RESUMO
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Proteína HMGA1a/antagonistas & inibidores , Proteína HMGA2/antagonistas & inibidores , Suramina/química , Adipogenia/efeitos dos fármacos , Motivos de Aminoácidos/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA2/química , Proteína HMGA2/genética , Ensaios de Triagem em Larga Escala , Humanos , Suramina/isolamento & purificação , Suramina/farmacologiaRESUMO
Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC(50) values ranging from 1 to 3 µM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.