules. Schiff bases play a crucial role in anticancer research because of their ease of synthesis and excellent antiproliferative effect against multiple cancer cell lines. Therefore, we started our research work with the discovery of resorcinol/4-chloro resorcinol derived Schiff bases as Hsp90 inhibitors, which resulted in the discovery of a viable anticancer lead molecule. OBJECTIVE: The objective of the study is to discover more promising lead molecules using our previously established drug discovery program, wherein the rational drug design is achieved by molecular docking studies. METHODS: The docking studies were carried out by using Surflex Geom X programme of Sybyl X-1.2 version software. The molecules with good docking scores were synthesized and their structures were confirmed by IR, 1H NMR and mass spectral analysis. Subsequently, the molecules were evaluated for their potential to attenuate Hsp90 ATPase activity by Malachite green assay. The anticancer effect of the molecules was examined on PC3 prostate cancer cell lines by utilizing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. RESULTS: Schiff bases 11, 12, 20, 23 and 27 exhibiting IC50 value below 1µM and 15µM, in malachite green assay and MTT assay, respectively, emerged as viable lead molecules for future optimization. CONCLUSION: The research work will pave the way for the rational development of cost-effective Schiff bases as Hsp90 inhibitors as the method employed for the synthesis of the molecules is simple, economic and facile.

Nuclear Receptors: A Historical Perspective.

In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands. Currently, members of this receptor superfamily are categorized in six groups, including "orphan receptors." Research in the area has resulted in several clinically used drugs and continues to reveal further previously unknown roles for these receptors paving the road toward more valuable discoveries in the future.

Reconstitution of the Steroid Receptor Heterocomplex.

Steroid receptors are members of a subfamily of the nuclear receptor superfamily. They play a dual role of steroid hormone receptors and transcription factors. Actually, these receptors are steroid-activated transcription factors. Classical soluble receptors exist as oligomeric complexes with the Hsp90-based chaperone machinery. The steroid receptor field was born and developed along with the molecular chaperone field. Chaperones are not exclusive partners associated to these receptors, but also comprise a large variety of heterocomplexes with other proteins involved in signal transduction. By using the glucocorticoid receptor (GR) as a standard model for most Hsp90-client proteins, in this chapter we describe the functional GR·Hsp90 heterocomplex assembly system from reticulocyte lysate or purified proteins.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

Fe3 O4 nanoparticles mediated synthesis of novel spirooxindole-dihydropyrimidinone molecules as Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is a validated molecular chaperone considered as the new key recipient for cancer intervention. The current study illustrates the synthesis of novel spirooxindole-dihydropyrimidinones (4a-j) by Fe3 O4 nanoparticles intervened synthesis and their Hsp90 ATPase inhibitory activity was investigated by the malachite green assay. All the compounds in the study demonstrated a moderate to potent ATPase inhibitory profile, with IC50 values ranging from 0.18 to 6.80 µM. Compounds 4j, 4h, 4f, and 4i exhibited maximum inhibitory potential with IC50 values of 0.18, 0.20, 0.35, and 0.55 µM, respectively. They were found to be better than the standard drug, geldanamycin (Hsp9 ATPase inhibition IC50 = 0.90 µM). Compounds 4h and 4j with IC50 values of 22.82 ± 0.532, 20.78 ± 0.234 and 21.32 ± 0.765, 28.43 ± 0.653 µM showed significantly greater potencies against the MCF-7 and HepG2 cell lines, respectively. Compound 4j showed good antioxidant activities in the DPPH test and H2 O2 assay (IC50 = 20.13.23 ± 0.32 and 23.27 ± 0.32 µg/mL) when compared with the standard ascorbic acid (IC50 = 19.16 ± 0.20 and 20.66 ± 1.09 µg/mL). A molecular docking study was performed to observe the binding efficiency and steric interactions of the lead moiety.

The Nuclear Receptor Field: A Historical Overview and Future Challenges.

