Inhibition of GLI Transcriptional Activity and Prostate Cancer Cell Growth and Proliferation by DAX1.

The Hedgehog (Hh) signaling pathway plays an essential role in the initiation and progression of prostate cancer. This is mediated by transcriptional factors belonging to the GLI (glioma-associated oncogene) family, which regulate downstream targets to drive prostate cancer progression. The activity of GLI proteins is tightly controlled by a range of mechanisms, including molecular interactions and post-translational modifications. In particular, mitogenic and oncogenic signaling pathways have been shown to regulate GLI protein activity independently of upstream Hh pathway signaling. Identifying GLI protein regulators is critical for the development of targeted therapies that can improve patient outcomes. This study aimed to identify a novel protein that directly regulates the activity of GLI transcription factors in prostate cancer. We performed gene expression, cellular analyses, and reporter assays to demonstrate that DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) interacts with GLI1 and GLI2, the master regulators of Hh signaling. Interestingly, DAX1 overexpression significantly inhibited Hh signaling by reducing GLI1 and GLI2 activity, prostate cancer cell proliferation, and viability. Our results shed light on a novel regulatory mechanism of Hh signaling in prostate cancer cells. The interaction between DAX1 and GLI transcription factors provides insight into the complex regulation of Hh signaling in prostate cancer. Given the importance of Hh signaling in prostate cancer progression, targeting DAX1-GLI interactions may represent a promising therapeutic approach against prostate cancer. Overall, this study provides new insights into the regulation of the Hh pathway and its role in prostate cancer progression. The findings suggest that DAX1 could serve as a potential therapeutic target for the treatment of prostate cancer.

One-Pot Synthesis of 1,3,4-Oxadiazines from Acylhydrazides and Allenoates.

The framework of 1,3,4-oxadiazine is crucial for numerous bioactive molecules, but only a limited number of synthetic methods have been reported for its production. In 2015, Wang's group developed a 4-(dimethylamino)pyridine (DMAP)-catalyzed [2 + 4] cycloaddition of allenoates with N-acyldiazenes, which provided an atom-efficient route for 1,3,4-oxadiazines. However, the practicality of this method was limited by the instability of N-acyldiazenes as starting materials. Building upon our ongoing research about the aerobic oxidation of hydrazides and their synthetic applications, we hypothesized that aerobic oxidative cycloadditions using acylhydrazides instead of N-acyldiazenes may provide a more practical synthetic route for 1,3,4-oxadiazines. In this manuscript, we describe a one-pot synthetic protocol for 1,3,4-oxadiazines from acylhydrazides and allenoates. The developed one-pot protocol consists of aerobic oxidations of acylhydrazides into N-acyldiazenes using NaNO2 and HNO3, followed by the DMAP-catalyzed cycloaddition of allenoate with the generated N-acyldiazenes. A variety of 1,3,4-oxadiazines were produced in good to high yields. In addition, the practicality of the developed method was demonstrated by a gram-scale synthesis of 1,3,4-oxadiazine.

Suppression of lytic replication of Kaposi's sarcoma-associated herpesvirus by autophagy during initial infection in NIH 3T3 fibroblasts.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the angioproliferative neoplasm Kaposi's sarcoma (KS). We first confirmed the susceptibility of NIH 3T3 fibroblasts to KSHV by infecting them with BCP-1-derived KSHV. Lytic replication of KSHV was confirmed by PCR amplification of viral DNA isolated from culture supernatants of KSHV-infected cells. The template from KSHV-infected NIH 3T3 cells resulted in an intense viral DNA PCR product. A time course experiment revealed the disappearance of KSHV-specific DNA in culture supernatant of NIH 3T3 cells during a period between 48 h and 72 h postinfection. Furthermore, 3 days postinfection, infected NIH 3T3 cells showed no evidence of latent or lytic transcripts, including LANA, vFLIP, vCyclin, and vIL-6. These results imply that KSHV infection in NIH 3T3 cells is unstable and is rapidly lost on subsequent culturing. Additionally, we detected an enhancement of autophagy early in infection with KSHV. More interestingly, inhibition of autophagy by Beclin 1 siRNA or 3-methyladenine significantly increased the amount of KSHV-specific DNA in the culture supernatant of NIH 3T3 cells when compared to the group treated with KSHV infection alone, implying that autophagy prevents lytic replication of KSHV. Taken together, our data suggest that autophagy could be one of the cellular mechanisms utilized by host cells to promote viral clearance.

