Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

Abstract

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

Conflict of interest statement

Competing Interests: SPP and DVF, authors of this manuscript have the following competing interests: employment, equity interest in, and research funding from Phoenicia Biosciences, Inc. GLW and RFW have received research funding from Phoenicia BioSciences, Inc. The other authors have declared that no competing interests exist. Drs. Perrine and Faller are Founders and have employment support from Phoenicia BioSciences. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Schema of the high-throughput screening assay (HTS).

(A.) A construct containing the 1.4-kilobase (kb) KpnI-BglII fragment of HS2 of the locus control region (LCR) linked to the γ-globin gene promoter driving the enhanced green fluorescent protein (EGFP) reporter gene stably transfected in K562 cells. Transfected cells were treated with compounds from diverse chemical libraries in an HTS format. (B.) Example of a hit in the high-throughput system is shown for compound MS-275. Fluorescent microphotographs of untreated cells are shown in the left panel; cells treated with MS-275 are shown in the right panel. (C.) Relative activity (relative fluorescence units) of new candidates identified by the HTS screen: Ambroxol (ABX), Desloratadine (DLT), MS-275, Resveratrol (RSV), Benserazide (BEN), NSC-95397 (NSC), and Idarubicin (IDA) are shown.

Fig 2. Structures of therapeutic candidates identified in high-throughput screening.

Structures of the drug candidates which induce the fetal globin gene promoter are shown. Butyric acid, a known inducer of the fetal globin gene promoter, is shown for comparison.

Fig 3. γ-globin mRNA induction in human erythroid progenitors treated with therapeutic candidates.

(A.) Mean fold-induction of γ-globin mRNA with addition of Ambroxol (ABX), resveratrol (RSV), Desloratadine (DLT), NSC-95397 (NSC), MS-275, or Benserazide (BEN), compared to vehicle-treated control (Con) in erythroid progenitors from the same subject and to arginine butyrate (AB)-treatment. The range in γ-globin mRNA induction from 2.4- to 4-fold, compared to the subject’s baseline was statistically significant for all candidates and exceeded induction of γ-globin mRNA compared by butyrate (AB) in the same cultures. Error bars indicate standard deviation (SD). (B.) Western blot demonstrating HbF protein induction in erythroid cells treated with therapeutic candidates. HbF protein relative to beta actin is shown below the bands. HbF increased above the ratios in control cells from the same source, by 3.2-fold with Benserazide treatment and by 2.5-fold with MS-275 treatment.

Fig 4. Fetal globin induction in an anemic baboon treated with MS-275 (class I HDAC inhibitor).

(A.) Treatment with MS-275 (0.2 mg/kg/dose three times/week), shown by the black bars above the graph, resulted in a 2.7-fold induction of γ-globin mRNA, (B.) 7% increase in F-cells, (C.) 30% increase in F reticulocytes, and (D.) a 1 g/dl rise in total hemoglobin despite daily phlebotomy of 6 ml/kg.

Fig 5. Fetal globin induction in an anemic baboon treated with Desloratidine (DLT).

(A.) Treatment with DLT (once per day, 5 days/week), shown by the black bars above the graph, resulted in 5-to 11-fold induction in γ-globin mRNA, (B.) a 15% increase in F-reticulocytes, and (C.) a 1.0 gm/dL increase in total hemoglobin.

Fig 6. Fetal globin induction in an anemic baboon treated with Benserazide.

(A.) Treatment with Benserazide resulted in a dose-dependent increase in γ-globin mRNA up to 34-fold. Two doses were tested, 1 mg/kg/dose (shown by the open squares) and 2 mg/kg/dose, (shown by the dark squares). (B.) F-reticulocytes increased by 15% during treatment with 2 mg/kg, and (C.) total hemoglobin increased by 1.5 gm/dl, despite the phlebotomy which exchanged the blood volume every 12 days.

Fig 7. Comparison of fetal globin induction in anemic baboons treated with lead and known candidates.

(A.) The magnitude of γ-globin mRNA induction with treatment with Hydroxyurea (HU), MS-275, DLT (mean 7-fold), and Benserazide (27-fold) are shown, and (B.) % F-reticulocytes with the drug treatments are shown. Error bars indicate standard deviation (SD).

Fig 8. Responses of transgenic mice to treatment with Benserazide or Hydroxyurea.

(A. & C.) Mice containing the human non-alpha globin genes in a YAC were treated with vehicle (water) as a control, Hydroxyurea (HU), or Benserazide. % F-cells and mean fluorescent intensity are shown pretreatment vs. 2 weeks after initiation of treatment, and (B. & D) pretreatment vs. 5 weeks after initiation of treatment. Changes with treatment were significant, (p<0.001 for Benserazide and p< 0.05 for Hydroxyurea). (E.) Total hemoglobin levels are shown pre- treatment vs. 2 weeks and 5 weeks after initiation of each treatment.