Identification of a selective inhibitor of transforming growth factor ß-activated kinase 1 by biosensor-based screening of focused libraries.

Transforming growth factor-ß activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, plays an essential role in mediating signals from various pro-inflammatory cytokines and therefore may be a good target for developing anti-inflammation agents. Herein, we report our efforts to identify TAK1 inhibitors with a good selectivity profile with which to initiate medicinal chemistry. Instead of resorting to a high-throughput screening campaign, we performed biosensor-based biophysical screening for a limited number of compounds by taking advantage of existing knowledge on kinase inhibitors. Rather than focusing on one specific inhibition mode, we searched for three different types, Type I (ATP-competitive, DFG-in), Type II (DFG-out), and Type III binders (non-ATP competitive) in parallel, and succeeded in identifying candidates in all three categories efficiently and rapidly. Finally, the biosensor-based binding kinetics for the active and inactive forms of TAK1 were measured to prioritize the Type I and Type II inhibitors. The effort resulted in the identification of a new TAK1-selective Type I compound with a thienopyrimidine scaffold that served as a good starting point for medicinal chemistry.

Discovery of a potent and highly selective transforming growth factor ß receptor-associated kinase 1 (TAK1) inhibitor by structure based drug design (SBDD).

A novel thienopyrimidinone analog was discovered as a potent and highly selective TAK1 inhibitor using the SBDD approach. TAK1 plays a key role in inflammatory and immune signaling, so TAK1 is considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). After the hit compound had been obtained, our modifications successfully increased TAK1 inhibitory activity and solubility, but metabolic stability was still unsatisfactory. To improve metabolic stability, we conducted metabolic identification. Although the obtained metabolite was fortunately a potent TAK1 inhibitor, its kinase selectivity was low. Subsequently, to achieve high kinase selectivity, we used SBDD to follow two strategies: one targeting unique amino acid residues in TAK1, especially the combination of Ser111 and Asn114; the other decreasing the interaction with Tyr106 at the hinge position in TAK1. As expected, our designed compound showed an excellent kinase selectivity profile in both an in-house and a commercially available panel assay of over 420 kinases and also retained its potent TAK1 inhibitory activity (TAK1 IC50=11nM).

Development of a Method for Converting a TAK1 Type I Inhibitor into a Type II or c-Helix-Out Inhibitor by Structure-Based Drug Design (SBDD).

We have developed a method for converting a transforming growth factor-ß-activated kinase 1 (TAK1) type I inhibitor into a type II or c-helix-out inhibitor by structure-based drug design (SBDD) to achieve an effective strategy for developing these different types of kinase inhibitor in parallel. TAK1 plays a key role in inflammatory and immune signaling, and is therefore considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). We have already reported novel type I TAK1 inhibitor, so we utilized its X-ray information to design a new chemical class type II and c-helix-out inhibitors. To develop the type II inhibitor, we superimposed the X-ray structure of our reported type I inhibitor onto a type II compound that inhibits multiple kinases, and used SBDD to design a new type II inhibitor. For the TAK1 c-helix-out inhibitor, we utilized the X-ray structure of a b-Raf c-helix-out inhibitor to design compounds, because TAK1 is located close to b-Raf in the Sugen kinase tree, so we considered that TAK1 would, similarly to b-Raf, form a c-helix-out conformation. The X-ray crystal structure of the inhibitors in complex with TAK1 confirmed the binding modes of the compounds we designed. This report is notable for being the first discovery of a c-helix-out inhibitor against TAK1.

Mechanophores with a reversible radical system and freezing-induced mechanochemistry in polymer solutions and gels.

Visualization and quantitative evaluation of covalent bond scission in polymeric materials are highly important for understanding failure, fatigue, and deterioration mechanisms and improving the lifetime, durability, toughness, and reliability of the materials. The diarylbibenzofuranone-based mechanophore radical system enabled, through electron paramagnetic resonance spectroscopy, in situ quantitative evaluation of scission of the mechanophores and estimation of mechanical energy induced along polymer chains by external forces. The coagulation of polymer solutions by freezing probably generated force but did not cleave the mechanophores. On the other hand, cross-linking led to efficient propagation of the force of more than 80 kJ mol(-1) to some mechanophores, resulting their cleavage and generation of colored stable radicals. This mechanoprobe concept has the potential to elucidate other debated issues in the polymer field as well.

Network reorganization of dynamic covalent polymer gels with exchangeable diarylbibenzofuranone at ambient temperature.

