Search

// Miriam S. Butler 1, *, # , Mani Roshan-Moniri 1, *, # , Michael Hsing 1, *, # , Desmond Lau 2, *, # , Ari Kim 1 , Paul Yen 1 , Marta Mroczek 1 , Mannan Nouri 1 , Scott Lien 1 , Peter Axerio-Cilies 1 , Kush Dalal 1 , Clement Yau 1 , Fariba Ghaidi 1 , Yubin Guo 1 , Takeshi Yamazaki 1 , Sam Lawn 1 , Martin E. Gleave 1 , Cheryl Y. Gregory-Evans 3 , Lawrence P. McIntosh 2, * , Michael E. Cox 1, * , Paul S. Rennie 1, * and Artem Cherkasov 1, * 1 Vancouver Prostate Centre and the Department of Urologic Sciences, University of British Columbia, Vancouver, BC V6H 3Z6, Canada 2 Department of Biochemistry and Molecular Biology, Department of Chemistry, Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 3 Department of Ophthalmology and Visual Sciences, Eye Care Centre, University of British Columbia, Vancouver, BC V5Z 3N9, Canada * These authors contributed equally to this work # Co-first authors Correspondence to: Michael E. Cox, email: mcox@prostatecentre.com Artem Cherkasov, email: acherkasov@prostatecentre.com Keywords: prostate cancer, ERG, rational drug design, small molecule inhibitor, TMPRSS2-ERG Received: July 29, 2016 Accepted: April 04, 2017 Published: April 15, 2017 ABSTRACT Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. Within the ETS family, high-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker or no detectable binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-binding sequences were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2–H5), resembling the SAM domain, appended with two additional helices (H0–H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

InterFeron-gamma Inducible protein 16 (IFI16) belongs to the interferon inducible HIN200 protein family that contains transcriptional regulators linked to cell cycle regulation and differentiation. All family members contain at most two domains of 200 amino acids, called HIN200, each containing two Oligonucleotide/Oligosaccharide Binding (OB) folds. IFI16 is involved in transcriptional repression and is a component of the DNA repair multi-protein complex known as BASC, which forms after UV-induced DNA damage. In this study, we used fold recognition and biophysical approaches as a tool to infer and validate functions to the HIN200 domain. Since the best template to model IFI16-HIN200 is Replication Protein A (RPA) in complex with single-stranded nucleic acids, we tested six RPA nucleic acid-binding characteristics for IFI16-HIN200. Our results indicate that IFI16-HIN200 is an RPA-like, OB-fold, nucleic acid-binding protein that binds to ssDNA with higher affinity than to dsDNA, recognizes ssDNA in the same orientation as RPA, oligomerizes upon ssDNA binding, wraps and stretches ssDNA, but does not destabilize dsDNA. We finally propose a framework model explaining how the HIN200 domain could prevent ssDNA from re-annealing.

Summary DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K D ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.