High-Throughput Fluorescence Anisotropy Screen for Inhibitors of the Oncogenic mRNA Binding Protein, IMP-1

Mahapatra, Lily; Mao, Chengjian; Andruska, Neal; Zhang, Chen; Shapiro, David J.

doi:10.1177/1087057113499633

Cited by 22 publications

(33 citation statements)

References 35 publications

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“…Although the mechanism by which CRD-BP exerts its oncogenic function may still be unknown, it has become increasingly clear that its ability to physically associate with its target mRNAs is one important criterion. To date, only a few studies have described the use of molecules capable of blocking the CRD-BP-RNA interaction (Coulis et al, 2000;Mao et al, 2006;King et al, 2014;Mahapatra et al, 2014), but none have specifically described potential inhibitors of the CRD-BP-GLI1 mRNA interaction. In this study, we further characterize the CRD-BP-GLI1 RNA interaction and report that a sense RNA oligonucleotide specifically designed on the basis of the two-stem-loop motif region mapped in GLI1 mRNA nts 320-380 is not only capable CRD-BP-GLI1 RNA Interaction Inhibited by Oligonucleotide of blocking CRD-BP-GLI1 RNA interaction in vitro but also able to inhibit GLI1 mRNA expression in a panel of colon and breast cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of GLI1 Expression by Targeting the CRD-BP–GLI1 mRNA Interaction Using a Specific Oligonucleotide

et al. 2016

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The stabilization of glioma-associated oncogene 1 (GLI1) mRNA by coding region determinant binding protein (CRD-BP) through the Wnt/b-catenin signaling pathway is implicated in the proliferation of colorectal cancer and basal cell carcinoma. Here, we set out to characterize the physical interaction between CRD-BP and GLI1 mRNA so as to find inhibitors for such interaction. Studies using CRD-BP variants with a point mutation in the GXXG motif at each KH domain showed that KH1 and KH2 domain are critical for the binding of GLI1 RNA. The smallest region of GLI1 RNA binding to CRD-BP was mapped to nucleotides (nts) 320-380. A 37-nt S1 RNA sense oligonucleotide, containing two distinct stem-loops present in nts 320-380 of GLI1 RNA, was found to be effective in blocking CRD-BP-GLI1 RNA interaction. Studies using various competitor RNAs with modifications to S1 RNA oligonucleotide further displayed that both the sequences and the structure of the two stem-loops are important for CRD-BP-GLI1 RNA binding. The role of the two-stem-loop motif in influencing CRD-BP-RNA interaction was further investigated in cells. The 29-O-methyl derivative of the S1 RNA oligonucleotide significantly decreased GLI1, c-myc, and CD44 mRNA levels, in a panel of colon and breast cancer cells. The results from this study demonstrate the potential importance of the two-stem-loop motif as a target region for the inhibition of the CRD-BP-GLI1 RNA interaction and Hedgehog signaling pathway. Such results pave the way for the development of novel inhibitors that act by destabilizing the CRD-BP-GLI1 mRNA interaction.

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Section: Discussionmentioning

confidence: 99%

Inhibition of GLI1 Expression by Targeting the CRD-BP–GLI1 mRNA Interaction Using a Specific Oligonucleotide

et al. 2016

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“…59 In most published FA-based screenings Z′ lies in the range 0.50–0.70, although some can go to almost 0.8. 60, 61 Assays with higher Z′ factors are preferable and it has been noted that taking the extra time and effort to optimize HTS assays for even slightly-improved Z′ factors reduces false positives and increases confidence in the screening data. 62 …”

Section: Steady-state Fluorescence Anisotropymentioning

confidence: 99%

Fluorescence anisotropy (polarization): from drug screening to precision medicine

Zhang

Berezin

2015

Expert Opinion on Drug Discovery

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Introduction Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format. Areas covered This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection, and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for Long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery. Expert opinion Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry, and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine.

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“…Recent reports nonetheless indicate meaningful progress. Examples include initial success via high throughput screens of small molecule inhibitors to the c-Myc RNA binding protein, IMP1 56 ; Musashi’s as a family of proto-oncogenic RNA binding proteins 57 ; and to the adipocytic DNA binding protein HMGA2 58 . This elevates prospects for targeting IRP1.…”

Section: ] Targeting Hif2a To Heighten Endogenous Epo Productionmentioning

confidence: 99%

Targeting EPO and EPO receptor pathways in anemia and dysregulated erythropoiesis

Rainville

Jachimowicz

Wojchowski

2015

Expert Opinion on Therapeutic Targets

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Introduction Recombinant human erythropoietin (rhEPO) is a first-line therapeutic for the anemia of chronic kidney disease, cancer chemotherapy, AIDS (Zidovudine therapy) and lower-risk myelodysplastic syndrome. However, rhEPO frequently elevates hypertension, is costly and may affect cancer progression. Potential high merit therefore exists for defining new targets for anti-anemia agents within EPO, and EPO receptor (EPOR) regulatory circuits. Areas covered EPO production by renal interstitial fibroblasts is subject to modulation by several regulators of hypoxia-inducible factor 2a (HIF2a) including Iron Response Protein-1, prolyl hydroxylases and HIF2a acetylases, each with potential as anti-anemia drug targets. The cell surface receptor for EPO (EPOR) preassembles as a homodimer (together with JAK2), and novel agents that trigger EPOR complex activation also remain attractive to develop (activating antibodies, mimetics, small molecule agonists). Additionally, certain downstream transducers of EPOR/JAK2 signaling may be drugable including Erythroferrone (a hepcidin regulator), a cytoprotective Spi2a serpin, and select EPOR-associated protein tyrosine phosphatases. Expert Opinion While rhEPO (and biosimilars) presently are important mainstay erythropoiesis-stimulating agents, impetus exists for studies of novel ESAs that fortify HIF2a’s effects, act as EPOR agonists, and/or bolster select downstream EPOR pathways to erythroid cell formation. Such agents could lessen rhEPO dosing, side effects and/or costs.

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High-Throughput Fluorescence Anisotropy Screen for Inhibitors of the Oncogenic mRNA Binding Protein, IMP-1

Cited by 22 publications

References 35 publications

Inhibition of GLI1 Expression by Targeting the CRD-BP–GLI1 mRNA Interaction Using a Specific Oligonucleotide

Inhibition of GLI1 Expression by Targeting the CRD-BP–GLI1 mRNA Interaction Using a Specific Oligonucleotide

Fluorescence anisotropy (polarization): from drug screening to precision medicine

Targeting EPO and EPO receptor pathways in anemia and dysregulated erythropoiesis

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