A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies

Peterson, Kenneth R.; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee; Chazelle, Allen; Zelenchuk, Lesya; Fonteles, Andrea; Dalal, Parmita; Roy, Anuradha; Chaguturu, Rathnam; Li, Biaoru; Pace, Betty S.

doi:10.1371/journal.pone.0107006

Cited by 21 publications

(22 citation statements)

References 42 publications

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“…[Decreasing the concentration of 2,3-diphosphoglycerate inhibits polymerization by two mechanisms-increasing oxygen affinity and increasing solubility of HbS (25).] Highthroughput screens have been developed for the induction of HbF (42)(43)(44)(45)(46). The only high-throughput screen for antipolymerization compounds up until now tests for increasing oxygen affinity on isolated hemoglobin solutions, but it does not address the question of cell permeability (47).…”

Section: Discussionmentioning

confidence: 99%

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

Henry

Hofrichter

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.sickle cell | drugs | hemoglobin S | treatment | screening assay

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Section: Discussionmentioning

confidence: 99%

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

Henry

Hofrichter

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Cells were cultured in HyClone Iscove's modified Dulbecco's medium (SH30005.02, Thermo Scientific) supplemented with 10% heat-inactivated FBS, 1ϫ penicillin/streptomycin, and the CID, 5 M CL-COB-II-293 (synthesized by the University of Kansas Centers of Biomedical Research Excellence Center for Cancer Experimental Therapeutics Core C Synthesis Lab; commonly called AP20187). The cells were passaged at 2 ϫ 10 5 cells/ml every 3 days in the presence of CL-COB-II-293 (55). WT ␤-YAC BMCs were treated with 10 M TMG for 4 days with fresh TMG and culture medium supplied daily.…”

Section: Cell Culturementioning

confidence: 99%

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter

Zhang

Costa²,

Tan

et al. 2016

Journal of Biological Chemistry

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One mode of ␥-globin gene silencing involves a GATA-1⅐FOG-1⅐Mi2␤ repressor complex that binds to the ؊566 GATA site relative to the A ␥-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the ؊566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the A ␥-globin promoter at the ؊566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ␤-globin locus yeast artificial chromosome (␤-YAC) bone marrow cells. When WT ␤-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2␤ at the A ␥-globin promoter is increased. In addition, OGT and Mi2␤ recruitment is increased at the A ␥-globin promoter when ␥-globin becomes repressed in postconception day E18 human ␤-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2␤ is modified with O-GlcNAc, and both OGT and OGA interact with Mi2␤, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of ␥-globin gene regulation mediated by modulating the assembly of the GATA-1⅐FOG-1⅐Mi2␤ repressor complex at the ؊566 GATA motif within the promoter.

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“…Hydroxyurea (HU), the single FDA-approved therapeutic has good activity in young patients and beneficial effects in approximately 50% of adults [5,7-10]. Additional non-cytotoxic HbF inducers, which could be used long-term, would be beneficial for treatment of patients with β-hemoglobinopathies [5, 63,67]. Clinical trials have shown significant HbF elevation, reduced anemia, and fewer hospital days in patients treated with arginine butyrate or decitabine, given parenterally [12,14].…”

Section: Discussionmentioning

confidence: 99%

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms

Dai

Sangerman

Luo

et al. 2016

Blood Cells, Molecules, and Diseases

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Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

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A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies

Cited by 21 publications

References 42 publications

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms

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