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Four hundred Phytochemical (bio-active) compounds having predictive activity for treating Sickle Cell Anemia were screened, using PASS online computational resource. Twenty-six compounds out of the four hundred compounds which showed high probability for treating sickle were further screened for pharmacokinetics profiles (ADMET properties) using SwissAdmet, AdmetSAR 2 and Pro-tox II online resources. Only thirteen compounds that displayed good ADMET properties from the twenty-six were further used for DFT calculations and molecular docking against carbonmonoxy sickle hemoglobin (PDB ID: 5E6E). Molecular docking analysis reinforced by DFT calculations showed that two compounds, phenanthrene-5,6-dione (A9) and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one (A13, Luteolin) had the best binding affinity of  - 8.3 and - 8.9 kcal/mol, respectively, compared to voxelotor (GBT-440), a drug use in treating sickle cell disease. Molecular dynamic simulations showed that 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one (A13, Luteolin) is highly stable with the protein than voxelotor., (© 2024. The Author(s).)

Sickle cell disease (SCD) is a monogenic blood disorder associated with a mutation in the hemoglobin subunit β gene encoding for the β-globin of normal adult hemoglobin (HbA). This mutation transcribes into a Glu-β6 → Val-β6 substitution in the β-globins, inducing the polymerization of this hemoglobin form (HbS) when in the T-state. Despite advances in stem cell and gene therapy, and the recent approval of a new antisickling drug, therapeutic limitations persist. Herein, we demonstrate through molecular dynamics and umbrella sampling, that (unrestrained) blockage of the hydrophobic pocket involved in the lateral contact of the HbS fibers by 5-mer cyclic peptides, recently proposed as SCD aggregation inhibitors (Neto, V.; J. Med. Chem. 2023, 66, 16062-16074), is enough to turn the dimerization of HbS thermodynamically unfavorable. Among these potential drugs, some exhibit an estimated pocket abandonment probability of around 15-20% within the simulations' time frame, and an impressive specificity toward the mutated Val-β6. Additionally, we show that the dimerization can be thermodynamically unfavored by blocking a nearby region while the pocket remains vacant. These results are compared with curcumin, an antisickling molecule and a pan-assay interference compound, with a good binding affinity for different proteins and protein domains. Our results confirm the potential of some of these cyclic peptides as antisickling drug candidates to reduce the concentration of aggregation-competent HbS.

Rationale: Sickle cell disease, a debilitating genetic disorder affecting numerous newborns globally, has historically received limited attention in pharmaceutical research. However, recent years have witnessed a notable shift, with the Food and Drug Administration approving three innovative disease-modifying medications. Voxelotor, also known as GBT440, is a promising compound that effectively prevents sickling, providing a safe approach to alleviate chronic hemolytic anemia in sickle cell disease. It is a novel, orally bioavailable small molecule that inhibits hemoglobin S polymerization by enhancing oxygen affinity to hemoglobin. The investigation demonstrated that voxelotor led to an unintended elevation of hemoglobin levels in healthy individuals by increasing serum erythropoietin levels., Methods: Voxelotor and its metabolites in an in vitro setting utilizing equine liver microsomes were discussed. Plausible structures of the identified metabolites were inferred through the application of liquid chromatography in conjunction with high-resolution mass spectrometry., Results: Under the experimental conditions, a total of 31 metabolites were detected, including 16 phase I metabolites, two phase II metabolites, and 13 conjugates of phase I metabolites. The principal phase I metabolites were generated through processes such as hydroxylation, reduction, and dissociation. The presence of glucuronide and sulfate conjugates of the parent drug were also observed, along with hydroxylated, reduced, and dissociated analogs., Conclusions: The data acquired will accelerate the identification of voxelotor and related compounds, aiding in the detection of their illicit use in competitive sports. It is crucial to emphasize that the metabolites detailed in this manuscript were identified through in vitro experiments and their detection in an in vivo study may not be guaranteed., (© 2023 John Wiley & Sons Ltd.)

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the β subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep β-globin locus would recapitulate SCD's complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to β 2 -Glu6 and β 1 -Asp73 (β 1 -Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep β-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease., (© 2024. The Author(s).)

Voxelotor (GBT440, OXBRYTA®) appeared recently as one of the possible treatments for sickle cell disease. This molecule, by binding the alpha globin of hemoglobin, causes hyperaffinity of the latter for oxygen and reduces its polymerization properties. Several therapeutic trials have been able to show its effectiveness on certain aspects of sickle cell disease; thus, the french HAS (High Authority of Health) college issued an early access authorization and, since 2021, this treatment can be offered to patients under a temporary authorization for use. Consequently, the laboratories that carry out the biological monitoring of sickle cell patients will be confronted with new profiles characteristic of the presence of hemoglobin combined with GBT440. This work presents a collection of images obtained by different techniques: HPLC, capillary electrophoresis, isoelectrofocusing, alkaline gel and acid agar gel electrophoresis in transfused or non-transfused sickle cell disease patients. The ability to observe the presence of GBT440 by these analyzes could be useful in order to characterize the therapeutic follow-up of patients.

