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

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation ßGlu6 → ßVal6 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.

Potential of isoquercitrin as antisickling agent: a multi-spectroscopic, thermophoresis and molecular modeling approach.

Sickle cell disease is an inherited disease caused by point mutation in hemoglobin (ß-globin gene). Under oxygen saturation, sickle hemoglobin form polymers, leading to rigid erythrocytes. The transition of the blood vessels is altered and initiated by the adhesion of erythrocytes, neutrophils and endothelial cells. Sickle Hemoglobin (HbS) polymerization is a major cause in red blood cells (RBC), promoting sickling and destruction of RBCs. Isoquercitrin, a medicinal bioactive compound found in various medicinal plants, has multiple health benefits. The present study examines the potential of isoquercitrin as an anti-sickle agent, showing a significant decrease in the rate of polymerization as well as sickling of RBCs. Isoquercitrin-induced graded alteration in absorbance and fluorescence of HbS, confirmed their interaction. A negative value of ΔG° strongly suggests that it is a spontaneous exothermic reaction induced by entropy. Negative ΔH° and positive ΔS° predicted that hydrogen and hydrophobic binding forces interfered with a hydrophobic microenvironment of ß6Val leading to polymerization inhibition of HbS. HbS-Isoquercitrin complex exhibits helical structural changes leading to destabilization of the HbS polymer as confirmed by CD spectroscopy. MST and DSC results indicate greater changes in thermophoretic mobility and thermal stability of sickle hemoglobin in the presence of isoquercitrin, respectively. These findings were also supported by molecular simulation studies using DOCK6 and GROMACS. Hence, we can conclude that isoquercitrin interacts with HbS through hydrogen bonding, which leads to polymerization inhibition. Consequently, isoquercitrin could potentially be used as a medication for the treatment of sickle cell disease.Communicated by Ramaswamy H. Sarma.

Effect of Hereditary Hemochromatosis Gene H63D and C282Y Mutations on Iron Overload in Sickle Cell Disease Patients.

OBJECTIVE: Hemochromatosis is an autosomal recessive disease that is one of the most important reasons for iron overload. Sickle cell disease is a hemoglobinopathy that occurs as a result of a homozygous mutation in the hemoglobin gene. Erythrocyte transfusion is frequently used in the treatment of this disease. Iron overload as a result of transfusion is important in the mortality and morbidity of sickle cell anemia patients as well as in other hemoglobinopathies. In this study, the effect of hemochromatosis gene (HFE) p.H63D and p.C282Y mutations on transfusion-related cardiac and liver iron overload in sickle cell disease patients who carry homozygous hemoglobin S mutation has been investigated. MATERIALS AND METHODS: This is a prospective single-center cross-sectional study in patients with homozygous hemoglobin S mutation between the years 2008 and 2013. The patients were divided into two groups. The first group (group A, n=31) was receiving chelation therapy and the second group (group B, n=13) was not. Direct and indirect iron loads were analyzed by magnetic resonance imaging and biochemically, respectively. HFE gene mutations were analyzed by polymerase chain reaction-restriction fragment length polymorphism method. Statistical analyses were performed by independent samples t-test. RESULTS: p.H63D mutation was detected in 10 (32.3%) patients in group A and in only 1 patient (7.7%) in group B. When the 2 groups were compared for iron overload, iron deposition in the liver was significantly higher in group B (p=0.046). In addition, in group A, iron deposition was significantly higher in HFE mutation carriers compared to patients without the mutation (p=0.05). CONCLUSION: Results of this study showed that HFE gene mutations are important in iron deposition in the liver in patients with sickle cell disease.

No improvement in suboptimal vitamin A status with a randomized, double-blind, placebo-controlled trial of vitamin A supplementation in children with sickle cell disease.

BACKGROUND: Suboptimal vitamin A status is prevalent in children with type SS sickle cell disease (SCD-SS) and is associated with hospitalizations and poor growth and hematologic status. The supplemental vitamin A dose that optimizes suboptimal vitamin A status in this population is unknown. OBJECTIVE: The efficacy of Recommended Dietary Allowance (RDA) doses (based on age and sex) of vitamin A (300, 400, or 600 µg retinyl palmitate/d) or vitamin A + zinc (10 or 20 mg zinc sulfate/d) compared with placebo to optimize vitamin A status was assessed in children aged 2.0-12.9 y with SCD-SS and a suboptimal baseline serum retinol concentration (<30 µg/dL). DESIGN: In this randomized, double-blind, placebo-controlled trial, vitamin A status (serum retinol, prealbumin, retinol-binding protein, and relative-dose-response test) and disease-related illness events were assessed. RESULTS: Twelve months of vitamin A supplementation at the doses recommended for healthy US children (based on age and sex) failed to improve serum retinol values in either group (vitamin A: n = 23; vitamin A + zinc: n = 18) compared with placebo (n = 21). By 12 mo, the increase (±SD) in serum retinol (3.6 ± 2.8 µg/dL) in those taking 600 µg vitamin A/d was significantly different from the decrease (±SD; -2.8 ± 2.4 µg/dL) in those taking 300 µg/d, which possibly suggests a dose-response relation (P < 0.05) with RDA doses. CONCLUSIONS: Compared with placebo, 12 mo of vitamin A supplementation at the RDA for healthy children did not improve serum retinol values in children with SCD-SS, which possibly suggests that higher doses are needed. However, the existence of alternative conclusions emphasizes the need for future research.

