A New Generation of γGlobin Gene Inducers Act on Novel Molecular and Cellular Targets.

High-potency, non-cytotoxic oral therapeutics to stimulate both fetal globin expression and erythropoiesis are needed for optimal definitive therapy of the beta-hemoglobinopathies and thalassemias. Butyrate and phenylbutyrate have been the most active of the non-chemotherapy inducing agents, but their efficacy is limited by anti-proliferative effects. A discovery program to identify more potent fetal-globin inducing compounds has been pursued utilizing pharmacophore and pseudoreceptor models to screen chemical libraries for additional inducing agents, based initially on short chain fatty acid derivatives. From a library of 10,000 compounds, our pseudoreceptor model predicted that 30 candidate compounds would bind a structural receptor which results in induction of the γ globin gene promoter. Of the 30 candidates, two compounds (RB 4 and 9) were found to have inducing activity comparable to butyrate in reporter gene assays (producing 2-fold induction), two candidates (RB7 and 29) had higher potency (3–4 fold induction), and 9 compounds had intermediate (lower) inducing activity. Second, five compounds identified through the molecular modeling stimulated proliferation of cord blood erythroid progenitors significantly, even in the absence of any added cytokines. Four of the novel compounds induced fetal globin mRNA (assayed by RT-PCR) in erythroid progenitors by 1.5 to 7-fold over control cells, at concentrations one log lower than previously identified inducers, while butyrate induced γ mRNA by 2-fold but reduced erythroid colony growth by 15%. Of compounds studied in vivo, three candidate compounds induced γ globin mRNA (by 2 to 6-fold) over basal levels in baboons, or by 2–15-fold in mice transgenic for the human γ globin genes. Molecular mechanisms of fetal globin induction by the high-potency compound RB7 resulted from displacement of an HDAC3/NCoR repressor complex from the γ globin promoter, with concurrent local histone acetylation, binding of a remodeling complex, and recruitment of RNA Pol II. These studies together identify 1) novel therapeutic targets, and 2) therapeutic candidates with multiple beneficial actions for treating beta globin disorders and other anemias. Development of a high-potency, orally tolerable therapeutic to benefit even severely anemic thalassemia patients should now be feasible.