High photodynamic activities of water-soluble inclusion complexes of 5,15-diazaporphyrins in cyclodextrin.

Water-soluble inclusion complexes of 5,15-diazaporphyrin derivatives in the cavities of two trimethyl-ß-cyclodextrins (TMe-ß-CDxs) were synthesised. In the 2 : 1 complexes, two aryl groups of the diazaporphyrins protruded from the upper rims of two TMe-ß-CDxs. The complexes displayed high photodynamic activity under photoirradiation at wavelengths longer than 620 nm. Although the substituents on the two aryl groups protruding from TMe-ß-CDx barely affected intracellular uptake by HeLa cells, the cellular uptake of these complexes was as high as that of a TMe-ß-CDx-tetra(4-hydroxyphenyl)porphyrin complex. Furthermore, the diazaporphyrins in the complexes with TMe-ß-CDxs were able to generate high levels of singlet oxygen because of their strong absorption of light with wavelengths greater than 620 nm.

Interleukin enhancer-binding factor 3/NF110 is a target of YM155, a suppressant of survivin.

Survivin is responsible for cancer progression and drug resistance in many types of cancer. YM155 selectively suppresses the expression of survivin and induces apoptosis in cancer cells in vitro and in vivo. However, the mechanism underlying these effects of YM155 is unknown. Here, we show that a transcription factor, interleukin enhancer-binding factor 3 (ILF3)/NF110, is a direct binding target of YM155. The enhanced survivin promoter activity by overexpression of ILF3/NF110 was attenuated by YM155 in a concentration-dependent manner, suggesting that ILF3/NF110 is the physiological target through which YM155 mediates survivin suppression. The results also show that the unique C-terminal region of ILF3/NF110 is important for promoting survivin expression and for high affinity binding to YM155.

Computational design, construction, and characterization of a set of specificity determining residues in protein-protein interactions.

Proteins interact with different partners to perform different functions and it is important to elucidate the determinants of partner specificity in protein complex formation. Although methods for detecting specificity determining positions have been developed previously, direct experimental evidence for these amino acid residues is scarce, and the lack of information has prevented further computational studies. In this article, we constructed a dataset that is likely to exhibit specificity in protein complex formation, based on available crystal structures and several intuitive ideas about interaction profiles and functional subclasses. We then defined a "structure-based specificity determining position (sbSDP)" as a set of equivalent residues in a protein family showing a large variation in their interaction energy with different partners. We investigated sequence and structural features of sbSDPs and demonstrated that their amino acid propensities significantly differed from those of other interacting residues and that the importance of many of these residues for determining specificity had been verified experimentally.

Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) complex, which is required for survivin expression.

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Excessive/Aberrant and Maladaptive Synaptic Plasticity: A Hypothesis for the Pathogenesis of Alzheimer's Disease.

The amyloid hypothesis for the pathogenesis of Alzheimer's disease (AD) is widely accepted. Last year, the US Food and Drug Administration considered amyloid-ß peptide (Aß) as a surrogate biomarker and approved an anti-Aß antibody, aducanumab, although its effectiveness in slowing the progression of AD is still uncertain. This approval has caused a great deal of controversy. Opinions are divided about whether there is enough evidence to definitely consider Aß as a causative substance of AD. To develop this discussion constructively and to discover the most suitable therapeutic interventions in the end, an alternative persuasive hypothesis needs to emerge to better explain the facts. In this paper, I propose a hypothesis that excessive/aberrant and maladaptive synaptic plasticity is the pathophysiological basis for AD.

Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

Improved Stability and Photodynamic Activity of Water-Soluble 5,15-Diazaporphyrins Incorporated in ß-(1,3-1,6)-d-Glucan with On-Off Switch.

5,15-Diazaporphyrins, which have a large absorption at wavelengths over 600 nm, were dissolved in water by complex formation with ß-(1,3-1,6)-d-glucans. Aqueous solutions of these complexes were relatively stable compared with their trimethyl-ß-cyclodextrin-complexed analogues. ß-Glucan-complexed diazaporphyrins showed quenched fluorescence and had low singlet-oxygen-generation abilities owing to random self-aggregation. However, external stimuli, such as the presence of liposomes or intracellular uptake, restored the fluorescence and singlet-oxygen-generation abilities of ß-glucan-complexed diazaporphyrins. Consequently, ß-glucan-complexed diazaporphyrins showed very high photodynamic activities toward HeLa cells.

Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models.