Reversible bonds and interactions have been utilized to build stimuli-responsive and reorganizable polymer networks that show recyclability, plasticity, and self-healing. In addition, reorganization of polymer gels at ambient temperature, such as room or body temperature, is expected to lead to several biomedical applications. Although these stimuli-responsive properties originate from the reorganization of the polymer networks, not such microscopic structural changes but instead only macroscopic properties have been the focus of previous work. In the present work, the reorganization of gel networks with diarylbibenzofuranone (DABBF)-based dynamic covalent linkages in response to the ambient temperature was systematically investigated from the perspective of both macroscopic and microscopic changes. The gels continued to swell in suitable solvents above room temperature but attained equilibrium swelling in nonsolvents or below room temperature because of the equilibrium of DABBF linkages, as supported by electron paramagnetic resonance measurements. Small-angle X-ray scattering measurements revealed the mesh sizes of the gels to be expanded and the network structures reorganized under control at ambient temperature.

Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

Heterointerface Created on Au-Cluster-Loaded Unilamellar Hydroxide Electrocatalysts as a Highly Active Site for the Oxygen Evolution Reaction.

The oxygen evolution reaction (OER) is a critical element for all sorts of reactions that use water as a hydrogen source, such as hydrogen evolution and electrochemical CO2 reduction, and novel design principles that provide highly active sites on OER electrocatalysts push the limits of their practical applications. Herein, Au-cluster loading on unilamellar exfoliated layered double hydroxide (ULDH) electrocatalysts for the OER is demonstrated to fabricate a heterointerface between Au clusters and ULDHs as an active site, which is accompanied by the oxidation state modulation of the active site and interfacial direct OO coupling ("interfacial DOOC"). The Au-cluster-loaded ULDHs exhibit excellent activities for the OER with an overpotential of 189 mV at 10 mA cm-2 . X-ray absorption fine structure measurements reveal that charge transfer from the Au clusters to ULDHs modifies the oxidation states of trivalent metal ions, which can be active sites on the ULDHs. The present study, supported by highly sensitive spectroscopy combining reflection absorption infrared spectroscopy and modulation-excitation spectroscopy and density functional theory calculations, indicates that active sites at the interface between the Au clusters and ULDHs promote a novel OER mechanism through interfacial DOOC, thereby achieving outstanding catalytic performance.

Convection-enhanced delivery of a synthetic retinoid Am80, loaded into polymeric micelles, prolongs the survival of rats bearing intracranial glioblastoma xenografts.

Prognosis for the patients with glioblastoma, the most common malignant brain tumor, remains dismal. A major barrier to progress in treatment of glioblastoma is the relative inaccessibility of tumors to chemotherapeutic agents. Convection-enhanced delivery (CED) is a direct intracranial drug infusion technique to deliver chemotherapeutic agents to the central nervous system, circumventing the blood-brain barrier and reducing systemic side effects. CED can provide wider distribution of infused agents compared to simple diffusion. We have reported that CED of a polymeric micelle carrier system could yield a clinically relevant distribution of encapsulated agents in the rat brain. Our aim was to evaluate the efficacy of CED of polymeric micellar Am80, a synthetic agonist with high affinity to nuclear retinoic acid receptor, in a rat model of glioblastoma xenografts. We also used systemic administration of temozolomide, a DNA-alkylating agent, which has been established as the standard of care for newly diagnosed malignant glioma. U87MG human glioma cells were injected into the cerebral hemisphere of nude rats. Rats bearing U87MG xenografts were treated with CED of micellar Am80 (2.4 mg/m(2)) on day 7 after tumor implantation. Temozolomide (200 mg/m(2)/day) was intraperitoneally administered daily for 5 days, starting on day 7 after tumor implantation. CED of micellar Am80 provided significantly longer survival than the control. The combination of CED of micellar Am80 and systemic administration of temozolomide provided significantly longer survival than single treatment. In conclusion, temozolomide combined with CED of micellar Am80 may be a promising method for the treatment of malignant gliomas.

Therapeutic efficacy of a polymeric micellar doxorubicin infused by convection-enhanced delivery against intracranial 9L brain tumor models.

Convection-enhanced delivery (CED) with various drug carrier systems has recently emerged as a novel chemotherapeutic method to overcome the problems of current chemotherapies against brain tumors. Polymeric micelle systems have exhibited dramatically higher in vivo antitumor activity in systemic administration. This study investigated the effectiveness of CED with polymeric micellar doxorubicin (DOX) in a 9L syngeneic rat model. Distribution, toxicity, and efficacy of free, liposomal, and micellar DOX infused by CED were evaluated. Micellar DOX achieved much wider distribution in brain tumor tissue and surrounding normal brain tissue than free DOX. Tissue toxicity increased at higher doses, but rats treated with micellar DOX showed no abnormal neurological symptoms at any dose tested (0.1-1.0 mg/ml). Micellar DOX infused by CED resulted in prolonged median survival (36 days) compared with free DOX (19.6 days; p = 0.0173) and liposomal DOX (16.6 days; p = 0.0007) at the same dose (0.2 mg/ml). This study indicates the potential of CED with the polymeric micelle drug carrier system for the treatment of brain tumors.

