Functional adult hemoglobin (Hb) comprises two α-globin (α1α2) and two β-globin (β1β2) subunits arranged around each other, resulting in a large central water cavity. The α- and β-clefts define the two entry points into the central water cavity. Structural hemoglobinopathies, a consequence of gene variants expressing dysfunctional globin proteins, are among the most common congenital genetic disorders worldwide (Modell & Bulyzhenkov, 1988
). Sickle-cell disease (SCD) is the consequence of an A→T substitution at codon 6 of the β-globin gene, resulting in a Glu→Val substitution in the expressed β-globin protein (Modell & Bulyzhenkov, 1988
; Bunn & Forget, 1968
). Intracellular polymerization of deoxygenated sickle Hb (deoxy-Hb S) into long, rigid and insoluble fibers causes the pathophysiology associated with SCD (Modell & Bulyzhenkov, 1988
; Bunn & Forget, 1968
), facilitating a cascade of adverse events that include compensatory vasoconstriction, an increase in neutrophil count and the adhesion of red blood cells (RBCs) to tissue endothelium. The clinical condition is characterized by chronic hemolytic anemia, frequent and severe painful crises and multi-system pathology that impacts nearly every organ. Although various supportive therapies have improved the quality of life of patients with SCD, an efficacious, well tolerated therapeutic option remains elusive. Lack of response in up to 30% of patients, poor tolerance and myelosuppression severely limit the use of hydroxyurea, the only drug that is widely used clinically for SCD therapy (Charache et al.
; Platt, 2008
A vast number of published studies (Nnamani et al.
; Abdulmalik et al.
; Safo et al.
; Abraham et al.
; Zaugg et al.
; Merrett et al.
; Beddell et al.
) have focused on the investigation of naturally occurring or synthetic aldehydes as potential therapeutic agents for the treatment of SCD. Two well known examples are 12C79 (or tucaresol; Merrett et al.
) and the food flavoring agent vanillin (Abraham et al.
) (see Fig. 1). These aldehydes form Schiff-base adducts with the N-terminal Val1α of Hb S to left-shift the oxygen-equilibrium curve (OEC), thereby increasing the the oxygen affinity of Hb. This left-shift of the OEC is advantageous because only the deoxygenated/unliganded form (tense or T state) of Hb S polymerizes, while the oxygenated/liganded form (relaxed or R state) is soluble.
Structures of vanillin, 12C79, 5-HMF and INN compounds.
Although relatively nontoxic, the large vanillin dose needed to elicit in vivo
antisickling therapeutic effects was not clinically acceptable. 12C79 (see Fig. 1), a more potent antisickling agent, required significantly lower therapeutic doses and underwent phase II clinical trials, but was terminated owing to immune-mediated toxicity. Another potent antisickling aldehyde, 5-hydroxymethyl-2-furfural (5-HMF; see Fig. 1), has recently been shown to protect transgenic sickle mice from death from acute pulmonary sequestration of sickle cells under hypoxic conditions (Abdulmalik et al.
). Preclinical and phase I clinical antisickling studies of 5-HMF for the treatment of sickle-cell anemia are currently at the planning stages. The atomic interactions between 5-HMF and deoxy-Hb and with liganded Hb (relaxed state in the R2 conformation) have also been reported and showed the compound bound in a symmetry-related fashion at the α-cleft of the central water cavity of the protein (Safo et al.
). The liganded R2 quaternary structure is one of several known relaxed-state Hb conformations, including the classical R state that exists in equilibrium (Jenkins et al.
; Safo & Abraham, 2005
; Safo et al.
; Silva et al.
; Schumacher et al.
With the aim of enhancing the antisickling potency of naturally occurring antisickling compounds and lowering the therapeutic dose requirements, we synthesized several pyridyl derivatives of vanillin (INN compounds). We have previously reported the synthesis of these compounds and their effect on the oxygen affinity of normal hemoglobin (Nnamani et al.
). In the current study, the functional and antisickling properties of two of the derivatives, INN-312 [5-methoxy-2-(pyridin-3-ylmethoxy)benzaldehyde] and INN-298 [4-methoxy-3-(pyridin-2-ylmethoxy)benzaldehyde] (see Fig. 1) have been further investigated. We have also studied the atomic interactions of INN-312 and INN-298 with both liganded and unliganded Hb to elucidate the structural basis of their potent and/or dual antisickling activities.