Osheiza Abdulmalik

5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells ,

In an attempt to find new types of anti‐sickling agents that specifically bind to intracellular sickle haemoglobin (HbS) without inhibition by plasma and tissue proteins or other undesirable consequences, we identified 5‐hydroxymethyl‐2‐furfural (5HMF), a naturally occurring aromatic aldehyde, as an agent that fulfils this criterion. Preliminary studies in vitro showed that 5HMF forms a high‐affinity Schiff‐base adduct with HbS and inhibits red cell sickling by allosterically shifting oxygen equilibrium curves towards the left. Further studies with transgenic (Tg) sickle mice showed that orally administered 5HMF was rapidly absorbed into the bloodstream from the gastrointestinal tract without being destroyed, traversed the red blood cell membrane and specifically bound with, and modified, HbS molecules at levels as high as 90%. Pretreatment of Tg sickle mice with 5HMF inhibited the formation of sickle cells and significantly prolonged survival time under severe hypoxia, compared with untreated mice, which died within 15 min because of sickling‐dependent pulmonary sequestration. These results indicate the feasibility of 5HMF as an attractive potential candidate for therapy of sickle cell disease.

An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

Erythrocyte precursors produce abundant alpha- and beta-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. alphaHb-stabilizing protein (AHSP) binds free alpha subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free alpha-globin caused by beta-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free alpha-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 alpha-globin genes (genotype Ahsp(-/-)alpha-globin*(alpha/alphaalpha)) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated alpha-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free alpha-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess alpha-globin, AHSP also acts as a molecular chaperone to stabilize nascent alpha-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.

Investigating the magnetic susceptibility properties of fresh human blood for noninvasive oxygen saturation quantification

Quantification of blood oxygen saturation on the basis of a measurement of its magnetic susceptibility demands knowledge of the difference in volume susceptibility between fully oxygenated and fully deoxygenated blood (Δχdo). However, two very different values of Δχdo are currently in use. In this work we measured Δχdo as well as the susceptibility of oxygenated blood relative to water, Δχoxy, by MR susceptometry in samples of freshly drawn human blood oxygenated to various levels, from 6 to 98% as determined by blood gas analysis. Regression analysis yielded 0.273 ± 0.006 and −0.008 ± 0.003 ppm (cgs) respectively, for Δχdo and Δχoxy, in excellent agreement with previous work by Spees et al. (Magn Reson Med 2001;45:533–542). Magn Reson Med, 2012.

Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy

OBJECTIVE Mixed hematopoietic chimerism after bone marrow transplantation can provide effective treatment for beta-thalassemia because of the selective advantage that exists for donor erythropoiesis. In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism, particularly when a selective advantage exists for donor cells. To investigate the biology of IUHSCTx in hemoglobinopathies, we performed fully allogeneic IUHSCTx in murine models of beta-thalassemia (Thal) and sickle cell disease (SCD). MATERIALS AND METHODS We serially assessed and compared levels of mononuclear cell (MNC) and erythroid chimerism after IUHSCTx of either adult bone marrow (BM)- or fetal liver (FL)-derived allogeneic donor cells in the two hemoglobinopathy models, which differ significantly in their degree of anemia (Thal>>SCD) and red cell half-life (Thal<<SCD). RESULTS The mean level of donor MNC chimerism was higher for SCD and Thal chimeras receiving FL- compared to adult BM-derived donor cells and tended to increase over time in the FL recipients. Donor hemoglobin (Hb) levels also were higher in all groups receiving FL compared to adult BM. Donor Hb levels in chimeric Thal mice were significantly higher than those in SCD or wild-type mice. Hematologic parameters such as Hb, hematocrit (Hct), mean cell volume (MCV), membrane-associated denatured Hb, and the oxygen equilibration curve were improved in chimeric hemoglobinopathy mice. However, the improvement in Hb, Hct, and MCV was not sustained despite stable levels of donor leukocyte engraftment. CONCLUSION The severity of the hemoglobinopathy being treated and the source of donor cells may be important determinants of success in the treatment of hemoglobinopathy by IUHSCTx.

