RESUMEN
Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.
Asunto(s)
Colestenos/farmacología , Colesterol/metabolismo , Receptores X del Hígado/química , Oxiesteroles/metabolismo , Colestenos/química , Colesterol/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Metabolismo de los Lípidos , Receptores X del Hígado/genética , Receptores X del Hígado/ultraestructura , Análisis por Micromatrices , Conformación Molecular , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Oxiesteroles/química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.
RESUMEN
BACKGROUND: Liver X receptors (LXRs) are transcription factors activated by cholesterol metabolites containing an oxidized side chain. Due to their ability to regulate lipid metabolism and cholesterol transport, they have become attractive pharmacological targets. LXRs are closely related to DAF-12, a nuclear receptor involved in nematode lifespan and regulated by the binding of C-27 steroidal acids. Based on our recent finding that the lack of the C-25 methyl group does not abolish their DAF-12 activity, we evaluated the effect of removing it from the (25R)-cholestenoic acid, a LXR agonist. METHODS: The binding mode and the molecular basis of action of 27-nor-5-cholestenoic acid were evaluated using molecular dynamics simulations. The biological activity was investigated using reporter gene expression assays and determining the expression levels of endogenous target genes. The in vitro MARCoNI assay was used to analyze the interaction with cofactors. RESULTS: 27-Nor-5-cholestenoic acid behaves as an inverse agonist. This correlates with the capacity of the complex to better bind corepressors rather than coactivators. The C-25 methyl moiety would be necessary for the maintenance of a torsioned conformation of the steroid side chain that stabilizes an active LXRß state. CONCLUSION: We found that a 27-nor analog is able to act as a LXR ligand. Interestingly, this minimal structural change on the steroid triggered a drastic change in the LXR response. GENERAL SIGNIFICANCE: Results contribute to improve our understanding on the molecular basis of LXRß mechanisms of action and provide a new scaffold in the quest for selective LXR modulators.