Non-electrostatic interactions associated with aggregate formation between polyallylamine and Escherichia coli.

Bacterial aggregation by mixing with polymers is applied as pretreatment to identify pathogens in patients with infectious diseases. However, the detailed interaction between polymers and bacteria has yet to be fully understood. Here, we investigate the interaction between polyallylamine and Escherichia coli by isothermal titration calorimetry. Aggregation was observed at pH 10 and the binding was driven by favorable enthalpic gain such as the electrostatic interaction. Neither aggregation nor the apparent heat of binding was observed at pH 4.0, despite the strong positive charge of polyallylamine. These results suggest that intermolecular repulsive forces of the abundant positive charge of polyallylamine cause an increased loss of conformational entropy by binding. Non-electrostatic interaction plays a critical role for aggregation.

Structural and interaction analysis of human lipocalin-type prostaglandin D synthase with the poorly water-soluble drug NBQX.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a secretory lipid-transporter protein that was shown to bind a wide variety of hydrophobic ligands in vitro. Exploiting this function, we previously examined the feasibility of using L-PGDS as a novel delivery vehicle for poorly water-soluble drugs. However, the mechanism by which human L-PGDS binds to poorly water-soluble drugs is unclear. In this study, we determined the solution structure of human L-PGDS and investigated the mechanism of L-PGDS binding to 6-nitro-7-sulfamoyl-benzo[f]quinoxalin-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. NMR experiments showed that human L-PGDS has an eight-stranded antiparallel ß-barrel structure that forms a central cavity, a short 310 -helix and two α-helices. Titration with NBQX was monitored using 1 H-15 N HSQC spectroscopy. At higher NBQX concentrations, some cross-peaks of the protein exhibited fast-exchanging shifts with a curvature, indicating at least two binding sites. These residues were located in the upper portion of the cavity. Singular value decomposition analysis revealed that human L-PGDS has two NBQX binding sites. Large chemical shift changes were observed in the H2-helix and A-, B-, C-, D-, H- and I-strands and H2-helix upon NBQX binding. Calorimetric experiments revealed that human L-PGDS binds two NBQX molecules with dissociation constants of 46.7 µm for primary binding and 185.0 µm for secondary binding. Molecular docking simulations indicated that these NBQX binding sites are located within the ß-barrel. These results provide new insights into the interaction between poorly water-soluble drugs and human L-PGDS as a drug carrier.

Thermodynamic stability of human lipocalin-type prostaglandin D synthase under various pH conditions.

Lipocalin-type prostaglandin D synthase (L-PGDS) binds various hydrophobic small molecules. Since we aim to use human L-PGDS as a carrier in a drug delivery system (DDS) for poorly water-soluble drugs, quality control of the protein is indispensable. In this study, we investigated the thermodynamic stability of human L-PGDS under various pH conditions. Differential scanning calorimetry revealed that the thermal unfolding of L-PGDS was an almost-reversible two-state transition between the native and unfolded states over the pH range from 2.5 to 7.4. The linear relationship of ΔH(Tm) to Tm in this pH range gave a heat capacity change (ΔCp) of 4.76 kJ/(K·mol), which was small compared to those commonly found in globular proteins. The temperature-dependent free energy of unfolding, ΔG(T), specified by Tm, ΔH(Tm) and ΔCp, showed a pH dependence with the highest value at pH 7.4 closest to the isoelectric point of 8.3. The small value of Cp resulted in a large value of ΔG(T), which contributed to the stability of the protein. Taken together, these results demonstrated that human L-PGDS is sufficiently thermostable for storage and practical use and can be useful as a delivery vehicle of protein-based DDS.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded ß-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Zinc mediates the interaction between ceruloplasmin and apo-transferrin for the efficient transfer of Fe(III) ions.

Fe(II) exported from cells is oxidized to Fe(III), possibly by a multicopper ferroxidase (MCF) such as ceruloplasmin (CP), to efficiently bind with the plasma iron transport protein transferrin (TF). As unbound Fe(III) is highly insoluble and reactive, its release into the blood during the transfer from MCF to TF must be prevented. A likely mechanism for preventing the release of unbound Fe(III) is via direct interaction between MCF and TF; however, the occurrence of this phenomenon remains controversial. This study aimed to reveal the interaction between these proteins, possibly mediated by zinc. Using spectrophotometry, isothermal titration calorimetry, and surface plasmon resonance methods, we found that Zn(II)-bound CP bound to iron-free TF (apo-TF) with a Kd of 4.2 µM and a stoichiometry CP:TF of ∼2:1. Computational modeling of the complex between CP and apo-TF predicted that each of the three Zn(II) ions that bind to CP further binds to an acidic amino acid residue of apo-TF to play a role as a cross-linker connecting both proteins. Domain 4 of one CP molecule and domain 6 of the other CP molecule fit tightly into the clefts in the N- and C-lobes of apo-TF, respectively. Upon the binding of two Fe(III) ions to apo-TF, the resulting diferric TF [Fe(III)2TF] dissociated from CP by conformational changes in TF. In human blood plasma, zinc deficiency reduced the production of Fe(III)2TF and concomitantly increased the production of non-TF-bound iron. Our findings suggest that zinc may be involved in the transfer of iron between CP and TF.

Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.

Solubility-Improved 10-O-Substituted SN-38 Derivatives with Antitumor Activity.