The drug voxelotor (commercially known as Oxbryta) has been approved by the US Food and Drug Administration for the treatment of sickle cell disease. It is known to reduce disease-causing sickling by inhibiting the transformation of the non-polymerizing, high-oxygen-affinity R quaternary structure of sickle hemoglobin into its polymerizing, low-affinity T quaternary structure. It has not been established whether the binding of the drug has anti-sickling effects beyond restricting the change of quaternary structure. By using a laser photolysis method that employs microscope optics, we have determined that fully deoxygenated sickle hemoglobin will assume the T structure. We show that the nucleation rates essential to generate the sickle fibers are not significantly affected by voxelotor. The method employed here should be useful for determining the mechanism of sickling inhibition for proposed drugs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in hemoglobin (Hb), the protein in the red blood cell (RBC) responsible for the transport of oxygen (O 2 ) throughout the body. The mutation leads to the expression of sickle cell hemoglobin (HbS). Both Hb and HbS exist in equilibrium between oxygenated and deoxygenated forms; however, deoxygenated HbS can polymerize to form long fibers which distort the shape of RBCs into the characteristic sickled shape. The misshapen RBCs can obstruct blood vessels and capillaries, resulting in a vaso-occlusive crisis. Vaso-occulsion deprives tissues and organs of O 2 and can cause intense pain which often results in hospitalization. Chronic organ damage is a major cause of reduced life expectancy for SCD patients.Allosteric effectors are molecules which regulate protein function. HbS allosteric effectors can be used to decrease polymerization by stabilizing the oxygenated form of HbS, which leads to an increase in O 2 uptake and a decrease in the sickling of RBCs. Allosteric effectors that have been evaluated for the treatment of SCD include vanillin, 5-hydroxymethyl furfural (5-HMF), and voxelotor, which was approved by the U.S. Food and Drug Administration (FDA) for the treatment of SCD in 2019. 5-HMF did not progress to phase III clinical trials since it suffered from rapid metabolic degradation. However, several derivatives of 5-HMF and vanillin have been synthesized and evaluated as potential candidates for SCD treatment. Derivatives of these compounds have shown promise, but their shortcomings, such as high levels of oxidative metabolism, have prevented them from progressing into marketable drugs. Our efforts have produced multiple 5-HMF derivatives which have been evaluated for their potential to treat SCD. Each derivative was evaluated for its ability to increase O 2 affinity (i.e., P 50 , the partial pressure at which hemoglobin is 50% saturated with O 2 ). The synthesized aryl ether derivatives were evaluated, and results suggest that compounds with multiple aromatic aldehydes may have enhanced biological properties. One such derivative, compound 5 , which features two furan aldehyde rings, exhibited increased O 2 affinity ( P 50 = 8.82 ± 1.87 mmHg) over that of unmodified Hb ( P 50 = 13.67 ± 0.22 mmHg). Future studies include obtaining crystal structures of the 5-HMF derivatives complexed with HbS to confirm the protein-allosteric effector interactions.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α 2 β 2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Sickle cell disease (SCD) is characterized by the production of sickle hemoglobin (HbS), which when deoxygenated, polymerizes leading to red blood cell damage and hemolytic anemia, a defining feature of SCD. Voxelotor (Oxbryta) is a small molecule inhibitor of HbS polymerization that disrupts the polymerization mechanism by binding HbS to increase HbS oxygen affinity. Voxelotor is approved in the United States for the treatment of SCD in patients greater than or equal to 12 years of age at a 1500 mg once-daily (q.d.) dose. These exposure-response analyses aimed to evaluate the relationships between voxelotor whole blood concentration and change from baseline (CFB) in clinical measures of anemia and hemolysis and between voxelotor whole blood and plasma concentrations and the incidence of selected safety end points to confirm the voxelotor mechanism of action and to support the clinical dose recommendation. In patients treated with voxelotor up to 72 weeks, CFB hemoglobin (Hb) increased linearly (p < 0.001) with increasing voxelotor concentration and percent Hb occupancy and increases in CFB Hb corresponded to improvements in measures of hemolysis. The target 1 g/dl increase in CFB Hb was achieved with 1500 mg voxelotor q.d. Significant relationships were observed between voxelotor exposures and grade greater than or equal to 1 increased alanine aminotransferase and decreased white blood cell count; however, most events were grade 1. No clinically important covariate effects on voxelotor efficacy or safety were observed. Overall, these analyses support 1500 mg q.d. as the therapeutic dose for voxelotor in adults and adolescents., (© 2022 Global Blood Therapeutics, Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Sickle cell disease (SCD) is a major medical problem in which mono-therapeutic interventions have so far shown only limited effectiveness. We studied the repurpose of genistein, which could prevent sickle hemoglobin from polymerizing under hypoxic conditions in this disease. Genistein an important nutraceutical molecule found in soybean. The present study examines the repurposing genistein as an anti- sickling agent. Genistein shows inhibition of Hb S polymerization as well as a sickle reversal. Also, we have explored the interaction of the genistein with sickle hemoglobin (Hb S), using fluorescence, far-UV-CD spectroscopy, MicroScale Thermophoresis (MST), FTIR, combined with molecular modeling computations. The quenching constant decreases with increasing temperature, a characteristic that coincides with the static type of quenching mechanism. Temperature-dependent fluorescence measurements and molecular modeling studies reveal that apart from the hydrogen bonding, electrostatic interactions also play a crucial role in genistein and Hb S complex formation. In silico , distribution prediction of adsorption, digestion, metabolism, excretion, and toxicity (ADME/Tox) based on physical and chemical properties show that genistein is nontoxic and has ideal drug properties. The helicity and thermophoretic mobility of Hb S was a change in the presence of genistein, which leads to the destabilizing the Hb S polymer was examined using CD and MST, respectively. Our results open up the possibility for a promising therapeutic approach for the SCD by repurposed genistein as an anti-sickling agent.Communicated by Ramaswamy H. Sarma.

