Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication

Abstract

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5′-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells

“…MB17 likely binds specifically to kinetoplast DNA, which results in the inhibition of DNA-dependent enzymes (10,11,31). Alternatively, MB17 may directly inhibit transcription and replication enzymes (11,31,(36)(37)(38). We propose that MB17 acts through its binding activity on the parasite DNA and not as a trypanocidal agent.…”

Deregulation of the Pit-1 transcription factor in human breast cancer cells promotes tumor growth and metastasis

“…The possibility of directly targeting Pit-1 has been opened by a recent in vitro study, showing a direct inhibition of the DNA-binding activity of Pit-1 and Brn3 by a small molecule (a phenyl-furanbenzimidazole dication, DB293) (52). In addition, our group has also demonstrated an inhibitory effect of 1,25-dihydroxyvitamin D3 (1,25 The in vitro data obtained from the experimental manipulation of breast cancer cell lines and the in vivo data from the SCID mouse studies indicate that Pit-1 induces EMT-associated changes and activates other cellular programs, such as cell survival and proliferation.…”

“…MCF-7 and MDA-MB-231 cells (2 × 10 4 cells) were transfected for 48 hours with the corresponding constructs, seeded in slides, and fixed for 10 minutes in 96% ethanol, before processing for immunohistochemistry, as described below for immunohistochemistry animal studies. Transient transfections were performed as previously described (52). Stable transfections were performed with the Cell Line Nucleofector Kit (Amaxa).…”

Surface Plasmon Resonance and Quartz Crystal Microbalance Methods for Detection of Molecular Interactions

“…An outstanding success of SPR detection was in the discovery of heterocyclic dications that bind in the DNA minor groove as dimers [54][55][56]. The dogma at the time was that dimers could not form with dications because of charge repulsion.…”