Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

Abstract

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.

NA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs.1 The RBP Hu antigen R (HuR, also known as HuA; Hu references the patient's initials from whom an anti-HuR, autoinflammatory antibody was first isolated2) is a member of the embryonic lethal abnormal vision-like (ELAVL) protein family that binds to adenine- and uridine-rich elements (ARE) mainly located in the mRNA 3′-untranslated region (UTR).1,3,4 HuR is elevated in a broad range of cancer tissues compared with the corresponding normal tissues.5 In early reports, upregulated HuR in brain and colon cancers was linked to the enhanced expression of COX-2, VEGF, TGF-β, IL-8, and other cancer-associated proteins,6,7 Subsequent studies revealed that HuR was broadly overexpressed in virtually all malignancies tested, including cancers of the colon,5,8,9 prostate,10,11 breast,12 brain,6 ovaries,13 pancreas,14 and lung.15 Elevated cytoplasmic accumulation of HuR correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in those cancers.3,4,16 HuR is proposed to play a causal role in tumor development. Cultured carcinoma cells with elevated HuR produced significantly larger tumors than those arising from control populations in a mouse xenograft model,5 while reducing HuR by siRNA or microRNA led to decreased tumor size.5,17

HuR contains three RNA recognition motifs (RRM), of which RRM1 and RRM2 are involved in RNA binding, whereas RRM3 does not contribute to RNA binding but is needed for cooperative assembly of HuR oligomers on RNA.18 Many cytokine and proto-oncogene mRNAs have been identified as containing AREs within their 3′-UTRs, which confer a short mRNA half-life.19 Cytoplasmic binding of HuR to these ARE-containing mRNAs is generally accepted as leading to mRNA stabilization and increased translation.20,21 HuR promotes tumorigenesis by interacting with cancer-associated mRNAs which encode proteins implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis.3,4,16 HuR also promotes the translation of several target mRNAs encoding proteins that are involved in cancer treatment resistance.16,22–24 Taken together, these findings suggest that HuR is an attractive target for developing novel cancer therapies.

RBPs have been considered “undruggable targets” due to the lack of a well-defined binding pocket for target mRNA. Indeed, there has globally been limited success in finding small molecules that directly disrupt the HuR interaction with AREs of target mRNAs, with limited reports indicating several active hits arising from screening for HuR inhibitors.25–27 Those reported hits are structurally independent, so they cannot provide information for later structure–activity relationship (SAR) analysis to design more potent and specific HuR inhibitors. Currently, the most potent hit reported (MS-444) acts via inhibition of HuR homodimerization, leading to disruption of the HuR–ARE interaction.25 Here, we try to identify HuR inhibitors, which competitively bind to HuR and directly disrupt the HuR–ARE interaction.

In this study, we optimized a fluorescent polarization-based (FP-based) binding assay using human full-length HuR protein and an ARE region of Musashi1 (Msi1) 3′-UTR mRNA. HuR binds to and stabilizes the mRNA of Msi128 allowing for oncogenic overexpression of Msi1 and negative regulation of Numb and adenomatous polyposis coli (APC), which are involved in controlling Notch and Wnt signaling pathways.29 Using this FP-based HTS, we screened a library of ~6000 compounds and identified a set of HuR–ARE disruptors, which were validated by AlphaLISA assay, SPR, RNP IP, and luciferase reporter functional studies. The discovery of these inhibitors and related inactive compounds provides the impetus for rational design of more potent and specific HuR–ARE disruptors.