Basic research on hemoglobin has been essential for understanding the origin and treatment of many hematological disorders due to abnormal hemoglobins. The most important of the hemoglobinopathies is sickle cell disease - Linus Pauling's "molecular disease" that gave birth to molecular medicine. In this review, I will describe the contributions of basic biophysical research on normal and sickle cell hemoglobin (HbS) to understanding the molecular pathogenesis of the disease and providing the conceptual basis for the various approaches to drug therapy that target HbS polymerization. Most prominent among these are the experimental results on the solubility of HbS as a function of oxygen saturation explained by the allosteric model of Monod, Wyman, and Changeux and the Gill-Wyman thermodynamic linkage relation between solubility and oxygen binding, the solubility of mixtures of HbS with normal or fetal hemoglobin explained by Minton's thermodynamic model, and the highly unusual kinetics of HbS polymerization explained by a novel double nucleation mechanism that also accounts for the aggregation kinetics of the Alzheimer's peptide. The HbS polymerization kinetics are of great importance to understanding the pathophysiology and clinical course, as well as guiding drug development for treating this common and severe disease. The article focuses primarily on experimental and theoretical results from my lab, so it is not a comprehensive review of the subject., (Published by Elsevier Ltd.)

It is well documented that caffeic acid (3,4-dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H 2 O 2 ), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe 4+ ). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H 2 O 2 (0.5-fold over heme), we observed a fivefold reduction in the amount of HbFe 4+ accumulated in a mixture of ferric and H 2 O 2 solution. Higher levels of H 2 O 2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe 4+ , respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5-molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to βCys 93 than to βCys 112 and αCys 104 . The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Sickle cell disease, a genetic disorder affecting a sizeable global demographic, manifests in sickle red blood cells (sRBCs) with altered shape and biomechanics. sRBCs show heightened adhesive interactions with inflamed endothelium, triggering painful vascular occlusion events. Numerous studies employ microfluidic-assay-based monitoring tools to quantify characteristics of adhered sRBCs from high resolution channel images. The current image analysis workflow relies on detailed morphological characterization and cell counting by a specially trained worker. This is time and labor intensive, and prone to user bias artifacts. Here we establish a morphology based classification scheme to identify two naturally arising sRBC subpopulations-deformable and non-deformable sRBCs-utilizing novel visual markers that link to underlying cell biomechanical properties and hold promise for clinically relevant insights. We then set up a standardized, reproducible, and fully automated image analysis workflow designed to carry out this classification. This relies on a two part deep neural network architecture that works in tandem for segmentation of channel images and classification of adhered cells into subtypes. Network training utilized an extensive data set of images generated by the SCD BioChip, a microfluidic assay which injects clinical whole blood samples into protein-functionalized microchannels, mimicking physiological conditions in the microvasculature. Here we carried out the assay with the sub-endothelial protein laminin. The machine learning approach segmented the resulting channel images with 99.1±0.3% mean IoU on the validation set across 5 k-folds, classified detected sRBCs with 96.0±0.3% mean accuracy on the validation set across 5 k-folds, and matched trained personnel in overall characterization of whole channel images with R2 = 0.992, 0.987 and 0.834 for total, deformable and non-deformable sRBC counts respectively. Average analysis time per channel image was also improved by two orders of magnitude (∼ 2 minutes vs ∼ 2-3 hours) over manual characterization. Finally, the network results show an order of magnitude less variance in counts on repeat trials than humans. This kind of standardization is a prerequisite for the viability of any diagnostic technology, making our system suitable for affordable and high throughput disease monitoring., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: UAG and Case Western Reserve University have financial interests in BioChip Labs Inc. (Cleveland, Ohio). BioChip Labs Inc. offers commercial clinical microfluidic biomarker assays for inherited or acquired blood disorders. Financial interests include licensed intellectual property, stock ownership, research funding, employment, and consulting. UAG and Case Western Reserve University have financial interests in Xatek Inc. (Cleveland, Ohio). Xatek Inc. offers point-of-care global assays to evaluate the hemostatic process. Financial interests include licensed intellectual property and stock ownership. UAG and Case Western Reserve University have financial interests in Hemex Health Inc. (Portland, Oregon). Hemex Health Inc. offers point-of-care diagnostics for hemoglobin disorders, anemia, and malaria. Financial interests include licensed intellectual property, stock ownership, research funding, employment, and consulting. UAG has financial interests in DxNow Inc. (Gaithersburg, MD). DxNow Inc. offers microfluidic and bio-imaging technologies for in vitro fertilization, forensics, and diagnostics. Financial interests include licensed intellectual property and stock ownership. Competing interests of Case Western Reserve University employees are overseen and managed by the Conflict of Interests Committee according to a Conflict-of-Interest Management Plan. A patent application has been filed related to the methods discussed in this work.

The issue of treating sickle cell disease with drugs that increase hemoglobin oxygen affinity has come to the fore with the US Food and Drug Administration approval in 2019 of voxelotor, the only antisickling drug approved since hydroxyurea in 1998. Voxelotor reduces sickling by increasing the concentration of the nonpolymerizing, high oxygen affinity R (oxy) conformation of hemoglobin S (HbS). Treatment of sickle cell patients with voxelotor increases Hb levels and decreases indicators of hemolysis, but with no indication as yet that it reduces the frequency of pain episodes. In this study, we used the allosteric model of Monod, Wyman, and Changeux to simulate whole-blood oxygen dissociation curves and red cell sickling in the absence and presence of voxelotor under the in vivo conditions of rapid oxygen pressure decreases. Our modeling agrees with results of experiments using a new robust assay, which shows the large, expected decrease in sickling from the drug. The modeling indicates, however, that the increase in oxygen delivery from reduced sickling is largely offset by the increase in oxygen affinity. The net result is that the drug increases overall oxygen delivery only at the very lowest oxygen pressures. However, reduction of sickling mitigates red cell damage and explains the observed decrease in hemolysis. More importantly, our modeling of in vivo oxygen dissociation, sickling, and oxygen delivery suggests that drugs that increase fetal Hb or decrease mean corpuscular hemoglobin concentration (MCHC) should be more therapeutically effective than drugs that increase oxygen affinity., (© 2021 by The American Society of Hematology.)

