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

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.

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp13 Helicase

“…Suramin is a large symmetrical molecule carrying two polysulfonated naphthyl urea groups containing six negative charges at physiological pH and therefore, the basis for its many targets is likely to be its ability to strongly bind positively charged regions in proteins such as polymerases (42,44). Indeed, we show that suramin and related compounds also inhibited the SARS-CoV-2 RdRp with micromolar IC50s without detergent sensitivity.…”

“…Therefore, it was excluded from further analysis. Suramin and suramin-related compounds (NF 023, PPNDS, Evans Blue and Diphenyl Blue) are heavily negatively charged molecules and may inhibit nucleic acid-binding proteins by binding to positively charged protein regions (40)(41)(42)(43)(44). They were also identified as hits in the RNAdependent RNA polymerase (RdRp) screen (Bertolin et al Biochem J, this issue) but not in other parallel screens, consistent with the ability of these polyanionic compounds to strongly inhibit certain nucleic acid-binding proteins (41,(45)(46)(47).…”

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

“…These compounds were also identified as inhibitors of SARS-CoV-2 nsp13 helicase in a parallel screen (Zeng et al [46]). Suramin and suramin-like molecules are polyanionic compounds that likely bind to positively charged patches found in a diverse array of proteins [55][56][57][58][59][60][61], including nucleic acid binding proteins like helicases [62] and viral polymerases [63][64][65][66][67]. In vitro validation experiments described in Zeng et al showed that all five compounds inhibit SARS-CoV-2 nsp13 helicase with IC 50 values between 0.5 and 6 mM and SARS-CoV-2 RdRp with IC 50 values between 0.5 and 5 mM.…”

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase

“…Suramin seems to inhibit multiple steps in viral infection and replication: it interferes with virus-receptor interaction and hence viral–host cell binding and uptake [ 66 , 67 ], it interferes with viral helicase activities ([ 48 ] and this work) and it interferes with viral RNA polymerase activity [ 49 , 68 ]. Suramin is a large symmetrical molecule carrying two polysulfonated naphthyl urea groups containing six negative charges at physiological pH and therefore, the basis for its many targets is likely to be its ability to strongly bind positively charged regions in proteins such as polymerases [ 44 , 46 ]. Indeed, we show that suramin and related compounds also inhibited the SARS-CoV-2 RdRp with micromolar IC 50 s without detergent sensitivity.…”