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.

Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma.

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.

CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses.

A wide range of antiviral drugs is currently available; however, drug-resistant viruses have begun to emerge and represent a potential public health risk. Here, we explored the use of compounds that inhibit or interfere with the action of essential host factors to prevent virus replication. In particular, we focused on the cyclin-dependent kinase 9 (CDK9) inhibitor, FIT-039, which suppressed replication of a broad spectrum of DNA viruses through inhibition of mRNA transcription. Specifically, FIT-039 inhibited replication of herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, and human cytomegalovirus in cultured cells, and topical application of FIT-039 ointment suppressed skin legion formation in a murine HSV-1 infection model. FIT-039 did not affect cell cycle progression or cellular proliferation in host cells. Compared with the general CDK inhibitor flavopiridol, transcriptome analyses of FIT-039-treated cells revealed that FIT-039 specifically inhibited CDK9. Given at concentrations above the inhibitory concentration, FIT-039 did not have a cytotoxic effect on mammalian cells. Importantly, administration of FIT-039 ameliorated the severity of skin lesion formation in mice infected with an acyclovir-resistant HSV-1, without noticeable adverse effects. Together, these data indicate that FIT-039 has potential as an antiviral agent for clinical therapeutics.

Effects of dronabinol on morphine-induced dopamine-related behavioral effects in animals.

The present study examined the effects of dronabinol, a United States FDA-approved synthetic cannabinoid receptor agonist, on morphine (a prototypic µ-opioid receptor agonist)-induced dopamine-related behaviors in animals. Dronabinol suppressed the rewarding effects of morphine in rats and its emetic effects in ferrets. Furthermore, the morphine-induced increase in dopamine release from the nucleus accumbens was significantly attenuated by dronabinol, which indicated that the suppressive effects of dronabinol on morphine-induced behaviors are at least in part mediated by regulation of the dopaminergic system. Since cannabinoid receptor agonists have been shown to enhance the antinociceptive effects of morphine, the use of dronabinol as an adjuvant could be useful for preventing the adverse effects of µ-opioid receptor agonists when used to control pain.

Change in microRNAs associated with neuronal adaptive responses in the nucleus accumbens under neuropathic pain.

Neuropathic pain is the most difficult type of pain to control, and patients lose their motivation for the purposive pursuit with a decrease in their quality of life. Using a functional magnetic resonance imaging analysis, we demonstrated that blood oxygenation level-dependent signal intensity was increased in the ipsilateral nucleus accumbens (N.Acc.) following peripheral nerve injury. microRNAs are small, noncoding RNA molecules that direct the post-transcriptional suppression of gene expression, and play an important role in regulating synaptic plasticity. In this study, we found that sciatic nerve ligation induced a drastic decrease in the expression of miR200b and miR429 in N.Acc. neurons. The expression of DNA methyltransferase 3a (DNMT3a), which is the one of the predicted targets of miR200b/429, was significantly increased in the limbic forebrain including N.Acc. at 7 d after sciatic nerve ligation. Double-immunolabeling with antibodies specific to DNMT3a and NR1 showed that DNMT3a-immunoreactivity in the N.Acc. was located in NR1-labeled neurons, indicating that increased DNMT3a proteins were dominantly expressed in postsynaptic neurons in the N.Acc. area under a neuropathic pain-like state. The results of these analyses provide new insight into an epigenetic modification that is accompanied by a dramatic decrease in miR200b and miR429 along with the dysfunction of "mesolimbic motivation/valuation circuitry" under a neuropathic pain-like state. These phenomena may result in an increase in DNMT3a in neurons of the N.Acc. under neuropathic pain, which leads to the long-term transcription-silencing of several genes.

Implication of dopaminergic projection from the ventral tegmental area to the anterior cingulate cortex in µ-opioid-induced place preference.