Background: Minority RBC donors are important to support the transfusion needs of patients with sickle cell disease. Testing of donors for sickle cell trait (SCT) is performed to avoid transfusion of hemoglobin S+ (HbS+) RBCs to specific patient groups and to investigate leukoreduction failures. A screening assay based on hemoglobin solubility is commonly used. The purpose of this study was to validate a DNA approach for HbS screening., Methods: Hemoglobin solubility screening (Pacific Hemostasis or SICKLEDEX) and PreciseType human erythrocyte antigen (HEA)-HbS (Immucor) targeting c.20A>T in the β-globin gene were performed according to manufacturer's directions. Resolution of differences in results included gene sequencing and high-performance liquid chromatography (HPLC)., Results: Initial validation of HEA-HbS performed by testing 60 known samples, 20 HbS/A, A/A, and S/S, gave expected results. However, in the subsequent parallel testing phase, 4/58 samples HbS+ by solubility assay tested negative by HEA-HbS; the negative results were confirmed by β-globin gene sequencing. Samples from donors self-identifying as White testing HbS+ by solubility assay (n = 60) were retested by HEA-HbS and HPLC. The HEA-HbS assay was concordant with HPLC which is recognized as the gold standard for hemoglobin variation., Conclusion: A DNA-based approach is an alternative to screen donors for SCT, found in approximately 7% of Black and 1.7% of our random donors. HEA-HbS correlated with HPLC results in all samples tested, supporting the use of HEA-HbS as the test of record. The method allows higher throughput screening and testing at the donor center allows association of the screening result with the donor record to avoid repeat testing., (© 2021 AABB.)

The β subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (βE6 V) provides significant resistance to oxidative stress by shielding βCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of βCys93 to hydrogen peroxide (H 2 O 2 ) in α subunit mutations in βE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/βE6 V; (2) αH50Q/βE6 V; (3) αH20Q/H50Q/βE6 V; (4) αH20R/βE6 V; and (5) αH20R/H50Q/βE6 V. Estimation by mass spectrometry of irreversible oxidation of βCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (βE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H 2 O 2 . Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Nitric oxide (NO) is an omnipresent signalling molecule in all vertebrates. NO modulates blood flow and neural activity. Nitrite anion is one of the most important sources of NO. Nitrite is reduced to NO by various physiological mechanisms including reduction by hemoglobin in vascular system. In this study, nitrite reductase activity (NRA) of hemoglobin is reported using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in a wide potential window from +0.3 V to -1.3 V (vs. Ag/AgCl). To the best of our knowledge, a detailed look into NRA of hemoglobin is proposed here for the first time. Our results indicated two different regimes for reduction of nitrite by hemoglobin in its Fe(II) and Fe(I) states. Both reactions showed a reversible behaviour in the time scale of the experiments. The first reduction displayed a normal redox behaviour, while the latter one had the characteristics of a catalytic electro-reduction/oxidation. The reduction in Fe(II) state was selected as a tool for comparing the NRA of hemoglobin (Hb) and hemoglobin-S (Hb-S) under native-like conditions in a didodecyldimethyl ammonium bromide (DDAB) liquid crystal film. These investigations lay the prospects and guidelines for understanding the direct electrochemistry of hemoglobin utilizing a simplified mediator-free platform., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Abnormal shapes of red blood cells (RBC) have been associated with various diseases. Diverse RBC shapes have also been intriguing for membrane biophysics. Here we focus on sickle shaped RBC which form due to abnormal growth of semi-rigid hemoglobin (HbS) fibers confined in RBC. Using the area difference elasticity (ADE) model for RBC and worm-like chain model for the confined HbS fibers, we explore shape deformations at equilibrium using Monte-Carlo simulations. We show that while a single HbS fiber is not rigid enough to produce sickle like deformation, a fiber bundle can do so. We also consider multiple disjoint filaments and find that confinement can generate multipolar RBC shapes and can even promote helical filament conformations which have not been discussed before. We show that the same model, when applied to microtubules confined in phospholipid vesicles, predicts vesicle tubulation. In addition we reproduce the tube collapse transition and tennis racket type vesicle shapes, as reported in experiments. We conclude that with a decrease in the surface area to volume ratio, and membrane rigidity, the vesicles prefer tubulation over sickling. The highlight of this work is several important non-axisymmetric RBC and vesicle shapes, which have never been explored in simulations.

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine., Competing Interests: The authors declare no competing interest.