With the objective of improving the poor water solubility of the potent antitumor compound SN-38, 10-O-substituted SN-38 derivatives were developed by the introduction of fluoroalkyl, fluorobenzoyl, or bromobenzoyl groups. The 10-O-fluoropropyl-substituted compound 2 {(S)-4,11-diethyl-9-(3-fluoropropoxy)-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione} was found to be 17-fold more soluble than SN-38 in phosphate-buffered saline, and it exhibited a level of biological activity ≈50 % that of SN-38 in a cytotoxicity assay using the prostate cancer cell line PC-3. Five other derivatives did not show solubility improvements to the same extent, but their activities in cytotoxicity assays were nearly the same as that of SN-38. In vivo studies of 2 with PC-3 tumor-bearing mice revealed that it has higher antitumor activity than SN-38, even at lower dosage. These results will promote the medicinal chemistry application of 10-O-modifications of SN-38 and help reestablish the potential this drug. Furthermore, the inclusion of fluoro and bromo substituents means that the synthetic strategy developed here may be used to obtain 18 F- or 76 Br-labeled SN-38 derivatives for in vivo positron emission tomography studies.

MEK and PI3K catalytic activity as predictor of the response to molecularly targeted agents in triple-negative breast cancer.

Hyper-activation of the MAPK and PI3K-AKT pathways is linked to tumour progression in triple-negative breast cancer (TNBC). However, clinically effective predictive markers for drugs targeted against protein kinases involved in these pathways have not been identified. We investigated the ability of MEK and PI3K catalytic activity to predict sensitivity to trametinib and wortmannin in TNBC. MEK and PI3K activities correlated strongly with each other only in cell lines showing wortmannin-specific sensitivity, as shown by a linear regression curve (R = 0.951). Accordingly, we created a new parameter that distinguishes trametinib and wortmannin sensitivity in vitro and in vivo. Our findings suggest that the catalytic activities of MEK and PI3K might predict the response of TNBC to trametinib and wortmannin.

Kinetics and polymorphs of yeast prion Sup35NM amyloidogenesis.

Amyloidogenic proteins often form many types of aggregates, which are a critical determinant of cytotoxicity and tissue specificity. However, the molecular mechanisms underlying the generation of distinct amyloids and their influence on cells remain largely unknown. We herein investigated the polymorphic amyloid formation of the yeast prion protein, Sup35NM, an intrinsically disordered N-terminal fragment of Sup35, under various conditions and its potential relationship to cytotoxicity. Sup35NM aggregated to amyloid fibrils with distinct kinetics, structures, morphologies, tinctorial properties, and conformational stabilities depending on the concentration of NaCl, pH, and temperature, indicating the polymorphic amyloidogenesis of Sup35NM. Detailed kinetic analyses of Sup35NM amyloid formation revealed a strong inverse correlation between the lag time and elongation rate without a correlation between kinetic and structural parameters. These results suggest that kinetic polymorphisms due to distinct nucleation and elongation rates result in structural polymorphs of amyloid fibrils, and also that conditions that enhance or inhibit the nucleation of Sup35NM promote or delay fibril growth. The deleterious effects of polymorphic Sup35NM amyloid fibrils on membrane integrity and cell vitality were minimal. We hypothesize that the innocuous polymorphic nature of Sup35NM amyloid fibrils may be beneficial for gaining time for prion infection prior to cell death.

Development of pH-Independent Drug Release Formulation Using Lipocalin-Type Prostaglandin D Synthase.

The purpose of this study was to develop a pH-independent drug release formulation using lipocalin-type prostaglandin D synthase, a member of the lipocalin superfamily, with the function of forming complexes together with various small lipophilic molecules. Dipyridamole, a poorly water-soluble drug, showing a pH-dependent solubility profile, was used as the model drug. The solubilization of dipyridamole was achieved by a simple complex formulation method with lipocalin-type prostaglandin D synthase. The complex formulation was produced successfully by spray drying, and the obtained powder formulation showed complete dissolution in fasted-state simulated gastric fluid (pH, 1.6) and phosphate-buffered solution (pH, 6.8). In addition, the potential stability of the complex formulation was assessed, and the dissolution profile of the produced powder at pH 6.8 was maintained after 4-week storage under several storage conditions. Furthermore, a pharmacokinetic study using hypochlorhydria model rats was performed to verify the improvement of the intestinal absorption behavior, and eventually the complex formulation overcame the problematic absorption profile of dipyridamole in the elevated gastric pH conditions. These results, taken together, demonstrate that the use of this well-designed drug-delivery carrier is feasible for the development of pH-independent drug release formulations.

Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 µM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke.

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.

Novel oral formulation approach for poorly water-soluble drug using lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS), a member of the lipocalin superfamily, possesses the function of forming complexes together with various small lipophilic molecules. In this study, we chose telmisartan as a model drug due to its pH dependent poor water solubility, and developed and characterized a novel solubilized formulation of telmisartan using a complex formulation with L-PGDS. The solid state of the complex formulation was prepared using a spray-drying process. The spray-dried formulation of telmisartan/L-PGDS powder showed a typical spray-dried particle without any change in the secondary and tertiary structures of L-PGDS. Furthermore, the complex formulation showed a high rate and level of drug release in pH 1.2, 5.0, and 6.8 solutions in comparison with the active pharmaceutical ingredient (API) and commercial product. To validate the potential for oral administration of the telmisartan/L-PGDS complex in vivo, the pharmacokinetic and pharmacodynamic profiles were assessed in spontaneous hypertensive rats. An animal study revealed that the complex formulation led to a significant improvement of AUC and Cmax as compared with API, and the prolonged pharmacologic effect on blood pressure reduction was comparable with the commercial product. These results, taken together, demonstrate that this novel approach is feasible for the solubilized solid oral formulation and it can be applied to poorly water-soluble drugs to enhance oral bioavailability.

Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands.

The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains. Gibbs energy changes strongly correlated with the number of hydrogen bond acceptors of ligand. Thus, the broad binding capability of L-PGDS for ligands should be viewed as hydrophilic interactions delicately tuned by enthalpy-entropy compensation using combined effects of hydrophilic and hydrophobic interactions.