Discovery of 11ß-hydroxysteroid dehydrogenase type 1 inhibitor.

Various adamantane sulfonamides showed potent inhibitory activity against 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). In continuation of our efforts to discover a more potent, selective and metabolically stable 11ß-HSD1 inhibitor in mice as well as in humans, we optimized the adamantane sulfonamide using structure-based molecular modeling. Compound 3, which has alkyl side chains on the linker, demonstrated a potent inhibitory activity against human and mouse 11ß-HSD1 (IC50 of 0.6 nM and 26 nM, respectively) and good physicochemical properties as a new anti-diabetes drug candidate.

3-Amino-N-adamantyl-3-methylbutanamide derivatives as 11ß-hydroxysteroid dehydrogenase 1 inhibitor.

Many adamantane derivatives have been demonstrated to function as 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors. 3-Amino-N-adamantyl-3-methylbutanamide derivatives were optimized by structure-based drug design. Compound 8j exhibited a good in vitro and ex vivo inhibitory activity against both human and mouse 11ß-HSD1.

Differential inhibitory effects of two Raf-targeting drugs, sorafenib and PLX4720, on the growth of multidrug-resistant cells.

B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade in human cancers, making it an important therapeutic target. Here, we describe the differential effects of two Raf-targeting drugs, sorafenib and PLX4720, on multidrug-resistant v-Ha-ras-transformed cells (Ras-NIH 3T3/Mdr). We demonstrate that the growth of the NIH 3T3/Mdr cell line was affected in a dose-dependent manner more significantly by the pan-Raf inhibitor sorafenib than by the selective mutant B-Raf inhibitor PLX4720. Despite their differential effects on LKB1/AMPK phosphorylation, both sorafenib and PLX4720 inhibited downstream mTOR signaling with concomitant induction of autophagy, implying that the differential effects of sorafenib and PLX4720 on multidrug-resistant cells might not be due to different levels of autophagy and apoptosis. Interestingly, sorafenib caused a dose-dependent increase in rhodamine 123 uptake and retention. More importantly, sorafenib reversed the resistance to paclitaxel in Ras-NIH 3T3/Mdr cells. Moreover, MEK/ERK signaling was hyperactivated by the selective mutant B-Raf inhibitor PLX4720 and inhibited by the pan-Raf inhibitor sorafenib. Our data suggest that sorafenib sensitivity in MDR cells is mediated through the inhibition of P-glycoprotein activity following strong inhibition of Raf/MEK/ERK signaling. Thus, Raf inhibition with sorafenib might be a promising approach to abrogate the multidrug resistance of cancer cells.

Comparative Analysis of Anticancer and Antibacterial Activities among Seven Trametes Species.

Species in the genus Trametes (Basidiomycota, Polyporales) have been used in natural medicine for a long time. Many studies reported that mycelia or fruiting bodies of Trametes spp. exhibited effects of antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. However, comparative analysis in this genus is scarce due to limitation of morphological identification and the sample number. In this study, the 19 strains of seven Trametes species were chosen to generate a five-gene-based phylogeny with the 31 global references. In addition, 39 culture extracts were prepared for 13 strains to test for anticancer and antibacterial activities. Strong anticancer activities were found in several extracts from T. hirsuta and T. suaveolens. Anticancer activities of T. suaveolens, T. cf. junipericola and T. trogii were first described here. The antibacterial ability of T. versicolor and T. hirsuta extracts has been confirmed. The antibacterial activities of T. suaveolens have been reported at the first time in this study. These results suggest an efficient application of the genus Trametes as the drug resources especially for anticancer agents.