The presence of hemoglobin A-S (HbAS) in erythrocytes has been related to the high production of reactive oxygen species (ROS) and an increased in intracellular oxidative stress that affects the progress of Plasmodium erythrocytic cycle life and attenuates its serious clinical symptoms. Nevertheless, oxidative effects on P. falciparum proteome across the intraerythrocytic cycle in the presence of HbAS traits have not been described yet. Here, an immune dot-blot assay was used to quantify the carbonyl index (C.I) on P. falciparum 3D7 proteome at the different asexual erythrocytic stages. Protein carbonylation on parasites cultivated in erythrocytes from two donors with HbAS increased 5.34 ± 1.42 folds at the ring stage compared to control grown in hemoglobin A-A (HbAA) red blood cells. Whereas at trophozoites and schizonts stages were augmented 2.80 ± 0.52 and 3.05 ± 0.75 folds, respectively. Besides proteins involved in processes of the stress response, recognition and invasion were identified from schizonts carbonylated bands by combining SDS-PAGE with MALDI-TOF-TOF analysis. Our results reinforce the hypothesis that such oxidative modifications do not appear to happen randomly, and the sickle cell trait affects mainly a small fraction of parasite proteins particularly sensitive to ROS.

Polymerization of hemoglobin S is a major cause of morbidity and mortality in sickle cell disease, which leads to sickling and destruction of red blood cell. Alizarin, a bioactive compound from Rubia cordifolia , is reported to be blood purifier. This study investigates the potential of alizarin as an anti-sickling agent, showing a significant decrease in the rate of polymerization, therefore inhibiting the rate of sickling with increasing concentration. Interaction studies indicated that the fluorescence intensity of sickle hemoglobin (Hb S) decreases gradually with increasing alizarin concentration. This suggests the static quenching, where binding constant and the number of binding sites were deduced at different temperatures. The negative values of Gibbs energy change (Δ G 0 ) strongly suggest that it is entropy-driven spontaneous and exothermic reaction. Negative enthalpy (Δ H 0 ) and positive entropy (Δ S 0 ) stipulated that hydrogen and hydrophobic bonding forces were interfering in a hydrophobic micro-environment of β6Val leading to Hb S polymerization inhibition. In circular dichroism (CD) spectra, Hb S in the presence of alizarin shows helical structural changes leading to destabilization of Hb S polymer. These findings were also supported by molecular docking simulation studies using DOCK6 and GROMACS. So, from these findings, we may conclude that alizarin interacts with Hb S through hydrogen bonding and leading to inhibition of Hb S polymerization. Consequently, alizarin may have potential use as an anti-sickle cell medication for sickle cell disorder. Communicated by Ramaswamy H. Sarma.

The complex, frequently devastating, multi-organ pathophysiology of sickle cell disease has a single root cause: polymerization of deoxygenated sickle hemoglobin. A logical approach to disease modification is, therefore, to interdict this root cause. Ideally, such interdiction would utilize small molecules that are practical and accessible for worldwide application. Two types of such small molecule strategies are actively being evaluated in the clinic. The first strategy intends to shift red blood cell precursor hemoglobin manufacturing away from sickle hemoglobin and towards fetal hemoglobin, which inhibits sickle hemoglobin polymerization by a number of mechanisms. The second strategy intends to chemically modify sickle hemoglobin directly in order to inhibit its polymerization. Important lessons have been learnt from the pre-clinical and clinical evaluations to date. Open questions remain, but this review summarizes the valuable experience and knowledge already gained, which can guide ongoing and future efforts for molecular mechanism-based, practical and accessible disease modification of sickle cell disease., (Copyright© 2019 Ferrata Storti Foundation.)

Sickle cell disease is induced by a mutation that converts normal adult hemoglobin to sickle hemoglobin (HbS) and engenders intracellular polymerization of deoxy-HbS and erythrocyte sickling. Development of anti-sickling therapies requires quantitative understanding of HbS polymerization kinetics under organ-specific conditions, which are difficult to assess with existing experimental techniques. Thus, we developed a kinetic model based on the classical nucleation theory to examine the effectiveness of potential anti-sickling drug candidates. We validated this model by comparing its predictability against prior in vivo and in vitro experimental results. We used the model to quantify the efficacy of sickling inhibitors and obtain results consistent with recent screening assays. Global sensitivity analysis on the kinetic parameters in the model revealed that the solubility, nucleation rate prefactor, and oxygen affinity are quantities that dictate HbS polymerization. This finding provides quantitative guidelines for the discovery of intracellular processes to be targeted by sickling inhibitors.

Cell sickling is the process in which intracellular polymerization of deoxygenated sickle hemoglobin (HbS) leads to distorted, rigid cells, resulting in abnormal blood rheology and painful vaso-occlusion. Current methods for detection of this process mainly rely on optical microscopy of cellular morphology and measurements of cell deformability and blood rheology. As electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics, it can be a promising marker for characterization of abnormal blood rheology and a means more convenient than optics to be integrated into point-of-care devices. In this work, a microfluidics-based electrical impedance sensor has been developed for characterizing the dynamic cell sickling-unsickling processes in sickle blood. The sensor is capable of measuring the continuous variation in the sickle cell suspension due to cyclic hypoxia-induced intracellular HbS polymerization and depolymerization. Simultaneous microscopic imaging of cell morphological change shows the reliability and repeatability of the electrical impedance-based measurements of cell sickling and unsickling processes. Strong correlation is found between the electrical impedance measurement and patients' hematological parameters such as levels of HbS and fetal hemoglobin. The combination of electrical impedance measurement and on-chip hypoxia control provides a promising method for rapid assessment of the dynamic processes of cell sickling and unsickling in patients with sickle cell disease.