Characterization of polymer-polymer type charge-transfer (CT) blend membranes for fuel cell application.

The data presented in this article are related to polymer-polymer type charge-transfer blend membranes for fuel cell application. The visible spectra of the charge-transfer (CT) blend membranes indicated formation of CT complex in the blend membranes, and behavior of CT complex formation by polymers was clarified by Job plot of the visible spectra. The effect of fluorine for membrane property and fuel cell performance of CT blend membranes were evaluated by 19F NMR and overvoltage analysis, respectively.

The depth of molecular recognition: voltage-sensitive blockage of synthetic multifunctional pores with refined architecture.

Voltage-sensitive blockage by ADP, ATP and phytate (IP6) demonstrates that active-site contraction toward the middle of newly synthesized rigid-rod beta-barrels provides a general strategy to rationally create and modulate the voltage sensitivity (and to increase the efficiency) of molecular recognition by synthetic multifunctional pores.

Diarylbibenzofuranone-Based Dynamic Covalent Polymer Gels Prepared via Radical Polymerization and Subsequent Polymer Reaction.

Diarylbibenzofuranone (DABBF) is a dynamic covalent bonding unit, which is in equilibrium with the corresponding radicals at room temperature, and polymers with DABBF linkages show notable properties such as self-healing. The preparation routes have been strictly limited, however, and no polymer with the linkages has been synthesized via radical polymerization because of the strong antioxidant activity of DABBF. Here we present a new method to prepare DABBF-containing polymers via radical polymerization of the precursor, arylbenzofuranone (ABF), and subsequent polymer reaction, dimerization of ABF units in the linear polymers. Polymer gels cross-linked by DABBF linkages were obtained against the relatively strong antioxidant activity of ABF and showed dynamic network reorganization at room temperature.

Mechanochromic Dynamic Covalent Elastomers: Quantitative Stress Evaluation and Autonomous Recovery.

Stress evaluation in polymeric materials is important in order to not only spot danger in them before serious failure, but also precisely interpret the destructive mechanism, which can improve the lifetime and durability of polymeric materials. Here, we are able to visualize stress by color changes, as well as quantitatively estimate the stress in situ, in segmented polyurethane elastomers with diarylbibenzofuranone-based dynamic covalent mechanophores. We prepared films of the segmented polyurethanes, in which the mechanophores were incorporated in the soft segments, and efficiently activated them by mechanical force. Cleavage of the mechanophores during uniaxial elongation and their recovery after the removal of the stress were quantitatively evaluated by in situ electron paramagnetic resonance measurements, accompanied by drastic color changes.

A facile preparation method of a PFC-containing nano-sized emulsion for theranostics of solid tumors.

Theranostics means a therapy conducted in a diagnosis-guided manner. For theranostics of solid tumors by means of ultrasound, we designed a nano-sized emulsion containing perfluoropentane (PFC5). This emulsion can be delivered into tumor tissues through the tumor vasculatures owing to its nano-size, and the emulsion is transformed into a micron-sized bubble upon sonication through phase transition of PFC5. The micron-sized bubbles can more efficiently absorb ultrasonic energy for better diagnostic images and can exhibit more efficient ultrasound-driven therapeutic effects than nano-sized bubbles. For more efficient tumor delivery, smaller size is preferable, yet the preparation of a smaller emulsion is technically more difficult. In this paper, we used a bath-type sonicator to successfully obtain small PFC5-containing emulsions in a diameter of ca. 200nm. Additionally, we prepared these small emulsions at 40°C, which is above the boiling temperature of PFC5. Accordingly, we succeeded in obtaining very small nano-emulsions for theranostics through a very facile method.

Vital microscopic analysis of polymeric micelle extravasation from tumor vessels: macromolecular delivery according to tumor vascular growth stage.

Particles larger than a specific size have been thought to extravasate from tumor vessels but not from normal vessels. Therefore, various nanoparticles incorporating anticancer drugs have been developed to realize selective drug delivery to solid tumors. However, it is not yet clear whether nanoparticles extravasate readily from all tumor vessels including vessels of microtumors. To answer this question, we synthesized new polymeric micelles labeled with fluorescein isothiocyanate (FITC) and injected them into the tail vein of rats with implanted skinfold transparent chambers. We also analyzed, by means of time-lapse vital microscopy with image analysis, extravasation of FITC micelles from tumor vessels at different stages of growth of Yoshida ascites sarcoma LY80. Polymeric micelles readily leaked from vessels at the interface between normal and tumor tissues and those at the interface between tumor tissues and necrotic areas. The micelles showed negligible extravasation, however, from the vascular network of microtumors less than 1 mm in diameter and did not accumulate in the microtumor. Our results suggest that we must develop a novel therapeutic strategy that can deliver sufficient nanomedicine to microtumors.