In this article we summarize the birth of the field of nuclear receptors, the discovery of untransformed and transformed isoforms of ligand-binding macromolecules, the discovery of the three-domain structure of the receptors, and the properties of the Hsp90-based heterocomplex responsible for the overall structure of the oligomeric receptor and many aspects of the biological effects. The discovery and properties of the subfamily of receptors called orphan receptors is also outlined. Novel molecular aspects of the mechanism of action of nuclear receptors and challenges to resolve in the near future are discussed.

Gene expression regulation by heat-shock proteins: the cardinal roles of HSF1 and Hsp90.

The ability to permit gene expression is managed by a set of relatively well known regulatory mechanisms. Nonetheless, this property can also be acquired during a life span as a consequence of environmental stimuli. Interestingly, some acquired information can be passed to the next generation of individuals without modifying gene information, but instead by the manner in which cells read and process such information. Molecular chaperones are classically related to the proper preservation of protein folding and anti-aggregation properties, but one of them, heat-shock protein 90 (Hsp90), is a refined sensor of protein function facilitating the biological activity of properly folded client proteins that already have a preserved tertiary structure. Interestingly, Hsp90 can also function as a critical switch able to regulate biological responses due to its association with key client proteins such as histone deacetylases or DNA methylases. Thus, a growing amount of evidence has connected the action of Hsp90 to post-translational modifications of soluble nuclear factors, DNA, and histones, which epigenetically affect gene expression upon the onset of an unfriendly environment. This response is commanded by the activation of the transcription factor heat-shock factor 1 (HSF1). Even though numerous stresses of diverse nature are known to trigger the stress response by activation of HSF1, it is still unknown whether there are different types of molecular sensors for each type of stimulus. In the present review, we will discuss various aspects of the regulatory action of HSF1 and Hsp90 on transcriptional regulation, and how this regulation may affect genetic assimilation mechanisms and the health of individuals.

Biological relevance of Hsp90-binding immunophilins in cancer development and treatment.

Immunophilins are a family of intracellular receptors for immunosuppressive drugs. Those immunophilins that are related to immunosuppression are the smallest proteins of the family, i.e., FKBP12 and CyPA, whereas the other members of the family have higher molecular weight because the show additional domains to the drug-binding site. Among these extra domains, the TPR-domain is perhaps the most relevant because it permits the interaction of high molecular weight immunophilins with the 90-kDa heat-shock protein, Hsp90. This essential molecular chaperone regulates the biological function of several protein-kinases, oncogenes, protein phosphatases, transcription factors and cofactors . Hsp90-binding immunophilins where first characterized due to their association with steroid receptors. They regulate the cytoplasmic transport and the subcellular localization of these and other Hsp90 client proteins, as well as transcriptional activity, cell proliferation, cell differentiation and apoptosis. Hsp90-binding immunophilins are frequently overexpressed in several types of cancers and play a key role in cell survival. In this article we analyze the most important biological actions of the best characterized Hsp90-binding immunophilins in both steroid receptor function and cancer development and discuss the potential use of these immunophilins for therapeutic purposes as potential targets of specific small molecules.

Synthetic pregnenolone derivatives as antiviral agents against acyclovir-resistant isolates of Herpes Simplex Virus Type 1.

The conventional therapy for the management of Herpes Simplex Virus Type 1 (HSV-1) infections mainly comprises acyclovir (ACV) and other nucleoside analogues. A common outcome of this treatment is the emergence of resistant viral strains, principally when immunosuppressed patients are involved. Thus, the development of new antiherpetic compounds remains as a central challenge. In this work we describe the synthesis and the in vitro antiherpetic activity of a new family of steroidal compounds derived from the endogenous hormone pregnenolone. Some of these derivatives showed a remarkable inhibitory effect on HSV-1 spread both on wild type and ACV-resistant strains. The results also show that these compounds seem to interfere with the late steps of the viral cycle.

Molecular docking study, synthesis and biological evaluation of Mannich bases as Hsp90 inhibitors.

