Synthesis and biological evaluation of pharbinilic acid and derivatives as NF-κB pathway inhibitors.

A 7-step synthesis of pharbinilic acid, a member of the gibberellin family of natural products and the first naturally occurring allogibberic acid, is reported. An efficient decarboxylative aromatization reaction enables the synthesis of pharbinilic acid and related analogs for evaluation as modulators of NF-κB activity. Remarkably, one analog displays a 2 µM IC50 in an NF-κB activity assay and inhibits an endogenous NF-κB-regulated pathway.

Phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis, in part, through regulation of the cancer-initiating cell (CIC) population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human protein kinase Cɛ at multiple residues, including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene-promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration and the CIC population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer-initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Towards a minimal motif for artificial transcriptional activators.

BACKGROUND: Most transcriptional activators minimally comprise two functional modules, one for DNA binding and the other for activation. Several activators also bear an oligomerization region and bind DNA as dimers or higher order oligomers. In a previous study we substituted these domains of a protein activator with synthetic counterparts [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. An artificial transcriptional activator, 4.2 kDa in size, comprised of a DNA binding hairpin polyamide tethered to a 20 residue activating peptide (AH) was shown to stimulate promoter specific transcription [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. The question arises as to the general nature and the versatility of this minimal activator motif and whether smaller ligands can be designed which maintain potent activation function. RESULTS: Here we have replaced the 20 amino acid AH peptide with eight or 16 residues derived from the activation domain of the potent viral activator VP16. The 16 residue activation module coupled to the polyamide activated transcription over two-fold better than the analogous AH conjugate. Altering the site of attachment of the activation module on the polyamide allowed reduction of the intervening linker from 36 atoms to eight without significant diminution of the activation potential. In this study we also exchanged the polyamide to target a different sequence without compromising the activation function further demonstrating the generality of this design. CONCLUSIONS: The polyamide activator conjugates described here represent a class of DNA binding ligands which are tethered to a second functional moiety, viz. an activation domain, that recruits elements of the endogenous transcriptional machinery. Our results define the minimal structural elements required to construct artificial, small molecule activators. If such activators are cell-permeable and can be targeted to designated sites in the genome, this series of conjugates may then serve as a tool to study mechanistic aspects of transcriptional regulation and eventually to modulate gene expression relevant to human diseases.

Activation of gene expression by small molecule transcription factors.

Eukaryotic transcriptional activators are minimally comprised of a DNA binding domain and a separable activation domain; most activator proteins also bear a dimerization module. We have replaced these protein modules with synthetic counterparts to create artificial transcription factors. One of these, at 4.2 kDa, mediates high levels of DNA site-specific transcriptional activation in vitro. This molecule contains a sequence-specific DNA binding polyamide in place of the typical DNA binding region and a nonprotein linker in place of the usual dimerization peptide. Thus our activating region, a designed peptide, functions outside of the archetypal protein context, as long as it is tethered to DNA. Because synthetic polyamides can, in principle, be designed to recognize any specific sequence, these results represent a key step toward the design of small molecules that can up-regulate any specified gene.