Ponasterone A

Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells.[Pubmed:12467649]

Int J Biochem Cell Biol. 2003 Jan;35(1):79-85.

The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by Ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of Ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster.[Pubmed:21958154]

BMC Genomics. 2011 Sep 29;12:475.

BACKGROUND: The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived Ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in Drosophila different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in Drosophila Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response. RESULTS: Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3rd instar larvae and we show that 20E-induced levels of EcR isoforms EcR-RA, ER-RC, and EcR-RD/E differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types. CONCLUSIONS: We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.

Classical and three-dimensional QSAR for the inhibition of [3H]ponasterone A binding by diacylhydrazine-type ecdysone agonists to insect Sf-9 cells.[Pubmed:15670941]

Bioorg Med Chem. 2005 Feb 15;13(4):1333-40.

The activity of 52 diacylhydrazine congeners was evaluated by measuring the inhibition of the incorporation of [3H]Ponasterone A into intact Sf-9 cells. Eleven compounds were newly synthesized in this study. Results showed that the substitution of the 2-CH3 or 3-OCH3 moiety of methoxyfenozide with other groups or the removal of either group was unfavorable to the activity. The activity was quantitatively analyzed using both classical QSAR (Hansch-Fujita) and three-dimensional QSAR methods (comparative molecular field analysis, CoMFA). Sterically favorable fields were observed at the 3- and 4-positions of the benzene ring opposite from the t-butyl group (B-ring), and a sterically unfavorable field was evidenced at the 2-position. Another sterically unfavorable field developed surrounding the favorable field observed at the 4-position of the B-ring. Electrostatically negative fields were observed near the CO moiety, above the benzene ring, and at the 4-position of the B-ring. The optimum hydrophobicity of compounds in terms of their logP values was calculated to be approximately 4.1. Results of the three dimensional structure-activity relationship analyses were consistent with those obtained from the previously reported classical QSAR for 2-chlorobenzoyl analogs containing various para-substituents. The high activity of potent insecticides such as tebufenozide and chromafenozide were rationalized by CoMFA. Thus, this CoMFA result will be useful in the design of new compounds and in understanding the molecular mechanism of the ligand-receptor interactions.

Synthesis of ponasterone A derivatives with various steroid skeleton moieties and evaluation of their binding to the ecdysone receptor of Kc cells.[Pubmed:18804484]

Steroids. 2008 Dec 22;73(14):1452-64.

A series of Ponasterone A (PNA) derivatives with various steroid moieties were synthesized to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity of compounds was evaluated by determining the concentration required to give the 50% inhibition (IC(50) in M) of the incorporation of [(3)H]PNA to Drosophila Kc cells. Compounds with no functional groups such as OH and CO group in the steroid skeleton moiety were inactive. By the introduction of functional groups such as the OH and the CO group in the steroidal structure, these compounds became active. Some compounds containing the A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active. The oxidation of CH(2) at 6-position to CO, enhanced the activity 19 times, but the activity was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5alpha-cholestan-6-one: pIC(50)=4.84] to cis [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5beta-cholestan-6-one: pIC(50)=7.23]. The latter cis-type compound which is the most potent among compounds synthesized in this study was equipotent to the natural molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.