Anti-sickling effect of MX-1520, a prodrug of vanillin: an in vivo study using rodents

Vanillin, a food additive, covalently binds with sickle haemoglobin (Hb S), inhibits cell sickling and shifts the oxygen equilibrium curve towards the left. These effects would potentially benefit patients with sickle cell disease (SCD). However, vanillin has no therapeutic effect if given orally because orally administered vanillin is rapidly decomposed in the upper digestive tract. To overcome this problem, a vanillin prodrug, MX‐1520, which is biotransformed to vanillin in vivo, was synthesized. Studies using transgenic sickle mice, which nearly exclusively develop pulmonary sequestration upon exposure to hypoxia, showed that oral administration of MX‐1520 prior to hypoxia exposure significantly reduced the percentage of sickled cells in the blood. The survival time under severe hypoxic conditions was prolonged from 6·6 ± 0·8 min in untreated animals to 28·8 ± 12 min (P < 0·05) and 31 ± 7·5 min (P < 0·05) for doses of 137·5 and 275 mg/kg respectively. Intraperitoneal injection of MX‐1520 to bypass possible degradation in the digestive tract showed that doses as low as 7 mg/kg prolonged the survival time and reduced the percentage of sickled cells during hypoxia exposure. These results demonstrate the potential for MX‐1520 to be a new and safe anti‐sickling agent for patients with SCD.

Pyridyl Derivatives of Benzaldehyde as Potential Antisickling Agents

Compounds that bind to sickle hemoglobin (Hb S) producing an allosteric shift to the high‐affinity Hb S that does not polymerize are being developed to treat sickle cell anemia (SCA). In this study, three series of pyridyl derivatives of substituted benzaldehydes (Classes I–III) that combine structural features of two previously determined potent antisickling agents, vanillin and pyridoxal, were synthesized. When analyzed with normal human whole blood, the compounds form Schiff‐base adducts with Hb and left shift the oxygen equilibrium curve (OEC) to the more soluble high‐affinity Hb, more than vanillin or pyridoxal. Generally, Class‐I compounds with an aromatic aldehyde located ortho to the pyridyl substituent are the most potent, followed by the Class‐II compounds with the aldehyde at the meta‐position. Class‐III compounds with the aldehyde at the para position show the weakest activity. The structure–activity studies of these pyridyl derivatives of substituted benzaldehydes demonstrate significant allosteric potency that may be useful for treating SCA.

Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers

Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.

Identification of a Small Molecule that Increases Hemoglobin Oxygen Affinity and Reduces SS Erythrocyte Sickling.

Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease.

New developments in anti-sickling agents: can drugs directly prevent the polymerization of sickle haemoglobin in vivo?

The hallmark of sickle cell disease is the polymerization of sickle haemoglobin due to a point mutation in the β‐globin gene (HBB). Under low oxygen saturation, sickle haemoglobin assumes the tense (T‐state) deoxygenated conformation that can form polymers, leading to rigid erythrocytes with impaired blood vessel transit, compounded or initiated by adhesion of erythrocytes to endothelium, neutrophils and platelets. This process results in vessel occlusion and ischaemia, with consequent acute pain, chronic organ damage, morbidity and mortality. Pharmacological agents that stabilize the higher oxygen affinity relaxed state (R‐state) and/or destabilize the lower oxygen affinity T‐state of haemoglobin have the potential to delay the sickling of circulating red cells by slowing polymerization kinetics. Relevant classes of agents include aromatic aldehydes, thiol derivatives, isothiocyanates and acyl salicylates derivatives. The aromatic aldehyde, 5‐hydroxymethylfurfural (5‐HMF) increases oxygen affinity of sickle haemoglobin and reduces hypoxia‐induced sickling in vitro and protects sickle cell mice from effects of hypoxia. It has completed pre‐clinical testing and has entered clinical trials as treatment for sickle cell disease. A related molecule, GBT440, has shown R‐state stabilization and increased oxygen affinity in preclinical testing. Allosteric modifiers of haemoglobin as direct anti‐sickling agents target the fundamental pathophysiological mechanism of sickle cell disease.