title: Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis.
abstract: Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Dominique C
authors.initials: DC
authors.lastname: Stephens
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Hye Mi
authors.initials: HM
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Arvind
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Abdelbasset A
authors.initials: AA
authors.lastname: Farahat
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Boykin
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA gpoon@gsu.edu.
authors.forename: Gregory M
authors.identifier: 0000-0001-5107-9458
authors.initials: GM
authors.lastname: Poon
mesh_terms: D000578:Amidines / Q000737:chemistry*; D001482:Base Composition / Q000235:genetics; D001562:Benzimidazoles / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D015534:Trans-Activators / Q000037:antagonists & inhibitors*; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D000578:Amidines; D001562:Benzimidazoles; D002413:Cations, Divalent; D004247:DNA; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C000621066:DB270 compound