Despite the importance of prefrontal cortical dopamine in modulating reward, little is known about the implication of the specific subregion of prefrontal cortex in opioid reward. We investigated the role of neurons projecting from the ventral tegmental area (VTA) to the anterior cingulate cortex (ACG) in opioid reward. Microinjection of the retrograde tracer fluorogold (FG) into the ACG revealed several retrogradely labelled cells in the VTA. The FG-positive reactions were noted in both tyrosine hydroxylase (TH)-positive and -negative VTA neurons. The released levels of dopamine and its major metabolites in the ACG were increased by either the electrical stimulation of VTA neurons or microinjection of a selective µ-opioid receptor (MOR) agonist, (D-Ala²,N-MePhe4,Gly-ol5) enkephalin (DAMGO), into the VTA. MOR-like immunoreactivity was seen in both TH-positive and -negative VTA neurons projecting to the ACG. The conditioned place preference induced by intra-VTA injection of DAMGO was significantly attenuated by chemical lesion of dopaminergic terminals in the ACG. The depletion of dopamine in the ACG induced early extinction of µ-opioid-induced place preference. The levels of phosphorylated DARPP32 (Thr34) and phosphorylated CREB (Ser133) were increased in the ACG of rats that had maintained the morphine-induced place preference, whereas the increases of these levels induced by morphine were blocked by pre-treatment of a selective dopamine D1 receptor antagonist SCH23390. These findings suggest that VTA-ACG transmission may play a crucial role in the acquisition and maintenance of µ-opioid-induced place preference. The activation of DARPP32 and CREB through dopamine D1 receptors in the ACG could be implicated in the maintenance of µ-opioid-induced place preference.

Interaction of BMP10 with Tcap may modulate the course of hypertensive cardiac hypertrophy.

Elevated wall stress by hypertension induces an adaptive myocardial hypertrophy via releasing prohypertrophic hormones such as angiotensin II. In this study, we investigated the involvement of bone morphogenetic protein-10 (BMP10) in hypertension-induced cardiac hypertrophy. Expression of BMP10 was increased in the hypertrophied ventricles from hypertensive rats. BMP10 localized on cell surface and at stretch-sensing Z disc of cardiomyocytes, where BMP10 interacted with a protein called titin-cap (Tcap). A rare variant of the human BMP10 gene, Thr326Ile, was found to be associated with hypertensive dilated cardiomyopathy. The variant BMP10 demonstrated decreased binding to Tcap and increased extracellular secretion. Conditioned medium from cells transfected with wild-type or variant BMP10 induced hypertrophy in rat neonatal cardiomyocytes, except that medium from variant BMP10-carrying cells showed an enhanced effect reflecting the increased secretion. These observations suggested that hypertension induced expression of prohypertrophic BMP10, and the hypertrophic effect of BMP10 was modulated, at least in part, by its binding to Tcap at the Z disc.

Angiotensin II-induced ionic currents and signalling pathways in submandibular ganglion neurons.

Angiotensin II (Ang II) is one of the most important vasoconstrictive hormones but is also known to act as a neuromodulator and a neurotransmitter in the central and peripheral nervous system. The submandibular ganglion (SMG) neuron is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. In this study, the effects of Ang II on SMG neurons were investigated using the whole-cell patch clamp technique. Membrane currents evoked by a ramp pulse from +50 to -100 mV (-150 mV/500 ms) were compared in both the absence and presence of Ang II. In eight neurons tested, 1 microM Ang II increased inward currents by 42.0+/-8.2%. The reversal potentials of the Ang II-induced current were 0.2+/-0.6 mV. These increase of inward currents by Ang II were antagonized by losartan, a selective antagonist of AT(1) receptors. Intracellular dialysis with 0.1mM guanosin 5'-O-(2-thiodiphosphate) (GDP-beta-S), a G-proteins blocker, and anti-G(q/11) antibody attenuated Ang II-induced ionic current. In addition, pretreatment of neurons with 10 microM staurosporine (stauro), a protein kinase C (PKC) inhibitor, 0.5 microM PMA, a PKC activator, and 10 microM KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaM K II) inhibitor, attenuated Ang II-induced ionic current in SMG neurons. The data presented here demonstrated that Ang II-induced ionic current via G(q/11)-proteins involving both PKC and CaM K II pathways in SMG neurons.