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

Optimizing the lyophilization of Lumbricus terrestris erythrocruorin

AbstractHaemorrhagic shock is a leading cause of death worldwide. Blood transfusions can be used to treat patients suffering severe blood loss but donated red blood cells (RBCs) have several limitations that limit their availability and use. To solve the problems associated with donated RBCs, several acellular haemoglobin-based oxygen carriers (HBOCs) have been developed to restore the most important function of blood: oxygen transport. One promising HBOC is the naturally extracellular haemoglobin (i.e. erythrocruorin) of Lumbricus terrestris (LtEc). The goal of this study was to maximise the portability of LtEc by lyophilising it and then testing its stability at elevated temperatures. To prevent oxidation, several cryoprotectants were screened to determine the optimum formulation for lyophilisation that could minimise oxidation of the haem iron and maximise recovery. Furthermore, samples were also deoxygenated prior to storage to decrease auto-oxidation, while resuspension in a solution containing ascorbic acid was shown to partially reduce LtEc that had oxidised during storage (e.g. from 42% Fe3+ to 11% Fe3+). Analysis of the oxygen equilibria and size of the resuspended LtEc showed that the lyophilisation, storage, and resuspension processes did not affect the oxygen transport properties or the structure of the LtEc, even after 6 months of storage at 40 °C. Altogether, these efforts have yielded a shelf-stable LtEc powder that can be stored for long periods at high temperatures, but future animal studies will be necessary to prove that the resuspended product is a safe and effective oxygen transporter in vivo

HeLa cell growth is not affected by doxycycline supplement.

<p>Values are the mean of three independent cultures; error bars indicate ±1 S.E.</p

Experimental schemata for transcriptional chase analyses of mRNA stability.

<p>(A) Conventional chase method. Identical aliquots cultured in doxycycline (dox)-supplemented media (thick line) are sacrificed at defined intervals (arrowheads). A hypothetical 80-hr chase experiment is illustrated. (B) Reverse-chase method. Identical aliquots cultured in dox-free media (thin line) are amended with dox at defined intervals, and sacrificed simultaneously at the conclusion of the experiment.</p

HeLa cell expansion under transcriptional chase conditions.

<p>Data from dox-supplementation experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g002" target="_blank">Fig. 2</a> (1 µg/mL) was regressed to an exponential function, and expansion factors defined [j = 0.0224 (h) or 0.0004 (min)].</p

Comparative transcriptional chase analyses of a long-lived test mRNA.

<p>(A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t₀, and sacrificed at defined intervals. Levels of β^WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β^WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β^WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t_1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t_1/2 values calculated following correction for interval cell expansion.</p

Values used in the current report.

<p>Values used in the current report.</p