scholarly journals Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

PLoS Genetics ◽  
2009 ◽  
Vol 5 (2) ◽  
pp. e1000376 ◽  
Author(s):  
Shondra M. Pruett-Miller ◽  
David W. Reading ◽  
Shaina N. Porter ◽  
Matthew H. Porteus
Keyword(s):  
Zinc Finger ◽  
Small Molecule ◽  
Zinc Finger Nuclease ◽  
Protein Levels

Towards Cell Therapy for FPD/AML with Patient-Specific IPS Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4677-4677
Author(s):  
Jon P Connelly ◽  
Marshall S Horwitz ◽  
Pu Paul Liu
Keyword(s):  
Stem Cells ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Small Molecule ◽  
Genetic Defect ◽  
Zinc Finger Nuclease ◽  
Zinc Finger Nucleases ◽  
Patient Specific ◽  
Hematopoietic Stem ◽  
Platelet Disorder

Abstract Abstract 4677 Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type. This makes them a valuable source of cells for treating genetic diseases. If the disease causing mutation can be corrected in patient-specific iPSCs and the cells differentiated to therapeutically relevant cell types and transplanted back, the symptoms of the disease may be alleviated or possibly cured. Hematopoietic diseases are an attractive target due to the ability of hematopoietic stem cells (HSCs) to reconstitute the entire hematopoietic system. However, directing the differentiation of iPSCs towards a HSC fate has proven difficult. Our lab aims to (1) Derive iPSCs from patients with a hematopoietic disease and correct the mutant gene through gene targeting. This will provide iPSCs that could be used in treating the patients. (2) Develop methods that direct the differentiation of iPSCs towards a HSC fate. This will be accomplished through novel iPSC reporter lines that we will use in small molecule and RNAi screens to identify factors that direct iPS cell differentiation towards a HSC fate. Familial Platelet Disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disorder caused by a mutation in the gene RUNX1 resulting in thrombocytopenia, platelet defects, and a predisposition to AML. Our first goal is to derive iPSCs from FPD/AML patients and correct the genetic defect using zinc finger nuclease mediated gene targeting. This will provide a potential source of cells for therapy. Towards this goal, we have obtained fibroblasts from four members of a FPD/AML family and derived iPSCs from two of the individuals. Currently we are correcting the RUNX1 mutation in these iPSC lines using zinc finger nuclease mediated gene targeting. We have developed a pair of zinc finger nucleases that bind and cleave RUNX1, resulting in gene targeting frequencies of 80–90% in 293 cells and iPSCs respectively. Our second goal is to develop RUNX1 iPSC reporter lines for use in screening small molecule and RNAi libraries to find methods of directing iPSC differentiation to the HSC fate. RUNX1 is a key player in the development of adult hematopoiesis and is expressed at the earliest stages of hematopoiesis. We have targeted fluorescent proteins and a luciferase cassette into the RUNX1 locus of wild type iPSC lines using the RUNX1 zinc finger nucleases at high efficiency. We will use these RUNX1 reporter iPSC lines in screens to identify inducers of RUNX1 expression soon. We believe such inducers have the potential to direct iPSCs towards a HSC fate. Disclosures: No relevant conflicts of interest to declare.

Progress in zinc finger nuclease engineering for targeted genome modification

2011 ◽  
Vol 33 (7) ◽  
pp. 665-683 ◽  
Author(s):  
An XIAO ◽  
Ying-Ying HU ◽  
Wei-Ye WANG ◽  
Zhi-Peng YANG ◽  
Zhan-Xiang WANG ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Nuclease ◽  
Genome Modification ◽  
Targeted Genome Modification

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

scholarly journals Zebrafish foxP2 Zinc Finger Nuclease Mutant Has Normal Axon Pathfinding

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43968 ◽  
Author(s):  
Lingyan Xing ◽  
Kazuyuki Hoshijima ◽  
David J. Grunwald ◽  
Esther Fujimoto ◽  
Tyler S. Quist ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Nuclease ◽  
Axon Pathfinding

scholarly journals Screening antiproliferative drug for breast cancer from bisbenzylisoquinoline alkaloid tetrandrine and fangchinoline derivatives by targeting BLM helicase

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wangming Zhang ◽  
Shuang Yang ◽  
Jinhe Liu ◽  
Linchun Bao ◽  
He Lu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Binding ◽  
Small Molecules ◽  
Cancer Cells ◽  
Small Molecule ◽  
Breast Cancer Cells ◽  
Dna Unwinding ◽  
Protein Levels ◽  
Blm Helicase ◽  
Bisbenzylisoquinoline Alkaloids

Abstract Background The high expression of BLM (Bloom syndrome) helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to screen potential antiproliferative small molecules from 12 small molecules (the derivatives of bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline) by targeting BLM642–1290 helicase. Then we explore the inhibitory mechanism of those small molecules on proliferation of MDA-MB-435 breast cancer cells. Methods Fluorescence polarization technique was used to screen small molecules which inhibited the DNA binding and unwinding of BLM642–1290 helicase. The effects of positive small molecules on the ATPase and conformation of BLM642–1290 helicase were studied by the malachite green-phosphate ammonium molybdate colorimetry and ultraviolet spectral scanning, respectively. The effects of positive small molecules on growth of MDA-MB-435 cells were studied by MTT method, colony formation and cell counting method. The mRNA and protein levels of BLM helicase in the MDA-MB-435 cells after positive small molecule treatments were examined by RT-PCR and ELISA, respectively. Results The compound HJNO (a tetrandrine derivative) was screened out which inhibited the DNA binding, unwinding and ATPase of BLM642–1290 helicase. That HJNO could bind BLM642–1290helicase to change its conformationcontribute to inhibiting the DNA binding, ATPase and DNA unwinding of BLM642–1290 helicase. In addition, HJNO showed its inhibiting the growth of MDA-MB-435 cells. The values of IC50 after drug treatments for 24 h, 48 h and 72 h were 19.9 μmol/L, 4.1 μmol/L and 10.9 μmol/L, respectively. The mRNA and protein levels of BLM helicase in MDA-MB-435 cells increased after HJNO treatment. Those showed a significant difference (P < 0.05) compared with negative control when the concentrations of HJNO were 5 μmol/L and 10 μmol/L, which might contribute to HJNO inhibiting the DNA binding, ATPase and DNA unwinding of BLM helicase. Conclusion The small molecule HJNO was screened out by targeting BLM642–1290 helicase. And it showed an inhibition on MDA-MB-435 breast cancer cells expansion.

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