Person:

Zapol, Warren

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.

Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca2+) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca2+ channel inhibitors, 2-ABP, and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca2+ signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca2+ in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.

Background. Children with cerebral malaria (CM) have high rates of mortality and neurologic sequelae. Nitric oxide (NO) metabolite levels in plasma and urine are reduced in CM. Methods. This randomized trial assessed the efficacy of inhaled NO versus nitrogen (N2) as an adjunctive treatment for CM patients receiving intravenous artesunate. We hypothesized that patients treated with NO would have a greater increase of the malaria biomarker, plasma angiopoietin-1 (Ang-1) after 48 hours of treatment. Results. Ninety-two children with CM were randomized to receive either inhaled 80 part per million NO or N2 for 48 or more hours. Plasma Ang-1 levels increased in both treatment groups, but there was no difference between the groups at 48 hours (P = not significant [NS]). Plasma Ang-2 and cytokine levels (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1β, IL-6, IL-10, and monocyte chemoattractant protein-1) decreased between inclusion and 48 hours in both treatment groups, but there was no difference between the groups (P = NS). Nitric oxide metabolite levels—blood methemoglobin and plasma nitrate—increased in patients treated with NO (both P < .05). Seven patients in the N2 group and 4 patients in the NO group died. Five patients in the N2 group and 6 in the NO group had neurological sequelae at hospital discharge. Conclusions. Breathing NO as an adjunctive treatment for CM for a minimum of 48 hours was safe, increased blood methemoglobin and plasma nitrate levels, but did not result in a greater increase of plasma Ang-1 levels at 48 hours.

Background: Volatile anesthetics increase levels of the neurotransmitter nitric oxide (NO) and the secondary messenger molecule cyclic guanosine monophosphate (cGMP) in the brain. NO activates the enzyme guanylyl cyclase (GC) to produce cGMP. We hypothesized that the NO-GC-cGMP pathway contributes to anesthesia-induced unconsciousness. Methods: Sevoflurane-induced loss and return of righting reflex (LORR and RORR, respectively) were studied in wild-type mice (WT) and in mice congenitally deficient in the GC-1α subunit (GC-1−/− mice). Spatial distributions of GC-1α and the GC-2α subunit in the brain were visualized by in situ hybridization. Brain cGMP levels were measured in WT and GC-1−/− mice after inhaling oxygen with or without 1.2% sevoflurane for 20 min. Results: Higher concentrations of sevoflurane were required to induce LORR in GC-1−/− mice than in WT mice (1.5 ± 0.1 vs. 1.1 ± 0.2%, respectively, n = 14 and 14, P < 0.0001). Similarly, RORR occurred at higher concentrations of sevoflurane in GC-1−/− mice than in WT mice (1.0 ± 0.1 vs. 0.8 ± 0.1%, respectively, n = 14 and 14, P < 0.0001). Abundant GC-1α and GC-2α mRNA expression was detected in the cerebral cortex, medial habenula, hippocampus, and cerebellum. Inhaling 1.2% sevoflurane for 20 min increased cGMP levels in the brains of WT mice from 2.6 ± 2.0 to 5.5 ± 3.7 pmol/mg protein (n = 13 and 10, respectively, P = 0.0355) but not in GC-1−/− mice. Conclusion: Congenital deficiency of GC-1α abolished the ability of sevoflurane anesthesia to increase cGMP levels in the whole brain, and increased the concentration of sevoflurane required to induce LORR. Impaired NO-cGMP signaling raises the threshold for producing sevoflurane-induced unconsciousness in mice.

An elevated intracellular NADH/NAD+ ratio, or “reductive stress,” has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain (ETC). As the intracellular NADH/NAD+ ratio can be in near-equilibrium with the circulating lactate/pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate into pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of recombinant LOXCAT to the media of cultured human cells with a defective ETC was able to decrease the extracellular lactate/pyruvate ratio, normalize the intracellular NADH/NAD+ ratio, upregulate glycolytic ATP production, and restore cellular proliferation. In mice, tail-vein injected LOXCAT reduced circulating lactate/pyruvate ratio, blunted a metformin-induced rise in blood lactate/pyruvate, and improved NADH/NAD+ balance in heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviate intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.

Chronic hypoxia induces pulmonary hypertension and right ventricular (RV) hypertrophy. Nitric oxide (NO) has been proposed to modulate the pulmonary vascular response to hypoxia. We investigated the effects of congenital deficiency of endothelial NO synthase (NOS3) on the pulmonary vascular responses to breathing 11% oxygen for 3-6 wk. After 3 wk of hypoxia, RV systolic pressure was greater in NOS3-deficient than in wild-type mice (35+/-2 vs 28+/-1 mmHg, x+/-SE, P < 0.001). Pulmonary artery pressure (PPA) and incremental total pulmonary vascular resistance (RPI) were greater in NOS3-deficient than in wild-type mice (PPA 22+/-1 vs 19+/-1 mmHg, P < 0.05 and RPI 92+/-11 vs 55+/-5 mmHg.min.gram.ml-1, P < 0.05). Morphometry revealed that the proportion of muscularized small pulmonary vessels was almost fourfold greater in NOS3-deficient mice than in wild-type mice. After 6 wk of hypoxia, the increase of RV free wall thickness, measured by transesophageal echocardiography, and of RV weight/body weight ratio were more marked in NOS3-deficient mice than in wild-type mice (RV wall thickness 0.67+/-0.05 vs 0.48+/-0.02 mm, P < 0.01 and RV weight/body weight ratio 2.1+/-0.2 vs 1.6+/-0.1 mg. gram-1, P < 0.05). RV hypertrophy produced by chronic hypoxia was prevented by breathing 20 parts per million NO in both genotypes of mice. These results suggest that congenital NOS3 deficiency enhances hypoxic pulmonary vascular remodeling and hypertension, and RV hypertrophy, and that NO production by NOS3 is vital to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress.