Structusl & Dynamics Studies of the Transcription Factor ERG and the Development of Small-Molecular Inhibitors

The Ets-Related Gene ERG belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant over-expression of ERG. We have identified regions flanking the ERG Ets domain responsible for auto-inhibition of DNA-binding and solved crystal structures of uninhibited, auto-inhibited, and DNA-bound ERG. Using a novel EPR method, we then determined that the auto-inhibitory regions are in conformational exchange, and by using PRE measurements and hydrogen/deuterium exchange mass spectrometry in conjunction with our structural data, we validated our crystal structures and concluded that ERG autoinhibition was allosteric. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the ms-s timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG auto-inhibition is mediated predominantly by the dynamic behavior of the Ets-domain, with the dynamic regime itself regulated by conformational exchange. Given this understanding of ERG function, we then sought to develop small-molecule inhibitors of ERG DNA binding to counter the effects of ERG over-expression. To this end, we screened a diverse compound fragment library and identified 70 hits, of which 22 were confirmed by NMR to bind ERG. Of these, six compounds showed specific activity against ERG while having minimal effects on a panel of related Ets family proteins. By optimizing these compounds, we hope to develop high-affinity, high-specificity drugs to be used in the treatment of prostate cancer, subsets of leukemia, and Ewing's sarcoma.

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