The ubiquitously expressed heat shock protein 90 is an encouraging target for the development of novel anticancer agents. In a program directed towards uncovering novel chemical scaffolds against Hsp90, we performed molecular docking studies using Tripos-Sybyl drug designing software by including the required conserved water molecules. The results of the docking studies predicted Mannich bases derived from 2,4-dihydroxy acetophenone/5-chloro 2,4-dihydroxy acetophenone as potential Hsp90 inhibitors. Subsequently, a few of them were synthesized (1-6) and characterized by IR, (1)H NMR, (13)C NMR and mass spectral analysis. The synthesized Mannich compounds were evaluated for their potential to suppress Hsp90 ATPase activity by the colorimetric Malachite green assay. Subsequently, the molecules were screened for their antiproilferative effect against PC3 pancreatic carcinoma cells by adopting the 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The activity profile of the identified derivatives correlated well with their docking results.

2,4-dihydroxy benzaldehyde derived Schiff bases as small molecule Hsp90 inhibitors: rational identification of a new anticancer lead.

Hsp90 is a molecular chaperone that heals diverse array of biomolecules ranging from multiple oncogenic proteins to the ones responsible for development of resistance to chemotherapeutic agents. Moreover they are over-expressed in cancer cells as a complex with co-chaperones and under-expressed in normal cells as a single free entity. Hence inhibitors of Hsp90 will be more effective and selective in destroying cancer cells with minimum chances of acquiring resistance to them. In continuation of our goal to rationally develop effective small molecule azomethines against Hsp90, we designed few more compounds belonging to the class of 2,4-dihydroxy benzaldehyde derived imines (1-13) with our validated docking protocol. The molecules exhibiting good docking score were synthesized and their structures were confirmed by IR, (1)H NMR and mass spectral analysis. Subsequently, they were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The antiproliferative effect of the molecules were examined on PC3 prostate cancer cell lines by adopting 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Finally, schiff base 13 emerged as the lead molecule for future design and development of Hsp90 inhibitors as anticancer agents.

NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.

Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.

Molecular docking study, synthesis and biological evaluation of Schiff bases as Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is an emerging attractive target for the discovery of novel cancer therapeutic agents. Docking methods are powerful in silico tools for lead generation and optimization. In our mission to rationally develop novel effective small molecules against Hsp90, we predicted the potency of our designed compounds by Sybyl surflex Geom X docking method. The results of the above studies revealed that Schiff bases derived from 2,4-dihydroxy benzaldehyde/5-chloro-2,4-dihydroxy benzaldehyde demonstrated effective binding with the protein. Subsequently, a few of them were synthesized (1-10) and characterized by IR, (1)HNMR and mass spectral analysis. The synthesized molecules were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The anticancer studies were performed by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The software generated results was in satisfactory agreement with the evaluated biological activity.

Molecular basis of mineralocorticoid receptor action in the nervous system.

The most relevant biological action of aldosterone in epithelial tissues is the regulation of sodium reabsorption through binding to the mineralocorticoid receptor (MR). Glucocorticoids also bind with high affinity to MR, which is usually protected by the enzyme 11ß-hydroxysteroid dehydrogenase. This activity prevents MR activation by cortisol despite the large prevalence of this steroid in plasma. Nonetheless, there are some aspects of the mechanism of action of MR that are not entirely explained by this competitive metabolic mechanism of protection. The picture is even more complicated in those tissues such as the nervous system where the enzyme is expressed at very low levels or is directly absent in various areas of the brain. Therefore, other cellular and molecular mechanisms must also intervene to allow specific aldosterone biological effects in the presence of overwhelming concentrations of glucocorticoids. In this article, we discuss some possible mechanisms that permit the specificity of action for each type of steroid, including those related to the recently discovered novel molecular mechanism of activation of corticosteroid receptors and the structural requirements of a given ligand to favor the mineralocorticoid action via MR. The relative contribution of these mechanisms may vary in different target cells allowing the fine tuning of cellular functions depending on the degree of cooperation between steroids, receptors, chaperones associated to receptors, and other factors. All these regulatory interactions can be altered in some pathophysiological situations, most of them related to stressing situations.