Targeting an “undruggable” RNA-binding protein: discovery of small molecule inhibitors of HuR for novel breast cancer therapy
Xiaoqing Wu1, Lan Lan1, Amber Smith1, Rebecca Marquez1, David Wilson2, Steven Rogers3, Philip Gao4, Scott Lovell 5, John Karanicolas1,6, Dan Dixon7, Jeffrey Aubé8, and Liang Xu1
1Department of Molecular Biosciences, 2Laboratory for Early Stage Translational Research, 3Center of Biomedical Research Excellence Medicinal Chemistry Core, 4COBRE-PSF Protein Purification Group, 5COBRE-PSF Protein Structure Core, 6Center for Bioinformatics, 8Department of Medicinal Chemistry, The University of Kansas; 7Department of Cancer Biology, The University of Kansas Medical Center.
Post-transcriptional gene regulation is essential for normal development, but when dysregulated, has many implications in disease conditions, including cancer. The RNA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs. Due to the lack of well-defined binding pocket, RBPs have been considered “undruggable targets”. The RBP Hu antigen R (HuR) is overexpressed in many types of cancer, including breast cancer. Elevated cytoplasmic HuR level correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in breast cancer. HuR promotes tumorigenesis by interacting with cancer-associated mRNAs, those mRNAs encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. HuR also modulates the sensitivity of breast cancer cells to chemotherapy. Our hypothesis is that small molecule compounds that disrupt the HuR-mRNA interaction will block HuR function, leading to the decay and reduced translation of mRNAs of the target genes critical for breast cancer cell growth and progression. High throughput screening (HTS) was carried out in several chemical libraries (~23,000 compounds) using fluorescence polarization (FP) assay and identified a series of initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit CMLD-2 and its analogs, specifically blocked HuR-mRNA interaction by competitive binding to HuR, subsequently shortened HuR target mRNAs’ half-life and decreased the level of the encoded proteins (Bcl-2, XIAP and Msi1/2). Moreover, those compounds selectively inhibited cancer cell proliferation but not normal cells with low HuR. We are testing the antitumor efficacy of those HuR inhibitors in mouse models. These results support future studies toward chemical probes for HuR function study and possibly a novel therapy for HuR-overexpressing cancers.