The pathophysiology associated with sickle cell disease (SCD) includes hemolytic anemia, vaso-occlusive events, and ultimately end organ damage set off by the polymerization of deoxygenated hemoglobin S (HbS) into long fibers and sickling of red blood cells (RBCs). One approach toward mitigating HbS polymerization is to pharmacologically stabilize the oxygenated (R) conformation of HbS and thereby reduce sickling frequency and SCD pathology. GBT440 is an α-subunit-specific modifying agent that has recently been reported to increase HbS oxygen binding affinity and consequently delay in vitro polymerization. In addition, animal model studies have demonstrated the potential for GBT440 to be a suitable therapeutic for daily oral dosing in humans. Here, we report an optimized method for detecting GBT440 intermediates in human patient hemolysate using a combination of HPLC and mass spectrometry analysis. First, oxygen dissociation curves (ODCs) analyzed from patient blood showed that oxygen affinity increased in a dose dependent manner. Second, HPLC and integrated mass spectrometric analysis collectively confirmed that GBT440 labeling was specific to the α N-terminus thereby ruling out other potential ligand binding sites. Finally, the results from this optimized analytical approach allowed us to detect a stable α-specific GBT440 adduct in the patient's hemolysate in a dose dependent manner. The results and methods presented in this report could therefore potentially help therapeutic monitoring of GBT440 induced oxygen affinity and reveal critical insight into the biophysical properties of GBT440 Hb complexes.

In sickle cell disease, the aberrant assembly of hemoglobin fibers induces changes in red blood cell morphology and stiffness, which leads to downstream symptoms of the disease. Therefore, understanding of this assembly process will be important for the treatment of sickle cell disease. By performing the highest spatiotemporal resolution measurements (55 nm at 1 Hz) of single sickle hemoglobin fiber assembly to date and combining them with a model that accounts for the multistranded structure of the fibers, we show that the rates of sickle hemoglobin addition and loss have been underestimated in the literature by at least an order of magnitude. These results reveal that the sickle hemoglobin self-assembly process is very rapid and inefficient (4% efficient versus 96% efficient based on previous analyses), where net growth is the small difference between over a million addition-loss events occurring every second.

Sickle cell disease is caused by the amino acid substitution of glutamic acid to valine, which leads to the polymerization of deoxygenated sickle hemoglobin (HbS) into long strands. These strands are responsible for the sickling of red blood cells (RBCs), making blood hyper-coagulable leading to an increased chance of vaso-occlusive crisis. The conformational changes in sickled RBCs traveling through narrow blood vessels in a highly viscous fluid are critical in understanding; however, there are few studies that investigate the origins of the molecular mechanical behavior of sickled RBCs. In this work, we investigate the molecular mechanical properties of HbS molecules. A mechanical model was used to estimate the directional stiffness of an HbS molecule and the results were compared to adult human hemoglobin (HbA). The comparison shows a significant difference in strength between HbS and HbA, as well as anisotropic behavior of the hemoglobin molecules. The results also indicated that the HbS molecule experienced more irreversible mechanical behavior than HbA under compression. Further, we have characterized the elastic and compressive properties of a double stranded sickle fiber using six HbS molecules, and it shows that the HbS molecules are bound to each other through strong inter-molecular forces.

Impact Statement: Sickle cell disease (SCD) is one of the most common inherited diseases and is associated with a reduced life expectancy and acute and chronic complications, including frequent painful vaso-occlusive episodes that often require hospitalization. At present, treatment of SCD is limited to hematopoietic stem cell transplant, transfusion, and limited options for pharmacotherapy, based principally on hydroxyurea therapy. This review highlights the importance of intracellular cGMP-dependent signaling pathways in SCD pathophysiology; modulation of these pathways with soluble guanylate cyclase (sGC) stimulators or phosphodiesterase (PDE) inhibitors could potentially provide vasorelaxation and anti-inflammatory effects, as well as elevate levels of anti-sickling fetal hemoglobin.

The polymerization of the mutant hemoglobin S upon deoxygenation to form fibers in red blood cells of patients suffering from sickle-cell anemia results in changes in cell shape and rigidity, also known as sickling, which underlie the pathology of the disease. While much has been learned about the fundamental physical chemistry of the polymerization process, transferring these insights to sickling of red cells under in vivo conditions requires being able to monitor, and ultimately predict, the time course of cellular sickling under physiological conditions of deoxygenation. To this end, we have developed an experimental technique for tracking the temporal evolution of the sickling of red blood cells under laboratory deoxygenation conditions, based on the automated analysis of sequences of microscope images and machine-learning analysis to characterize cell morphology. As an aid in the quantitative understanding of these experiments, we have developed a computational framework for simulating the time dependence of sickling in populations of red blood cells which incorporates the current theoretical and empirical understanding of the physical chemistry of the sickling process. In order to apply these techniques to our experiments, we have theoretically determined the time course of deoxygenation by solving the diffusion equation for oxygen in our experimental geometry. With this combined description, we are able to reproduce our experimentally observed kinetics of sickling, suggesting that our theoretical approach should be applicable to physiological deoxygenation scenarios.

Proteins that function in aqueous solution can be perturbed by the solvent. Here we present experimental studies on two such interactions in the hemoglobin molecule. (1) Hemoglobin's oxygen binding is altered by introduction of crowding species or osmoticants, such as sucrose, through the linked binding of ions such as Cl or CO 2 , but not otherwise. This rules out a significant role of buried surface in the allosteric energetics. (2) Sickle hemoglobin (HbS) polymerizes more readily in high concentrations of phosphate buffer. Such polymerization is analyzed quantitatively here for the first time in terms of the double nucleation mechanism. The changes in solubility are found to account for the increase in monomer addition rates and nucleation rates without requiring additional parameter adjustments. In the analysis, we also show how the analytical formulation of HbS nucleation may be adapted to include water that occupies the interstices between the assembled molecules. While such a "correction" has been applied to the equilibrium process, it has not previously been applied to the nucleation process.

