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.

Biological abilities of rice bran-derived antioxidant phytochemicals for medical therapy.

Rice bran contains important bioactive phytochemicals. Among these phytochemicals, steryl ferulates including γ-oryzanol and its major components such as cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferulate (24-mCAF), ß-sitosteryl ferulate (ß-SF), and campesteryl ferulate have been intensively studied due to their crucial roles in pathological processes. On the basis of experimental studies published during the last decade in relation to antioxidant, anti-inflammatory, anti-ulcerogenic, hypolipidemic, anti-neoplastic, anti-diabetic, and anti-allergic phenomena, these bioactive phytochemicals are reviewed in this paper. Particularly, in vivo and in vitro studies have clarified that rice bran phytosteryl ferulates mediate anti-inflammatory effects by down-regulating the inflammatory transcription factor, nuclear factor κB (NF-κB), which in turn reduces expression of inflammatory enzymes such as COX-2 and iNOS, and proinflammatory cytokines such as IL-1ß, IL-6 and TNF-α. Moreover, rice bran phytosteryl ferulates up-regulate blood adiponectin levels via indirect activation of peroxisomal proliferator-activated receptor γ (PPARγ) through NF-κB inhibition. In this review, we discuss potential pharmacological aspects of rice bran phytosteryl ferulates in the clinical setting.

Increase in hypothalamic aromatase in macaque monkeys treated with anabolic-androgenic steroids: PET study with [11C]vorozole.

In an earlier study in rodents, we showed that the aromatase that converts androgens to estrogens in the preoptic area and bed nucleus of stria terminalis was significantly increased in concentration after exposure to anabolic-androgenic steroids. To confirm whether this occurs in primates, we conducted a positron emission tomographic study using macaque monkeys. Male rhesus monkeys were treated with nandrolone decanoate for 3 weeks. To measure aromatase concentrations, we performed positron emission tomographic imaging using a 11C-labeled specific aromatase inhibitor, [11C]vorozole. After treatment with nandrolone, significant increase in [11C]vorozole binding was observed in the hypothalamus but not other areas including the amygdala, which is also aromatase enriched. These findings in monkeys are consistent with those we obtained earlier in rats. These findings strongly suggest that aromatase in the hypothalamus may play a crucial role in the emotional instability of anabolic-androgenic steroids abusers.

Palladium(0)-mediated rapid methylation and fluoromethylation on carbon frameworks by reacting methyl and fluoromethyl iodide with aryl and alkenyl boronic acid esters: useful for the synthesis of [11C]CH(3)--C- and [18F]FCH2--C-Containing PET tracers (PET=positron emission tomography).

A new synthetic methodology for the rapid methylation and fluoromethylation on aryl and alkenyl frameworks by using methyl and fluoromethyl iodide with an organoboronic acid ester has been developed under the simple and mild conditions of [Pd(2)(dba)(3)]/P(o-CH(3)C(6)H(4))(3)/K(2)CO(3) (dba= dibenzylideneacetone) in DMF at 60 degrees C for 5 min (see scheme). This boron protocol provides a firm chemical basis for the synthesis of (11)C- and (18)F-incorporated PET tracers.The rapid methylation and fluoromethylation on aryl and alkenyl carbon frameworks by reacting methyl and fluoromethyl iodide with aryl and alkenyl boronates have been studied with the focus on the realization of the synthesis of [(11)C]CH(3)- and [(18)F]FCH(2)-labeled positron emission tomography (PET) tracers. The coupling of methyl iodide and pinacol phenylboronate (40 equiv) is accomplished in >91 % yield within 5 min at 60 degrees C under the conditions of [Pd(2)(dba)(3)]/P(o-CH(3)C(6)H(4))(3)/K(2)CO(3) (0.5:2:2; dba=dibenzylideneacetone) in DMF. The reaction shows a high generality and is applicable to various types of aryl and alkenyl boronates, giving the corresponding methylated derivatives in high yields (80-99 %). This reaction is also useful for the rapid incorporation of the fluoromethyl group. Thus, this boron protocol provides a firm chemical basis for the synthesis of (11)C- and (18)F-incorporated PET tracers and can be used as a complementary method for [(11)C]methylation together with our previous and ongoing processes using organotributylstannanes.