The combretastatin derivative (Cderiv), a vascular disrupting agent, enables polymeric nanomicelles to accumulate in microtumors.

A previous study found almost no leakage of polymeric nanomicelles from vessels in microtumors. If such vessels become leaky, sufficient nanomedicines may be delivered to microtumors and large tumors. To create leaky vessels, a combretastatin derivative (Cderiv), a vascular disrupting agent, was used. Via vital microscopy with fluorescein isothiocyanate (FITC)-labeled nanomicelles, the effect of Cderiv pretreatment on changes in micelle extravasation was investigated. Whether such treatment would prolong microtumor retention of micelles was also examined. FITC-albumin was used for comparison. The degree of extravasation from intact vessels in microtumors (rat sarcoma LY80) was extremely low and comparable to that from normal vessels. Cderiv pretreatment (1 or 3 days before administration of FITC-labeled compounds) markedly enhanced extravasation of such nanomicelles and albumin from vessels that survived treatment and had restored blood flow. A high concentration of extravasated macromolecules remained even 24 h later in tissue areas whose microcirculatory function had collapsed. Tumors receiving 10 Gy irradiation 3 days before the macromolecules evidenced gradual removal of extravasated macromolecules, which did not accumulate in those areas, despite extravasation from tumor vessels. Our results strongly suggest that pretreatment with Cderiv is quite effective for maintaining microtumor concentrations of nanomicelles and albumin associated with anticancer or diagnostic drugs.

Histological study on side effects and tumor targeting of a block copolymer micelle on rats.

Histological examinations were performed with polymeric micelle-injected rats for evaluations of possible toxicities of polymeric micelle carriers. Weight of major organs as well as body weight of rats was measured after multiple intravenous injections of polymeric micelles forming from poly(ethylene glycol)-b-poly(aspartate) block copolymer. No pathological toxic side effects were observed at two different doses, followed only by activation of the mononuclear phagocyte system (MPS) in the spleen, liver, lung, bone marrow, and lymph node. This finding confirms the absence of--or the very low level of--in vivo toxicity of the polymeric micelle carriers that were reported in previous animal experiments and clinical results. Then, immunohistochemical analyses with a biotinylated polymeric micelle confirmed specific accumulation of the micelle in the MPS. The immunohistochemical analyses also revealed, first, very rapid and specific accumulation of the micelle in the vasculatures of tumor capsule of rat ascites hepatoma AH109A, and second, the micelle's scanty infiltration into tumor parenchyma. This finding suggests a unique tumor-accumulation mechanism that is very different from simple EPR effect-based tumor targeting.

Particle size-dependent triggering of accelerated blood clearance phenomenon.

A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2 nm in diameter) or PEGylated liposomes (119, 261 or 795 nm) were preadministered into BALB/c mice. Three days after the preadministration [(3)H]-labeled PEGylated liposomes (127 nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2-795 nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7-31.5 nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.

A cost-effective method for the optical transduction of chemical reactions. Application to hyaluronidase inhibitor screening with polyarginine-counteranion complexes in lipid bilayers.

We report the systematic elaboration of a cost-effective, interference-minimized assay for the label-free optical transduction of chemical reactions. Recently, we have found that certain complexes formed by arginine-rich cell-penetrating peptides (CPPs) and amphiphilic counteranions can act as synergistic anion carriers in lipid bilayer membranes. Application of this discovery to rapid and reversible cytosolic CPP delivery has been described (Futaki, S.; et al. ACS Chem. Biol. 2006, 1, 299). Here, we report the complementary use of polyarginine (pR)-counteranion complexes as general optical transducers of chemical reactions. Counterion screening revealed dodecyl phosphate (DP) as an ideal pR activator. Carboxyfluorescein (CF)-loaded vesicles with a shelf life of 3.5 years served best for the detection of fluorogenic CF release by pR-DP complexes with the naked eye. Inactivation of pR-DP complexes by counterion exchange with hyaluronan (HA) caused no CF emission, while HA removal by hyaluronidase (HAase) did. pR-DP complexes were further compatible with the optical detection of HA immobilization on solid support as well as inhibitor screening for HAase (cromolyn, heparin) with and without substrate immobilization. Controls concerning binary ATP/ADP discrimination for naked-eye kinase detection are mentioned to delineate scope but also limitations of this simple and quite universal method.