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Red Cell Biology & its Disorders
In vitro and in vivo induction of fetal hemoglobin with a reversible and selective
DNMT1 inhibitor
Ferrata Storti Foundation
Haematologica 2021 Volume 106(7):1979-1987
Aidan G. Gilmartin,1 Arthur Groy,1 Elizabeth R. Gore,1 Charity Atkins,1
Edward R. Long,1 Monica N. Montoute,1 Zining Wu,1 Wendy Halsey,1
Dean E. McNulty,1 Daniela Ennulat,1 Lourdes Rueda,1 Melissa B. Pappalardi,1 Ryan G. Kruger,1 Michael T. McCabe,1 Ali Raoof,2 Roger Butlin,2
Alexandra Stowell,2 Mark Cockerill,2o Ian Waddell,2o Donald Ogilvie,2o
Juan Luengo,1o Allan Jordan2o and Andrew B. Benowitz1
1GlaxoSmithKline, Collegeville, Pennsylvania, PA, USA and 2Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Alderley Park, Manchester, UK
oMC current address: MediTech Media, Manchester, UK; IW current address: Charles River Laboratories, Saffron Walden, UK; DO current address: Framingham Consulting Limited, Manchester, UK; JL current address: Prelude Therapeutics, Newark, DE, USA; AJ current address: Sygnature Discovery Limited, Nottingham, UK
ABSTRACT
Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of β-hemoglo- binopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce HbF expression in both preclin- ical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exem- plified by GSK3482364. This molecule potently inhibits the methyltrans- ferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells, GSK3482364 decreased overall DNA methylation resulting in de- repression of the γ-globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF lev- els and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.
Introduction
Adult hemoglobin (HbA) is a tetramer composed of two a-globin and two β-globin
polypeptide chains (a 2
) with four coordinated heme molecules, which is encoded 2
β producing sickle hemoglobin (a s
β
by genes HBA1, HBA2 and HBB. Various mutations in the β-globin gene HBB cause the β-hemoglobinopathies sickle cell disease (SCD) and β-thalassemia, the most com- mon heritable blood disorders in the world.1 In sickle cell anemia, the primary form of SCD, a missense mutation in both alleles of HBB results in an E6V substitution,
; HbS). In its deoxygenated state, the E6V mutant 2
2
β-globin proteins in the HbS tetramer enable hydrophobic interactions with mutant
β-globin proteins in neighboring HbS tetramers, resulting in hemoglobin aggregates. These aggregates grow into rods that distort the cell into a characteristic sickle shape, increase erythroid cell rigidity, and ultimately result in cell membrane damage and
Correspondence:
ANDREW B. BENOWITZ
Andrew.B.Benowitz@GSK.com
Received: January 30, 2020. Accepted: June 18, 2020. Pre-published: June 25, 2020.
https://doi.org/10.3324/haematol.2020.248658
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