Although homozygous sickle cell disease is often clinically severe, the corresponding heterozygous state, sickle cell trait, is almost completely benign despite the fact that there is only a modest difference in sickle hemoglobin levels between the two conditions. In both conditions, hypoxia can lead to polymerization of sickle hemoglobin, changes in red cell mechanical properties, and impaired blood flow. Here, we test the hypothesis that differences in the oxygen-dependent rheological properties in the two conditions might help explain the difference in clinical phenotypes. We use a microfluidic platform that permits quantification of blood rheology under defined oxygen conditions in physiologically sized microchannels and under physiologic shear rates. We find that, even with its lower sickle hemoglobin concentration, sickle trait blood apparent viscosity increases with decreasing oxygen tension and may stop flowing under completely anoxic conditions, though far less readily than the homozygous condition. Sickle cell trait blood flow becomes impaired at significantly lower oxygen tension than sickle cell disease. We also demonstrate how sickle cell trait can serve as a benchmark for sickle cell disease therapies. We characterize the rheological effects of exchange transfusion therapy by mixing sickle blood with nonsickle blood and quantifying the transfusion targets for sickle hemoglobin composition below which the rheological response resembles sickle trait. These studies quantify the differences in blood flow phenotypes of sickle cell disease and sickle cell trait, and they provide a potentially powerful new benchmark for evaluating putative therapies in vitro., (© 2018 Wiley Periodicals, Inc.)

Increasing the affinity of hemoglobin for oxygen represents a feasible and promising therapeutic approach for sickle cell disease by mitigating the primary pathophysiological event, i.e. the hypoxia-induced polymerization of sickle hemoglobin (Hb S) and the concomitant erythrocyte sickling. Investigations on a novel synthetic antisickling agent, SAJ-310, with improved and sustained antisickling activity have previously been reported. To further enhance the biological effects of SAJ-310, a structure-based approach was employed to modify this compound to specifically inhibit Hb S polymer formation through interactions which perturb the Hb S polymer-stabilizing αF-helix, in addition to primarily increasing the oxygen affinity of hemoglobin. Three compounds, TD-7, TD-8 and TD-9, were synthesized and studied for their interactions with hemoglobin at the atomic level, as well as their functional and antisickling activities in vitro. X-ray crystallographic studies with liganded hemoglobin in complex with TD-7 showed the predicted mode of binding, although the interaction with the αF-helix was not as strong as expected. These findings provide important insights and guidance towards the development of molecules that would be expected to bind and make stronger interactions with the αF-helix, resulting in more efficacious novel therapeutics for sickle cell disease.

Protein aggregation is associated with various diseases, including Alzheimer and Parkinson as well as sickle cell disease (SCD). From a molecular point of view, protein aggregation depends on a complex balance of electrostatic and hydrophobic interactions mediated by water. An impressive manifestation of the importance of this balance concerns the human hemoglobin (HbA) mutant, HbS (sickle cell Hb), where a single substitution at the 6th position of HbA β-chains, from glutamic acid to valine, causes the polymerization of deoxygenated HbS (deoxy-HbS), responsible for SCD. HbS polymerization is believed to occur via a double nucleation mechanism initiated by the formation of HbS fibers (homogeneous nucleation), followed by fiber growth. Furthermore, it was proposed that homogeneous nucleation proceeds through a two-step mechanism, where metastable dense clusters play the role of nucleation precursors. Thus, hindering or delaying the formation of such precursors could represent a potential SCD therapeutic route. Here, we study, through molecular dynamics, the binding free energy and protein-protein contacts involved in the deoxy-HbS dimer aggregation and stabilization process. A binding free energy of ∼-14.0 ± 1 kcal/mol is estimated from a one-dimensional potential of mean force. Analysis of protein-protein interactions shows that both electrostatic and van der Waals interactions play an important role on the aggregation of HbS. With respect to the former, our results indicate that aggregation is largely favored by the formation of salt bridges (SB), mostly, Lys-Glu, Lys-Asp, and Heme-Lys SB, which outweigh electrostatic repulsions involving similar residues. Thus, our results suggest that a potential antisickling drug could be one with the ability to weaken or hinder the formation of a few SB between carboxylate and ammonium groups.

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of βVal6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of βCys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of βVal6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The mutation of β6 from glu to val in hemoglobin is responsible for the polymer formation that leads to vaso-occlusion, and a range of severe consequences in sickle cell disease. The treatment of the disease can be addressed in many ways, but the prevention of polymer formation is one of the most fundamental approaches one can take. Such prevention includes affecting the polymer structure, or dilution of the fraction of polymerizable hemoglobin. The latter approach includes (1) induction of HbF, which does not itself, nor in hybrid form, join sickle polymers, or (2) restricting the allosteric change in hemoglobin that occurs in oxygen delivery, and which is required for polymer formation. These approaches will be critically reviewed, as well as the most recent developments that show the benefits of simply swelling the volume of the red cell., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A new variant of HbS, HbS-Einstein with a deletion of segment α 23-26 in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain. The design of HbS-Einstein has been undertaken to map the influence of perturbation of molecular surface topology and the flexibility of surface residues in the polymerization. The internal deletion exerts a strong inhibitory influence on Val-6 (β)-dependent polymerization, comparable to single contact site mutations and not for complete neutralization of Val-6(β)-dependent polymerization. The scaffold modification in cis-dimer is inhibitory, and is without any effect when present on the trans dimer. The flexibility changes in the surface topology in the region of scaffold modification apparently counteracts the intrinsic polymerization potential of the molecule. The inhibition is close to that of Le Lamentin mutation [His-20 (α) → Gln] wherein a mutation engineered without much change in flexibility of the contact domain. Interestingly, the chimeric HbS with swine-human chimeric α chain with multiple non-conservative mutations completely inhibits the Val-6(β)-dependent polymerization. The deformabilities of surface topology of chimeric HbS are comparable to HbS in spite of the multiple contact site mutations in the α-chain. We conclude that the design of antisickling Hbs for gene therapy of sickle cell disease should involve multiple mutations of intermolecular contact sites.

