VZHE-039, a novel antisickling agent that prevents erythrocyte sickling under both hypoxic and anoxic conditions

Abstract

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation βGlu6 → βVal6 that changes normal Hb (HbA) into sickle Hb (HbS). Under hypoxia, HbS polymerizes into rigid fibers, causing red blood cells (RBCs) to sickle; leading to numerous adverse pathological effects. The RBC sickling is made worse by the low oxygen (O₂) affinity of HbS, due to elevated intra-RBC concentrations of the natural Hb effector, 2,3-diphosphoglycerate. This has prompted the development of Hb modifiers, such as aromatic aldehydes, with the intent of increasing Hb affinity for O₂ with subsequent prevention of RBC sickling. One such molecule, Voxelotor was recently approved by U.S. FDA to treat SCD. Here we report results of a novel aromatic aldehyde, VZHE-039, that mimics both the O₂-dependent and O₂-independent antisickling properties of fetal hemoglobin. The latter mechanism of action-as elucidated through crystallographic and biological studies-is likely due to disruption of key intermolecular contacts necessary for stable HbS polymer formation. This dual antisickling mechanism, in addition to VZHE-039 metabolic stability, has translated into significantly enhanced and sustained pharmacologic activities. Finally, VZHE-039 showed no significant inhibition of several CYPs, demonstrated efficient RBC partitioning and high membrane permeability, and is not an efflux transporter (P-gp) substrate.

Figure 1

Structures of aromatic aldehydes.

Scheme 1

Synthetic route for the synthesis of VZHE-039

Figure 2

Structure of Hb in the R2 conformation in complex with two molecules of VZHE-039 bound at the α-cleft. For clarity, not all binding site residues are shown, but described in the text. Hb subunits are shown as ribbons (α1 subunit in pink, α2 in cyan). (A) A pair of bound VZHE-039 (yellow sticks) at the α-cleft of Hb showing the close hydrogen-bond interaction with the nitrogen atom of Met76 of the αF-helix. (B) Final 2F_o-F_c electron density map of VZHE-039 (yellow stick) contoured at 1.0σ. (C) Two-dimensional contacts between one VZHE-039 molecule, the protein, and the second VZHE-039 molecule as described in the text. (D) Superposition of TD-7 (green) and VZHE-039 (yellow) molecules at the α-cleft of Hb. (A–D) were generated using the Pymol graphic software (The Pymol Molecular Graphics System, version 1.7.4, Schrödinger, LLC; https://pymol.org/2/support.html). (C) was generated with LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions, version 2.2 (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/).

Figure 3

Time-dependent P₅₀-shift of HbA in normal blood incubated with 2 mM test compound.

Figure 4

Dose-dependent effect of VZHE-039 on SS cells sickling, Hb adduct formation and Hb O₂-affinity in vitro (Hct of 20%). (A) Morphology of SS cells before and after incubation with VZHE-039 under 2.5% O₂. (B) Representative HPLC chromatograms (Hb modification analyses) of lysates from the antisickling study. (C) Representative OEC curves of lysates from the antisickling study.

Figure 5

Antisickling effects of VZHE-039 on sickling of SS cells under various conditions. (A) Comparison of inhibition of SS cell sickling under 2.5% O₂ gas and 100% N₂ gas, at 2 mM concentrations, for untreated (control), Vanillin, TD-7, GBT-440 (Voxelotor), and VZHE-039. We observe identical antisickling effects for VZHE-039 under either hypoxia or anoxia, conclusively demonstrating an O₂-independent effect, which is not present or much less prominent for the comparator compounds. (B) pO₂-dependent sickling of SS RBCs under controlled conditions in a Hemox Analyzer (Control, VZHE-039 and Voxelotor. Note the delay in initiation of sickling, as well as reduction in the total number of sickled cells at 1 mM concentration, while a complete inhibition of sickling at the lowest recorded pO₂ is seen at 2 mM for VZHE-039.

Figure 6

RBC partitioning and cell permeability by VZHE-039. (A) Partitioning of VZHE-039 into RBC compartment (n = 4) (B) Bi-directional permeability through caco-2 cell monolayers.