Tamanaha, M. d. l. C.; Cabrera, E. F.; Sargeant, J.; Gershon, P. D.; Russell, P. P. S.; Cocco, M. J.

Voxelotor (Oxbryta, GBT440) is a first-in-class drug, FDA-approved to treat sickle cell disease in 2019 but withdrawn from market in 2024. This drug acts as an allosteric modulator, designed to shift the equilibrium to the oxygenated R conformation. The drug was shown to both limit the accumulation of deoxygenated T-conformation sickle cell Hb fibers and increase Hb oxygen affinity. X-ray crystallography previously showed one-to-one Voxelotor binding stoichiometry for Hb, with the drug molecule bound to N-terminus of an alpha subunit. Here we use NMR spectroscopy to assess the structure of Voxelotor-bound hemoglobin in solution and mass spectrometry (MS) to determine stoichiometry and sites of binding. We find that the structure and stoichiometry of binding are far more heterogeneous than previously described. The addition of Voxelotor to R-conformation Hb induces an NMR signal found in the T-conformation of Hb. In addition, MS shows that the drug binds Hb at multiple sites, including the N-terminus of the beta subunit. The properties of Hb with Voxelotor bound at secondary sites have not been explored but should be considered at high doses. Heterogeneous binding should be assessed in other drugs of this class including GBT(021)601 currently in clinical trial.