Recently, nanoparticles have attracted much attention as new scaffolds for hemoglobin-based oxygen carriers (HBOCs). Indeed, the development of bionanotechnology paves the way for the rational design of blood substitutes, providing that the interaction between the nanoparticles and hemoglobin at a molecular scale and its effect on the oxygenation properties of hemoglobin are finely controlled. Here, we show that human hemoglobin has a high affinity for silica nanoparticles, leading to the adsorption of hemoglobin tetramers on the surface. The adsorption process results in a remarkable retaining of the oxygenation properties of human adult hemoglobin and sickle cell hemoglobin, associated with an increase of the oxygen affinity. The cooperative oxygen binding exhibited by adsorbed hemoglobin and the comparison with the oxygenation properties of diaspirin cross-linked hemoglobin confirmed the preservation of the tetrameric structure of hemoglobin loaded on silica nanoparticles. Our results show that silica nanoparticles can act as an effector for human native and mutant hemoglobin. Manipulating hemoglobin oxygenation using nanoparticles opens the way to the design of novel HBOCs., (© 2018 by The American Society of Hematology.)

The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential. We used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to screen for HbS-binding RNA aptamers modified with nuclease-resistant 2'-fluoropyrimidines. Polymerization assays were employed to identify aptamers with polymerization-inhibitory properties. Two noncompeting aptamers, DE3A and OX3B, were found to bind hemoglobin, significantly increase the delay time, and reduce the rate of polymerization of HbS. These modifiable, nuclease-resistant aptamers are potential new therapeutic agents for SCD.

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Hemoglobin, the vital O 2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

Understanding of intracellular polymerization of sickle hemoglobin (HbS) and subsequent interaction with the membrane of a red blood cell (RBC) is important to predict the altered morphologies and mechanical properties of sickle RBCs in sickle cell anemia. However, modeling the integrated processes of HbS nucleation, polymerization, HbS fiber interaction, and subsequent distortion of RBCs is challenging as they occur at multispatial scales, ranging from nanometers to micrometers. To make progress toward simulating the integrated processes, we propose a hybrid HbS fiber model, which couples fine-grained and coarse-grained HbS fiber models through a mesoscopic adaptive resolution scheme (MARS). To this end, we apply a microscopic model to capture the dynamic process of polymerization of HbS fibers, while maintaining the mechanical properties of polymerized HbS fibers by the mesoscopic model, thus providing a means of bridging the subcellular and cellular phenomena in sickle cell disease. At the subcellular level, this model can simulate HbS polymerization with preexisting HbS nuclei. At the cellular level, if combined with RBC models, the generated HbS fibers could be applied to study the morphologies and membrane stiffening of sickle RBCs. One important feature of the MARS is that it can be easily employed in other particle-based multiscale simulations where a dynamic coarse-graining and force-blending method is required. As demonstrations, we first apply the hybrid HbS fiber model to simulate the interactions of two growing fibers and find that their final configurations depend on the orientation and interaction distance between two fibers, in good agreement with experimental observations. We also model the formation of fiber bundles and domains so that we explore the mechanism that causes fiber branching., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by the isopropyl group of βVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date. In the present study, we have attempted to understand the molecular mechanism of inhibition of polymerization by exploiting the exchange of backbone amide hydrogens of HbS with deuterated solvent. Hydrogen/deuterium exchange kinetics of peptide amide hydrogens of both oxy and deoxy form of HbS were monitored through ESI mass spectrometry. Upon oxygenation changes in the conformational flexibility across different regions of α and β globin chains in the tetrameric HbS molecule were investigated. It was observed that oxygenation led to perturbation in the conformation of several residues around the hydrophobic patch, groove of a tetramer and axial, lateral contacts across the double strands that are involved in HbS polymerization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients., (© 2016 John Wiley & Sons Ltd.)

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., Competing Interests: Dr M.K. Safo is a co-owner of a patent for the use of 5-HMF in sickle cell disease, and receives research funding from AesRx, LLC, a licensee for 5-HMF (Aes-103/Bax-555). Dr G.J. Kato has collaborated with and received research funding from AesRx, LLC, through a Clinical Trials Agreement between AesRx, LLC and the National Heart, Lung and Blood Institute, and has received consulting fees from Baxalta, current holder of the license for 5-HMF (Aes-103/Bax-555), and research funding from Bayer HealthCare Pharmaceuticals Inc., (© 2016 John Wiley & Sons Ltd.)

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells

Background: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (βA) and HbS beta chain (βS) for the most common Hb variant trait (HbAS) are examined., Methods: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms., Results: Glycation of βS chains was significantly higher (p<0.001) than βA chains; this difference was consistent across subjects. Total (α+β) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA., Conclusion: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of βS and βA. Although βS glycation was significantly higher than that of βA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS., (Copyright © 2016 Elsevier B.V. All rights reserved.)