Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors.

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Contribution of disulfide bonds and calcium to Molluscan hemocyanin stability.

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.

Influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the antigen-presenting activity of dendritic cells.

We have previously shown that exposure of mice to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induces activation-like changes in splenic dendritic cells (DC) in the absence of antigen challenge. Since activation of DC reduces their ability to phagocytize antigen, we examined the effects of TCDD on the ability of DC to process and present antigen to antigen-specific T cells and to internalize latex beads. Additionally, the expression of costimulatory and adhesion molecules was examined on DC from TCDD-treated mice injected with allogeneic tumor cells. The ability of DC from C57Bl/6 mice to induce proliferation of keyhole limpet hemocyanin (KLH)-specific 10.5.17 T cells and production of IL-4 was not significantly altered by TCDD exposure, either when KLH was added in vitro or when the mice were injected with KLH prior to DC isolation. In contrast, ovalbumin (OVA) presentation by DC from TCDD-treated Balb/c mice induced enhanced proliferation of OVA-specific D011.10 T cells, although the production of IL-2 and IFN-gamma was not affected. Enhanced in vivo proliferation of adoptively transferred, CFSE-labeled DO11.10 T cells was also observed in TCDD-treated Balb/c mice that were challenged with OVA. TCDD treatment modulated the expression of major histocompatibility complex (MHC) class II, CD24, ICAM-1, CD40, and LFA-1 on splenic DC from C57Bl/6 mice injected with allogeneic tumor cells; however, the effects of TCDD were identical to changes seen previously in nonimmune mice, suggesting that these effects were not antigen-dependent. Finally, TCDD treatment did not affect the ability of splenic DC to internalize latex beads administered in vivo. Taken together, these results suggest that the activation-like changes induced in DC by TCDD do not suppress the ability of DC to process and present antigen, but may enhance their ability to provide activation signals to T cells. This, in turn, may alter the survival of the T cells, the DC, or both, and might lead to dysregulation of the immune response.

Study on the oxyhemocyanin, deoxyhemocyanin, oxygen affinity and acid-base balance of Marsupenaeus japonicus following exposure to combined elevated nitrite and nitrate.

Marsupenaeus japonicus (11.47+/-0.71 g) exposed individually to six different nitrite and nitrate regimes [(0.002 (control), 0.359 and 1.456 mM nitrite combined with 0.005 (control) and 7.458 mM nitrate)] in salinity of 30 ppt (parts per thousand) were examined for hemocyanin oxygen affinity, the fractionation of oxyhemocyanin and deoxyhemocyanin, and the acid-base balance after 24 h. Ambient nitrite at concentration of 0.359 mM caused reduction of oxyhemocyanin and protein by 27 and 11%, respectively, whereas ambient nitrate as high as 7.458 mM caused reduction of oxyhemocyanin and protein by 10 and 7%. Ambient nitrite at concentration of 1.456 mM caused increases of P(50) (indicating reduced oxygen affinity) and pO(2), but caused reduction in hemolymph pCO(2), pH, HCO(3)(-) and TCO(2). Following exposure to combined solutions of 1.456 mM nitrite and 7.458 mM nitrate there were no further changes in oxyhemocyanin, protein, hemolymph P(50), pO(2), pCO(2), HCO(3)(-) and TCO(2), but there was a significant reduction of pH.

Active-site disruption in native Limulus hemocyanin and its subunits by disulfide-bond reductants: a chemical probe for the study of structure-function relationships in the hemocyanins.

The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins.

Extreme thermostability of tarantula hemocyanin.

Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24-meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90 degrees C. Our results demonstrate that the cation-mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.

Atrazine toxicity on transport properties of hemocyanin in the crab Oziotelphusa senex senex.

The current study reports the possible changes in the copper metabolism of the crab Oziotelphusa senex senex exposed to a sublethal concentration of atrazine. The alterations observed in the synthesis of pigment, hemocyanin, and its affinity to oxygen suggest the existence of respiratory distress in the crab as a consequence of atrazine toxicity.

DNA adducts of nitropyrene detected by specific antibodies.

Rabbit antisera containing polyclonal antibodies specific for the 1-nitropyrene derivatives, (1-acetylaminopyrene, 1-acetylamino-6-nitropyrene, 1-acetylamino-8-nitropyrene) and the major nitropyrene-DNA adduct, C-8-aminopyrene-deoxyguanosine, were obtained from New Zealand White male rabbits that were immunized with 1-nitrosopyrene-modified keyhole limpet hemocyanin (KLH). The affinity constants of the rabbit antisera for these derivatives ranged from 1 to 3 x 10(8) liters/mole. The ability of the antisera to detect 1-nitrosopyrene and the parent 1-nitropyrene was 25- to 30-fold less than the sensitivity to other metabolites. Female BALB/c and AJ mice were also immunized with 1-nitrosopyrene-modified KLH and 4 out of 18 surviving animals produced a low titer response when measured by an [3H] acetylaminopyrene-based radioimmunoassay. Mice that were immunized with a diazotized derived 1-aminopyrene bovine gamma globulin, 1-nitrosopyrene adducted bovine gamma globulin, and 1-nitrosopyrene-bound bovine serum albumin, produced very low immune responses. Spleen cells from selected mice were fused with myeloma cells but failed to produce stable clones that secreted nitropyrene-specific monoclonal antibodies. Therefore, the use of a 1-nitrosopyrene modified keyhole limpet hemocyanin to elicit an immune response specific for the nitropyrene moiety in one animal species (rabbit) was successful in producing a specific antisera. The immune response produced in mice and rabbits was much lower when compared to that produced by other chemically derived antigens we have used, such as the aflatoxins and 4-aminobiphenyl. The rabbit data encourages a continued attempt to produce monoclonal antibodies specific for nitropyrene. Such antibodies can be used in the development of preparative and analytical techniques to isolate and quantify nitropyrenes in biological samples from exposed human populations.