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.

Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.

Cross-linked hemoglobin-azides react with a bis-alkyne to form a bis-tetramer through sequential "click" reactions where the second step is promoted by the first.

The clinical evaluation of stabilized tetrameric hemoglobin as alternatives to red cells revealed that the materials caused significant increases in blood pressure and related problems and this was attributed to the scavenging of nitric oxide and extravasation. The search for materials with reduced vasoactivity led to the report that conjugates of hemoglobin tetramers and polyethylene glycol (PEG) chains did not elicit these pressor effects. However, this material does not deliver oxygen efficiently due to its lack of cooperativity and high oxygen affinity, making it unsuitable as an oxygen carrier. It has been recently reported that PEGconjugated hemoglobin converts nitrite to nitric oxide at a faster rate than does the native protein, which may compensate for the scavenging of nitric oxide. It is therefore important to alter hemoglobin in order to enhance nitrite reductase activity while retaining its ability to deliver oxygen. If the beneficial effect of PEG is associated with the increased size reducing extravasation, this can also be achieved by coupling cross-linked tetramers to one another, giving materials with appropriate oxygen affinity and cooperativity for use as circulating oxygen carriers. In the present study it is shown that cross-linked bis-tetramers with good oxygen delivery potential have enhanced nitrite reductase activity with k obs = 0.70 M -1 s -1 (24 °C), compared to native protein and cross-linked tetramers, k obs = 0.25 M -1 s -1 and k obs = 0.52 M -1 s -1 , respectively, but are less active in reduction of nitrite than Hb-PEG5K 2 (k obs = 2.5 M -1 s -1 ). However, conjugation of four PEG chains to the bis-tetramer (at each β-Cys-93) produces a material with greatly increased nitrite reductase activity (k obs = 1.8 M -1 s -1 ) while retaining cooperativity (P 50 = 4.1, n 50 = 2.4). Thus, PEGylated bistetramers combine increased size and enhanced nitrite reductase activity expected for decreased vasoactivity with characteristics of an acceptable HBOC.

The enlarged Hb derivatives do not evoke unfavorable circulatory responses that have been noted to result from infusion of Hb derivatives. These results suggest that a compromise between the P(50) , n(50) , and nitrite reductase activity of a Hb derivative can serve as the basis for producing HBOCs that can be tested for vasoactivity.

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Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be finetuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.antibody therapeutic | Frizzled receptors | Wnt signaling | X-ray crystallography | protein engineering W nt signaling is an evolutionarily conserved signaling cascade that plays a critical role in diverse biological processes, including embryonic development, tissue differentiation, organogenesis, stem cell maintenance, and normal adult tissue homeostasis (1-3). Activation of the well-characterized β-catenindependent canonical pathway is initiated by the binding of secreted Wnt proteins to Frizzled (FZD) receptors and coreceptors such as LRP5 and LRP6 (4-8). To date, 19 human Wnt and 10 FZD receptors have been identified that mediate differential cellular functions (9, 10). FZD receptors interact with Wnt through their N-terminal extracellular cysteine-rich domain (CRD). A Wnt residue, Ser187, (position number according to Xenopus Wnt8) is posttranslationally modified, leading to its palmitoleation, which mediates interactions with the FZD-CRD (site 1); a second site of interaction between FZD and Wnt is located at the opposing end of Wnt (11).Wnt/FZD signaling is essential for normal cell function, but aberrations in the pathway are frequently found in cancers, fibrosis, and degenerative diseases (12,13). Abnormal activation of the Wnt pathway is an essential driver of primary tumor formation and metastasis in multiple cancer types (14-18). Inactivating mutations in E3 ubiquitin ligase RNF43 inhibit the downmodulation of FZD expression on the cell surface and sensitize tumor cells to Wnt-dependent growth. These mutations have been identified in pancreatic, biliary duct, and colorectal cancers (19-21). FZD5 expression is up-regulated in renal cell carcinoma (22), prostate cancer (23), and pancreatic tumors (16); aberrant FZD7 expression is observed in hepatocellular carcinoma and colorectal and triple negative breast cancer (14,24,25); FZD8 is upregulated in acute lymphoblastic leukemia and lung cancer (17, 26); and FZD4 is elevated and drives epithelial-to-mesenchy...

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