Yuka Koike

Palladium-Catalyzed Cyanation of CarbonCarbon Triple Bonds Under Aerobic Conditions†

Essential oxygen: The title transformation involves two different modes of cyanation, syn and anti cyanopalladation, as the key steps in this catalytic reaction. These processes enable successful dicyanative cyclization of diyne and enyne derivatives (see scheme).

Catalytic dicyanative [4+2] cycloaddition triggered by cyanopalladation of conjugated enynes under aerobic conditions.

A palladium-catalyzed dicyanative [4+2] cycloaddition reaction using dienynes with TMSCN under aerobic conditions is described. This new reaction triggered by the cyanopalladation of terminal alkynes includes regioselective direct cyanation to C-C triple bonds by TMSCN to give pi-allyl Pd intermediates, which promotes 5-exo followed by 6-endo cyclization. This protocol enables (1) the formation of four C-C bonds through one operation, (2) the construction of highly functionalized cyclohexene rings, and (3) the generation of five contiguous stereogenic centers in one operation. The intermolecular cycloaddition reaction between a conjugated enyne and methyl acrylate also proceeded in a regioselective fashion to give multifunctionalized carbocycles.

Catalytic Dicyanative [4 + 2] Cycloaddition Triggered by Cyanopalladation Using Ene−Enynes and Cyclic Enynes with Methyl Acrylate

Palladium-catalyzed dicyanative [4 + 2] cycloaddition using various ene-enynes was investigated. The key species in this process is a cyanoallene intermediate that is obtained by the cyanopalladation of conjugated enynes followed by 5-exo-cyclization. To achieve an efficient [4 + 2] cycloaddition reaction, both the smooth generation of this species and critical control of regioselectivity in the 6-endo-cyclization step are quite important. A study of the substrate scope revealed that the reaction is strongly affected by the steric bulk of the substituents on the enyne and alkene units and prefers to give trans-fused cycloadducts. The stereochemistry of olefins was reasonably transferred to the corresponding products. Further study proved that this transformation includes not a thermal [4 + 2] cycloaddition process via 1,2-dicyanoalkenes generated in situ but rather a palladium-mediated stepwise cyclization sequence to control a maximum of five contiguous stereogenic centers in a single operation. An intermolecular version using methyl acrylate with conjugated cyclic enynes and TMSCN also gave the corresponding [4 + 2] cycloadducts in a regioselective manner.

Selective inhibition of the kinase DYRK1A by targeting its folding process

Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.

Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer’s disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.

Implementing Feedback Error Learning for FES control

Walking assist using Functional Electrical Stimulation (FES) has been studied for a quite long time to help paraplegic persons overcome their walking impairment. One of the problems of these devices is how to cope with individual dependent, time-varying, and nonlinear users characteristics and external disturbances. In this study, Feedback Error Learning (FEL), a scheme that integrates feedback and feedforward control, was applied to FES control. As a first step, an inverted-pendulum model was used to examine the usefulness of this scheme. Next, the control of a swinging motion, using a leg model simulation, was performed. After the verification in the simulation models, an FES experiment was to investigate the applicability of this system. The data obtained from the FES experiments were used to construct a new simulation model for investigating the effect of FES in different condition. The results showed that, usability of the FEL scheme for different FES control strategies.

Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. In our previous study, we reported TG003, an inhibitor of CDC2-like kinase 1 (CLK1), as a splice-modifying compound for exon-skipping therapy; however, its metabolically unstable feature hinders clinical application. Here, we show an orally available inhibitor of CLK1, named TG693, which promoted the skipping of the endogenous mutated exon 31 in DMD patient-derived cells and increased the production of the functional exon 31-skipped dystrophin protein. Oral administration of TG693 to mice inhibited the phosphorylation of serine/arginine-rich proteins, which are the substrates of CLK1, and modulated pre-mRNA splicing in the skeletal muscle. Thus, TG693 is a splicing modulator for the mutated exon 31 of the dystrophin gene in vivo, possibly possessing therapeutic potential for DMD patients.

Probabilistic Information Structure of Human Walking

Recently, the area of healthcare has been tremendously benefited from the advent of high performance computing in improving quality of life. Different processing techniques have been developed to understand the hidden complexity of the time series and will help clinicians in diagnosis and treatment. Analysis of human walking helps to study the various pathological conditions affecting balance and the elderly. In an elderly subjects, falls and paralysis are major problems, in terms of both frequency and consequences. Correct postural balance is important to well being and its effects will be felt in every movement and activity. In this paper, Bayesian Network (BN) was applied to recorded muscle activities and joint motions during walking, to extract causal information structure of normal walking and different impaired walking. The aim of this study is to use different BNs to express normal walking and various impaired walking, and identify the most important causal pairs that characterize specific impaired walking, through comparing the BNs for different walking.

Quantification of receptor activation by oxytocin and vasopressin in endocytosis-coupled bioluminescence reduction assay using nanoKAZ.

Oxytocin (OXT) and arginine vasopressin (AVP) are structurally similar neuropeptide hormones that function as neurotransmitters in the brain, and have opposite key roles in social behaviors. These peptides bind to their G protein-coupled receptors (OXTR and AVPRs), inducing calcium ion-dependent signaling pathways and endocytosis of these receptors. Because selective agonists and antagonists for these receptors have been developed as therapeutic and diagnostic agents for diseases such as psychiatric disorders, facile methods are in demand for the evaluation of selectivity between these receptors. In this study, we developed a quantitative assay for OXT- and AVP-induced endocytosis of their receptors. The mutated Oplophorus luciferase, nanoKAZ, was fused to OXTR and AVPRs to enable rapid quantification of agonist-induced endocytosis by bioluminescence reduction. Agonist stimulation significantly decreases bioluminescence of nanoKAZ-fused receptors in living cells. Using this system, we evaluated clinically used OXTR antagonist atosiban and a reported pyrazinyltriazole derivative, hereby designated as PF13. Atosiban acted as an antagonist of AVPR1a, as well as an agonist for AVPR1b, whereas PF13 antagonized OXTR more selectively than atosiban, as reported previously. This paper shows a strategy for quantification of agonist-induced endocytosis of OXTR and AVPRs, and confirms its potent utility in the evaluation of agonists and antagonists.