Identification and characterization of mechanistically distinct inducers of g-globin transcription

Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia. Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy. In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of g-globin gene expression in erythroid cells. We measured g-globin transcription in K562 cells transfected with either g promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2g) or a b-yeast artificial chromosome in which the luciferase reporter gene was recombined into the g-globin coding sequences (gYAC). Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells. Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase g gene transcription in either HS2g or gYAC models. Eleven distinct classes of compounds were identified, the majority of which were active within 24–48 hr. The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr. The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity. In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 mM. This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia. # 2003 Elsevier Inc. All rights reserved.