Novel Selective HDAC1/2 Inhibitor 2-Aminobenzamide Derivative Potently Induces Fetal Hemoglobin

Introduction: Induction of fetal hemoglobin (HbF) is a validated therapeutic strategy for sickle cell disease (SCD). Currently, affordable treatments for SCD are limited. Hydroxyurea (HU) is the only approved drug for inducing HbF, but its variable patient response and limited availability in African countries restrict its widespread use. Additionally, there is a lack of cost-effective and safe molecules for HbF induction. The silencing of γ-globin genes (HBG1/2) in adults involves epigenetic mechanisms, including the action of histone deacetylases 1 (HDAC1) and HDAC2, which confer a closed chromatin conformation at the γ-globin promoters. Inhibiting HDAC1 and HDAC2 has been demonstrated to induce HbF without altering cell cycle or proliferation. Therefore, targeting HDAC1/2 with selective inhibitors represents a refined therapeutic strategy for the treatment of SCD.

Methods: We investigated the effects of a new heterocyclic 2-aminobenzamide derivative, PTF, on HbF induction in HUDEP-2 cells, an erythroid precursor line. Cells were treated with 0.8 µM and 1 µM PTF for 72 and 96 hours. For comparison, cells were also treated with 100 µM HU. The percentage of HbF-positive cells was assessed by flow cytometry using anti-HbF antibodies, and HBG1/2 mRNA levels were quantified by qPCR. HDAC1/2 inhibition assays were performed using recombinant human enzymes. Additionally, absorption, distribution, metabolism and excretion (ADME) properties of PTF were evaluated.

Results: Treatment with PTF significantly increased the percentage of HbF-positive cells. At 72 hours, 0.8 µM and 1 µM PTF increased HbF-positive cells to 7.06 ± 0.92% and 7.48 ± 0.55%, respectively, compared to 0.83 ± 0.12% in controls (p<0.0001, n=4). At 96 hours, these values were 11.35 ± 1.15% and 12.78 ± 0.34%, compared to 0.91 ± 0.12% in controls (p<0.0001, n=4), representing an approximately 12.5-fold and 14-fold increase, respectively. qPCR analysis revealed substantial upregulation of HBG1/2 mRNA. At 72 hours, 0.8 µM and 1 µM PTF increased HBG1/2 mRNA levels by 58-fold and 63.03-fold, respectively (CTRL = 0.012 ± 0.002 arbitrary units (A.U) vs. 0.8 µM PTF = 0.68 ± 0.16 A.U and 1 µM PTF = 0.73 ± 0.17 A.U, p<0.0001, n=4). At 96 hours, increases were 49-fold and 52-fold, respectively (CTRL = 0.022 ± 0.014 A.U vs. 0.8 µM PTF = 1.1 ± 0.19 A.U and 1 µM PTF = 1.17 ± 0.28 A.U, p<0.0001, n=4). In contrast, HU at 100 µM induced an increase in HbF-positive cells to 5.65 ± 0.59% at 72 hours and 5.73 ± 0.52% at 96 hours, compared to 0.66 ± 0.23% and 0.64 ± 0.15% in controls (p<0.0001, n=2), reaching a maximum 10-fold increase in HBG1/2 mRNA expression at 72 hours (n=2).

In vitro HDAC assays showed that PTF inhibited HDAC1 activity by 91% and HDAC2 activity by 71% at 1 µM. ADME analysis indicated favorable properties for PTF, with a logD of 2.51 at pH 7.4 and high permeability (Pe 14.61 x 10^-6 cm/s). In human liver microsomes (HLM), PTF demonstrated high metabolic stability with a half-life of 266.6 minutes and a clearance rate of 10.4 µL/min/mg.

Conclusion: PTF, a selective HDAC1/2 inhibitor, effectively derepresses the HBG1/2, leading to significant HbF induction in HUDEP-2 cells, outperforming HU, the current standard treatment for SCD. These findings highlight PTF as a promising therapeutic candidate. The compound not only strongly inhibits HDAC1 and HDAC2 but also possesses favorable ADME properties, supporting its further development as a potential oral therapeutic.