Targeting Mitochondrial Oxidative Stress as a Strategy to Treat Aging and Age-Related Diseases.

Mitochondria are one of the organelles undergoing rapid alteration during the senescence process. Senescent cells show an increase in mitochondrial size, which is attributed to the accumulation of defective mitochondria, which causes mitochondrial oxidative stress. Defective mitochondria are also targets of mitochondrial oxidative stress, and the vicious cycle between defective mitochondria and mitochondrial oxidative stress contributes to the onset and development of aging and age-related diseases. Based on the findings, strategies to reduce mitochondrial oxidative stress have been suggested for the effective treatment of aging and age-related diseases. In this article, we discuss mitochondrial alterations and the consequent increase in mitochondrial oxidative stress. Then, the causal role of mitochondrial oxidative stress on aging is investigated by examining how aging and age-related diseases are exacerbated by induced stress. Furthermore, we assess the importance of targeting mitochondrial oxidative stress for the regulation of aging and suggest different therapeutic strategies to reduce mitochondrial oxidative stress. Therefore, this review will not only shed light on a new perspective on the role of mitochondrial oxidative stress in aging but also provide effective therapeutic strategies for the treatment of aging and age-related diseases through the regulation of mitochondrial oxidative stress.

The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts.

In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.

Identification of chalcones as potent and selective PDE5A1 inhibitors.

Chalcones have an affinity for many receptors, enzymes, and transcription factors as flavonoid analogues. Their most studied pharmacological action is that of vasodilatation due to inhibition of phosphodiesterase 5A1 (PDE5A1). To this end, we have established a recursive partitioning model with 3 chemical descriptors for the prediction of compounds that can inhibit PDE5A1. This model was able to predict active compounds with an accuracy of 82.8%. Compound 4 was found to be a potent and selective inhibitor, with a relatively low IC(50) value. The binding mechanism of this compound was also investigated through molecular docking studies.

Targeting regulation of ATP synthase 5 alpha/beta dimerization alleviates senescence.

Senescence is a distinct set of changes in the senescence-associated secretory phenotype (SASP) and leads to aging and age-related diseases. Here, we screened compounds that could ameliorate senescence and identified an oxazoloquinoline analog (KB1541) designed to inhibit IL-33 signaling pathway. To elucidate the mechanism of action of KB1541, the proteins binding to KB1541 were investigated, and an interaction between KB1541 and 14-3-3ζ protein was found. Specifically, KB1541 interacted with 14-3-3ζ protein and phosphorylated of 14-3-3ζ protein at serine 58 residue. This phosphorylation increased ATP synthase 5 alpha/beta dimerization, which in turn promoted ATP production through increased oxidative phosphorylation (OXPHOS) efficiency. Then, the increased OXPHOS efficiency induced the recovery of mitochondrial function, coupled with senescence alleviation. Taken together, our results demonstrate a mechanism by which senescence is regulated by ATP synthase 5 alpha/beta dimerization upon fine-tuning of KB1541-mediated 14-3-3ζ protein activity.

Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening.

We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 µM, respectively. In addition, compound 1 also exhibited replicon activity with EC(50) value of 2.16 µM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.

Discovery of potent, selective, and orally bioavailable PDE5 inhibitor: Methyl-4-(3-chloro-4-methoxybenzylamino)-8-(2-hydroxyethyl)-7-methoxyquinazolin-6-ylmethylcarbamate (CKD 533).