We describe in vitro properties and in vivo neuroprotective effects of a newly synthesized, high-affinity, selective allosteric metabotropic glutamate receptor type 1 (mGluR(1)) antagonist, N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074). YM-202074 bound an allosteric site of rat mGluR(1) with a K(i) value of 4.8+/-0.37 nM. YM-202074 also inhibited the mGluR(1)-mediated inositol phosphates production in rat cerebellar granule cells with an IC(50) value of 8.6+/-0.9 nM, while showing selectivity over mGluR(2-7). When YM-202074 was infused intravenously at an initial dose of 20 mg/kg/h for 0.5 h followed by a dose of 5 mg/kg/h for 7.5 h, the free concentration of YM-202074 in the brain rapidly (<12 min) reached approximately 0.3 microM, reaching a steady-state phase within 1.5 h. We first treated rats such that they developed transient middle cerebral artery (MCA) occlusion. Results clearly demonstrate a dose-dependent improvement of neurological deficit and reduction of the infarct volume in both the hemisphere and cortex when YM-202074 was infused intravenously immediately after occlusion at a dose of 10 or 20 mg/kg/h for 0.5 h followed by a dose of 2.5 or 5 mg/kg/h for 23.5 h, respectively. Significant neuroprotection was maintained even when the administration of drugs was delayed by up to 2 h following the onset of ischemia. Furthermore, the improvement of neurological deficit and the reduction of infarct volume were sustained for 1 week following the onset of ischemia. These results suggest that YM-202074 exhibits great potential as a novel neuroprotective agent for the treatment of stroke.

Supramolecular polymer formation by a de novo hemoprotein with a synthetic diheme compound.

Proteins are attractive materials for supramolecular chemistry due to their multifunctionality and self-organization ability. In this work, we synthesized a diheme compound, in which two iron-protoporphyrin IX molecules are associated via a linker chain, and introduced it into a de novo designed four-helix bundle protein with two heme-binding sites. The protein gradually bound the diheme compound by bis-histidyl ligation and formed supramolecular polymers. Polymer formation was observed by atomic force microscopy (AFM), which revealed the highly branched, dendritic forms of the fibrous architecture. The present results may open a pathway toward nanowire construction with de novo heme-proteins.

Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

Radioligand binding properties and pharmacological characterization of 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198), a high-affinity, selective, and noncompetitive antagonist of metabotropic glutamate receptor type 1.

Metabotropic glutamate receptor type 1 (mGluR1) is thought to play important roles in the neurotransmission and pathogenesis of several neurological disorders. Here, we describe the radioligand binding properties and pharmacological effects of a newly synthesized, high-affinity, selective, and noncompetitive mGluR1 antagonist, 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198). YM-298198 inhibited glutamate-induced inositol phosphate production in mGluR1-NIH3T3 cells with an IC50 of 16 +/- 5.8 nM in a noncompetitive manner. Its radiolabeled form, [3H]YM-298198, bound to mGluR1-NIH3T3 cell membranes with a KD of 32 +/- 8.5 nM and a Bmax of 2297 +/- 291 fmol/mg protein. In ligand displacement experiments using rat cerebellum membrane, an existing noncompetitive mGluR1 antagonist 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) competitively displaced [3H]YM-298198 binding, although glutamate and other mGluR1 ligands acting on a glutamate site failed to inhibit [3H]YM-298198 binding, suggesting that YM-298198 binds to CPCCOEt (allosteric) binding sites but not to glutamate (agonist) binding sites. Specificity was demonstrated for mGluR1 over mGluR subtypes 2 to 7, ionotropic glutamate receptors, and other receptor, transporter, and ion channel targets. In in vivo experiments, orally administered YM-298198 showed a significant analgesic effect in streptozotocin-induced hyperalgesic mice at doses (30 mg/kg) that did not cause Rotarod performance impairment, indicating that it is also useful even for in vivo experiments. In conclusion, YM-298198 is a newly synthesized, high-affinity, selective, and noncompetitive antagonist of mGluR1 that will be a useful pharmacological tool due to its highly active properties in vitro and in vivo. Its radiolabeled form [3H]YM-298198 will also be a valuable tool for future investigation of the mGluR1.

Exclusive association and simultaneous appearance of congophilic plaques and AT8-positive dystrophic neurites in Tg2576 mice suggest a mechanism of senile plaque formation and progression of neuritic dystrophy in Alzheimer's disease.

Progression of neuritic dystrophy is a histological hallmark of Alzheimer's disease (AD) in addition to amyloid deposition and neurofibrillary tangle formation. Dystrophic neurites (DNs) are abnormal neurites, and are closely associated with amyloid deposits. To clarify the process of DN formation, we immunohistochemically investigated phosphorylated tau (AT8 and Ser396)-positive DNs and plaques in Tg2576 mice overexpressing human beta-amyloid precursor protein (APP) with the Swedish type mutation (K670N/M671L). AT8-positive DNs were exclusively associated with the Congo red-positive plaques examined, and all Abeta(1-40)-positive plaques appeared to be associated with AT8-positive DNs, whereas there were no AT8-positive DNs with Abeta(1-42)-positive/Abeta(1-40)-negative plaques. Since we have previously shown that Abeta(1-42)-positive plaque precede Abeta(1-40) deposition, the appearance of congophilic structures is also late. Quantitative analyses were performed on AT8-positive DNs that were associated with congophilic plaques in the cerebral cortex and hippocampus (more than 1,000 plaques). The number of congophilic plaques increased dramatically with age. The area of DNs in the cerebral cortex and hippocampus increased 120- and 60-fold from 11-13 to 20.5 months of age, respectively. Interestingly, the mean ratio of DN area to congophilic plaque area in every plaque was unchanged, approximately 10%, through the ages examined. The mean plaque size was stable with age in both the cortex and hippocampus. These data suggest that the formation of AT8-positive DNs is simultaneous with Congo red-positive plaque development, and that the event may be closely related in the pathological progression of AD.