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1.
J Biol Chem ; 286(12): 10126-36, 2011 Mar 25.
Article in English | MEDLINE | ID: mdl-21220421

ABSTRACT

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.


Subject(s)
Enzyme Inhibitors/pharmacology , Exons , Fibroblasts/metabolism , RNA Precursors/metabolism , RNA Splicing/drug effects , Survival of Motor Neuron 1 Protein/metabolism , Cells, Cultured , Child , Humans , Male , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Protein Phosphatase 1/antagonists & inhibitors , Protein Phosphatase 1/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/metabolism , RNA Precursors/genetics , RNA Splicing/genetics , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolism
2.
Steroids ; 75(12): 825-33, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20685325

ABSTRACT

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.


Subject(s)
Estrogens/chemical synthesis , Estrogens/pharmacology , Steroids/chemical synthesis , Steroids/pharmacology , Dose-Response Relationship, Drug , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/chemistry , Female , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Oximes/chemistry , Protein Multimerization/drug effects , Protein Structure, Quaternary , Steroids/chemistry , Substrate Specificity , Transcriptional Activation/drug effects
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