Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays

Linjia Su,Alyssa Garabedian,Federica D’Alessio,Stefan Vasile,Jeremy Chambers,Francisco Fernandez-Lima,Prem P Chapagain,Juliano Freitas,Lidia Kos,Miriam Romano,Fenfei Leng,Nadezda Bryan,Layton Smith,Fabiana Falanga,Sabrina Battista,Alfredo Fusco

doi:10.1038/s41598-020-75890-0

Abstract

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.

Highlights

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
The disruption of Hmga[2] gene dramatically reduced obesity of leptindeficient mice (Lepob/Lepob)[2]. These results suggest that HMGA2 is a potential target for the treatment of obesity
We developed an AlphaScreen-based assay of HMGA2 binding to DNA that is amenable to automated ultra high-throughput screening (HTS) in a high-density plate format

Summary

Introduction

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. We report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. Our results show that the inhibition is likely through suramin binding to the “AT-hook” DNA-binding motifs and preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. HMGA2 is an intrinsically disordered protein (IDP29) When it binds to AT-rich DNA sequences, the “AT-hook” DNA binding motifs adopt defined structures[30]. This disordered-to-ordered conformational transition allows HMGA2 to adapt to different AT-rich DNA sequences and to participate in different nuclear a ctivities[20,21] These results suggest that HMGA2-DNA interactions could be chemically intervened for therapeutic p urposes[31]. We report the establishment of the assay, and the identification of several HMGA2 inhibitors including suramin, a century-old antiparasitic drug

Methods

Results

Conclusion