title: A small molecule inhibitor of Pot1 binding to telomeric DNA.
abstract: Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres 1), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of Schizosaccharomyces pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ∼20 000 compounds yielded a single inhibitor, which we found interacted tightly with sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Altschuler
authors.forename: Johnny E
authors.initials: JE
authors.lastname: Croy
authors.forename: Deborah S
authors.initials: DS
authors.lastname: Wuttke
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D020650:Combinatorial Chemistry Techniques; D003224:Congo Red / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D008958:Models, Molecular; D010446:Peptide Fragments; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D029702:Schizosaccharomyces pombe Proteins / Q000037:antagonists & inhibitors*; D000089804:Shelterin Complex; D054852:Small Molecule Libraries; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000037:antagonists & inhibitors*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D029702:Schizosaccharomyces pombe Proteins; D000089804:Shelterin Complex; D054852:Small Molecule Libraries; D034501:Telomere-Binding Proteins; C499082:pot1 protein, S pombe; D003224:Congo Red