In a continuing effort to discover novel PDE5 inhibitors, we have successfully found quinazolines with 4-benzylamino substitution as potent and selective PDE5 inhibitors. Initial lead compound (1) was found to be easily metabolized when incubated with human liver microsomes mainly through C6 amide hydrolysis. Blocking of this metabolic hot spot led to discovery of 10 (CKD533) which is highly potent, selective and orally efficacious in conscious rabbit model for erectile dysfunction and now is undergoing preclinical toxicology study.

Discovery of a potent tubulin polymerization inhibitor: synthesis and evaluation of water-soluble prodrugs of benzophenone analog.

Prodrugs have proven to be very useful in enhancing aqueous solubility of sparingly water-soluble drugs, thereby increasing in vivo efficacy without a need of special excipients. In vitro and in vivo evaluations of a number of amino acid prodrugs of 1, a previously identified potent tubulin polymerization inhibitor and cytotoxic against various cancer cell lines led to the discovery of 3·HCl (l-valine attached) which is highly efficacious in mouse xenografts bearing human cancer. Pharmacokinetic analysis in rats revealed that compound 1 was released immediately upon administration of 3·HCl intravenously, with rapid clearance of 3·HCl indicating the effective cleavage of prodrug. Compound 3·HCl (CKD-516) has now been progressed to phase 1 clinical trial.

Structure-based virtual screening of novel tubulin inhibitors and their characterization as anti-mitotic agents.

Microtubule cytoskeletons are involved in many essential functions throughout the life cycle of cells, including transport of materials into cells, cell movement, and proper progression of cell division. Small compounds that can bind at the colchicine site of tubulin have drawn great attention because these agents can suppress or inhibit microtubule dynamics and tubulin polymerization. To find novel tubulin polymerization inhibitors as anti-mitotic agents, we performed a virtual screening study of the colchicine binding site on tubulin. Novel tubulin inhibitors were identified and characterized by their inhibitory activities on tubulin polymerization in vitro. The structural basis for the interaction of novel inhibitors with tubulin was investigated by molecular modeling, and we have proposed binding models for these hit compounds with tubulin. The proposed docking models were very similar to the binding pattern of colchicine or podophyllotoxin with tubulin. These new hit compound derivatives exerted growth inhibitory effects on the HL60 cell lines tested and exhibited strong cell cycle arrest at G2/M phase. Furthermore, these compounds induced apoptosis after cell cycle arrest. In this study, we show that the validated derivatives of compound 11 could serve as potent lead compounds for designing novel anti-cancer agents that target microtubules.

Discrimination of Adzuki Bean (Vigna angularis) Geographical Origin by Targeted and Non-Targeted Metabolite Profiling with Gas Chromatography Time-of-Flight Mass Spectrometry.

As international food trade increases, consumers are becoming increasingly interested in food safety and authenticity, which are linked to geographical origin. Adzuki beans (Vigna angularis) are cultivated worldwide, but there are no tools for accurately discriminating their geographical origin. Thus, our study aims to develop a method for discriminating the geographical origin of adzuki beans through targeted and non-targeted metabolite profiling with gas chromatography time-of-flight mass spectrometry combined with multivariate analysis. Orthogonal partial least squares discriminant analysis showed clear discrimination between adzuki beans cultivated in Korea and China. Non-targeted metabolite profiling showed better separation than targeted profiling. Furthermore, citric acid and malic acid were the most notable metabolites for discriminating adzuki beans cultivated in Korea and China. The geographical discrimination method combining non-targeted metabolite profiling and pareto-scaling showed excellent predictability (Q2 = 0.812). Therefore, it is a suitable prediction tool for the discrimination of geographical origin and is expected to be applicable to the geographical authentication of adzuki beans.

Quinazolines as potent and highly selective PDE5 inhibitors as potential therapeutics for male erectile dysfunction.