Reduced risk for placental malaria in iron deficient women.

BACKGROUND: Nutritional iron deficiency may limit iron availability to the malaria parasite reducing infection risk, and/or impair host immunity thereby increasing this risk. In pregnant women, there is evidence of an adverse effect with iron supplementation, but the few reported studies are strongly confounded. METHODS: A case control study in pregnant Malawian women was undertaken in Chikhwawa southern Malawi in order to describe iron status in relation to placental malaria controlling for several confounding factors. Pregnancy characteristics were obtained and a blood sample at delivery. A full blood count was performed and serum ferritin and transferrin receptor quantified by enzyme-linked immunoassay. DNA analysis was used to identify genetic polymorphisms for ABO phenotype, hemoglobin HbS, and glucose -6 phosphate dehydrogenase deficiency. Placental tissue was obtained and malaria histology classified as active, past or no malaria infection. RESULTS: 112 cases with placental malaria were identified and 110 women with no evidence of placental infection. Iron deficiency was less frequent in women with placental Plasmodium falciparum infection. In those with acute, chronic or past placental infections the odds ratio for iron deficiency was 0.4, 95% CI 0.2-0.8, p = 0.01; for acute and chronic infections 0.4, 0.2-0.8, p = 0.006; for acute infection 0.3, 0.1-0.7, p = 0.001. The association was greater in multigravidae. CONCLUSION: Women with either acute, or acute and chronic placental malaria were less likely to have iron deficiency than women without placental malaria infection There is a priority to establish if reversing iron deficiency through iron supplementation programs either prior to or during pregnancy enhances malaria risk.

Newborn sickle cell disease screening: the Jamaican experience (1995-2006).

OBJECTIVES: The aim of this study was to evaluate the existing newborn sickle haemoglobinopathy screening programme in Jamaica. METHODS: A retrospective analysis of infants screened during the period 8 November 1995 to 22 July 2006 was performed. Patient data for analyses was restricted to patients with homozygous (Hb SS) sickle cell disease. Published data from the Jamaican Sickle Cell Cohort Study was used to make comparisons with the study sample. RESULTS: The study sample consisted of 435 patients with Hb SS disease. Acute chest syndrome was the most common clinical (non-death) event accounting for approximately 50% of all events. Acute splenic sequestration, no longer a significant cause of mortality, was responsible for approximately 32% of clinical events. Seven deaths (1.8%) occurred during the study period compared with 17.6% to the same age in the Jamaican Sickle Cell Cohort Study. There was a lower proportion of hospital admissions and episodes of serious illness in the study group compared with controls. CONCLUSIONS: Survival estimates for the study sample showed improvement compared with the Jamaican Sickle Cell Cohort Study. This study continues to demonstrate the benefits of, and as such shows support for, newborn screening and early interventions in sickle cell disease. In addition, it highlights some of the areas for continued focus and research development. Although the current system is providing an essential and beneficial service, the study emphasizes the need for newborn screening programmes to be comprehensive care systems to be fully effective.

Arginine supplementation improves rotorod performance in sickle transgenic mice.

Patients with sickle cell disease (SCD) have been shown to have impaired visual-motor speed and coordination. Sensorimotor deficits in mice can be investigated by motor coordination tests that require whole body movements such as the rotorod. A sickle transgenic mouse model (S+S-Antilles) that expresses human alpha, human beta(S) and human beta(S-Antilles), is homozygous for the mouse beta(major) deletion, and has low plasma arginine was compared to control C57BL/6J mice and S+S-Antilles mice supplemented with 5% arginine on the rotorod. The rotorod consists of a 2.5 cm diameter, grooved rod turning at constant acceleration, requiring postural adjustments on the part of the mice to maintain equilibrium. C57BL mice on Purina mouse chow had an average latency to fall of S+S-Antilles mice on Purina mouse chow had an average of 127+/-56 s S+S-Antilles mice after 5% arginine supplementation had a mean latency of Arginine may improve rotorod performance in sickle transgenic mice by increasing NO synthesis thereby improving vasodilatation and blood flow with reversal of ischemia in brain and/or muscle. In conclusion, impaired rotorod performance in sickle transgenic mice presents an opportunity to apply this simple task to provide an efficient method to screen some types of therapeutic regimens for efficacy in SCD.

Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.

A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

Mutational analysis of sickle haemoglobin (Hb) gelation.

The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants.

Role of hydrophobic amino acids at beta85 and beta88 in stabilizing F helix conformation of hemoglobin S.

Three Hb S variants containing Glu substitutions at Phe-beta85 and/or Leu-beta88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S. Helix stability of tetrameric Hb S betaF85E,betaL88E was measured by CD and compared with those of Hb S betaF85E, Hb S betaL88E, Hb A, and Hb S. The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10 degrees C. However, changes in ellipticity at 222 nm for Hb S betaF85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S betaL88E and Hb S betaF85E,betaL88E were similar and about 30-fold greater than Hb S. Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S betaF85E,betaL88E was similar to that of Hb S betaL88E rather than Hb S betaF85E. These results suggest that Glu insertion at both beta85 and beta88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S betaF85E, betaL88E is similar to Hb S betaL88E rather than Hb S betaF85E. Furthermore, these results suggest that both Phe-beta85 and Leu-beta88 are critical for F helix stabilization and that Glu insertion at beta88 leads to more destabilization than insertion at beta85.

Treatment with oral clotrimazole blocks Ca(2+)-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease.

Prevention of red cell K+ and water loss is a therapeutic strategy for sickle cell disease. We have investigated in vitro and in vivo the effects of clotrimazole (CLT) and miconazole (MIC) on transgenic mice red cells expressing hemoglobin SAD. CLT blocked the Gardos channel (ID50 75 +/- 22 nM; n = 3) and the A23187-induced dehydration of Hbbs/Hbbthal SAD 1 mouse erythrocytes in vitro. Oral treatment with CLT (160 mg/kg per d) and MIC (100 mg/kg per d) inhibited the Gardos channel in both SAD 1 and control (Hbbs/Hbbthal) mice. In the SAD 1 mice only, cell K+ content increased, and mean corpuscular hemoglobin concentration and cell density decreased. After 7 d of treatment, the hematocrit of SAD 1, CLT-treated animals also increased. All changes were fully reversible. Long-term treatments of SAD 1 mice with oral CLT (80 mg/kg per d for 28 d) lead to sustained increases in cell K+ content and hematocrit and sustained decreases in mean corpuscular hemoglobin concentration and cell density, with no changes in animals treated with vehicle alone. Thus, CLT and MIC can reverse dehydration and K+ loss of SAD 1 mouse erythrocytes in vitro and in vivo, further supporting the potential utility of these drugs in the treatment of sickle cell anemia.

Phosphorus-31 nuclear magnetic resonance studies of human red blood cells.

Phosphorus-31 nuclear magnetic resonance spectroscopy is used to monitor the changes in the concentrations of 2,3-diphosphoglycerate and adenosine-5' triphosphate as a function of time and to measure the intracellular pH of normal and abnormal red blood cells in the presence of 25% oxygen, 5.6% carbon dioxide, and 69.4% nitrogen at 37 degrees C. Under these conditions, the intracellular pH values of normal AA, sickle SS, AS, SC, AC, and CC red cells are 7.24 +/- 0.07, 7.13 +/- 0.04, 7.15 +/- 0.03, 7.16 +/- 0.03, 7.24 +/- 0.05, and 7.14 +/- 0.03, respectively. The intracellular pH of SS red cells is about 0.1 pH unit more acidic than that of AA red cells. Time-dependent changes in the concentration of 2,3-diphosphoglycerate of normal human red cells show an initial lag phase, followed by a slow linear decrease. The duration of the initial lag phase decreases in the following order: AA approximately equal to AS approximately equal to AC greater than SC greater than SS approximately equal to CC red cells. The decay of 2,3-diphosphoglycerate is much faster in SS and CC red cells compared to other red cells studied. The time-dependent depletion of adenosine-5' triphosphate in these red cells is similar in nature to that of 2,3-diphosphoglycerate. The linewidths of 2-P and 3-P resonances of 2,3-diphosphoglycerate for fully oxygenated SS red cells are broader (approximately 20 Hz) than those for other red cells (approximately 10 Hz). However, the linewidths of 2-P and 3-P resonances of 2,3-diphosphoglycerate in the lysates of these red cells are narrower (approximately 4.5 Hz) than those in the intact red cells and are very similar in all types of red cells studied. The linewidths of the 31P resonances of adenosine-5' triphosphate are also similar (approximately 30-40 Hz) in all red cells studied. In addition, we have investigated the effect of carbamylation on the metabolism of 2,3-diphosphoglycerate and the intracellular pH in SS and AA red cells and have found that neither one is affected by this process. Our results provide further evidence that phosphorus-31 nuclear magnetic resonance spectroscopy offers a direct, non-invasive way to investigate the intracellular environment and the metabolism of phosphorylated metabolites in intact red blood cells.