In an effort to minimize side effects associated with low selectivity against PDE isozymes, we have successfully identified a series of 6,7,8-substituted quinzaolines as potent inhibitors of PDE5 with high level of isozyme selectivity, especially against PDE6 and PDE11. PDE5 potency and isozyme selectivity of quinazolines were greatly improved with substitutions both at 6- and 8-position. The synthesis, structure-activity relationships and in vivo efficacy of this novel series of potent PDE5 inhibitors are described.

The efficacy and safety of clopidogrel resinate as a novel polymeric salt form of clopidogrel.

A novel polymeric salt of clopidogrel, clopidogrel resinate, was prepared as a anticoagulant drug. To prove the feasibility as a new active substance, clopidogrel resinate was evaluated for its efficacy and safety. In accelerated stability tests, the clopidogrel resinate tablet (Pregrel) showed less brown discoloration and fewer impurities than the clopidogrel bisulfate tablets under open and closed conditions. In toxicity tests, no deaths occurred after a single dose of up to 2000 mg/kg/day and 13-week repeated doses of up to 625 mg/kg/day in rats without abnormal symptoms compared to clopidogrel bisulfate. When clopidogrel resinate was treated onto Caco-2 cell monolayers, clopidogrel, but not the resin, permeated across the cells with a hight permeation coefficient (Papp) of 13.5 +/- 1.13 x 10(-6) cm/sec. Clopidogrel resinate and clopidogrel bisulfate showed similar pharmacokinetics following oral administration to beagle dogs. A single oral administration of clopidogrel resinate dose-dependently inhibited ADP-induced ex vivo aggregation up to 30 mg/kg in rats. In conclusion, clopidogrel resinate was proved to be an efficient and safe polymeric salt as a candidate for a new clopidogrel salt.

A novel highly potent and selective 11ß-hydroxysteroid dehydrogenase type 1 inhibitor, INU-101.

11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a cortisol regenerating enzyme that amplifies tissue glucocorticoid levels, especially in the liver and adipose tissue. Knockout mice or a selective inhibitor of 11ß-HSD1 improves metabolic syndrome parameters in preclinical models and human clinical trials. Here, we evaluated the therapeutic potential of INU-101, a potent and selective oral inhibitor of 11ß-HSD1. The in vitro activity of 11ß-HSD1 was measured using the homogeneous time-resolved fluorescence (HTRF) assay. Differentiated adipocytes were used to evaluate the cellular 11ß-HSD1 activity. To determine the inhibitory effects on 11ß-HSD1 in tissues, we performed ex vivo studies using liver and adipose tissue isolated from C57BL/6 J mice and Cynomolgus monkeys. KKAy mice, ob/ob mice and ZDF rats were administered INU-101 to evaluate whether this compound ameliorated metabolic abnormalities in obese and diabetic animals. INU-101 had highly potent inhibitory activity in mouse, monkey and human 11ß-HSD1, derived from liver microsomes. The oral administration of INU-101 significantly inhibited 11ß-HSD1 activity in the liver and adipose tissue of mice and monkeys. In KKAy mice, ob/ob mice and ZDF rats, the oral administration of INU-101 enhanced insulin sensitivity and lowered the fasting blood glucose level. Furthermore, INU-101 treatment decreased the body weight and ameliorated an improved lipid profile in the diabetic mouse model. These results suggest that the 11ß-HSD1 inhibitor, INU-101 may serve as a novel drug candidate for the treatment of type 2 diabetes and metabolic syndrome.

Prediction of GPCR-Ligand Binding Using Machine Learning Algorithms.

We propose a novel method that predicts binding of G-protein coupled receptors (GPCRs) and ligands. The proposed method uses hub and cycle structures of ligands and amino acid motif sequences of GPCRs, rather than the 3D structure of a receptor or similarity of receptors or ligands. The experimental results show that these new features can be effective in predicting GPCR-ligand binding (average area under the curve [AUC] of 0.944), because they are thought to include hidden properties of good ligand-receptor binding. Using the proposed method, we were able to identify novel ligand-GPCR bindings, some of which are supported by several studies.