Identification of LEM-14 inhibitor of the oncoprotein NSD2.

The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC50 of 132 µM and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC50 of 418 µM (NSD1), IC50 of 111 µM (NSD2) and IC50 of 60 µM (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors.

Characterization of in vitro and in vivo metabolism of leelamine using liquid chromatography-tandem mass spectrometry.

Leelamine is a diterpene compound found in the bark of pine trees and has garnered considerable interest owing to its potent anticancer properties. The aim of the present study was to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono-hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSn fragmentation obtained by collision-induced dissociation using LC-MS/MS in HLMs. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite, whereas CYP2C19, CYP1A1, and CYP3A4 contributed to some extent. Moreover, we identified only one metabolite M1, in the urine, but none in the feces. In conclusion, leelamine was metabolized to a mono-hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.

A novel benzamide derivative protects ligature-induced alveolar bone erosion by inhibiting NFATc1-mediated osteoclastogenesis.

Since elevated osteoclast formation and/or activity by inhibitory responses against pathogens leads to diverse osteolytic bone diseases including periodontitis, inhibition of osteoclast differentiation and bone resorption has been a primary therapeutic strategy. In this study, we investigated the therapeutic potential of a novel benzamide-linked molecule, OCLI-070, for preventing alveolar bone loss in mice with ligature-induced experimental periodontitis. OCLI-070 inhibited osteoclast formation by acting on both early and late stages of differentiation, and attenuated the induction of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific genes. In addition, OCLI-070 significantly suppressed the formation of actin rings and resorption pits. Analysis of the inhibitory action of OCLI-070 showed that it markedly suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced extracellular signal-regulated kinase (ERK) and NF-κB signaling cascades. Moreover, OCLI-070 prevented ligature-induced alveolar bone erosion in mice by suppressing osteoclast formation. These findings demonstrate that OCLI-070 attenuated osteoclast differentiation and function as well as ligature-induced bone erosion by inhibiting RANKL-mediated ERK and NF-κB signaling pathways.

Isolation, Synthesis, and Antisepsis Effects of a C-Methylcoumarinochromone Isolated from Abronia nana Cell Culture.

Only a few isoflavones have been isolated from plants of the genus Abronia. The biological properties of compounds isolated from Abronia species have not been well established, and their antisepsis effects have not been reported yet. In the present study, a new C-methylcoumarinochromone, was isolated from Abronia nana suspension cultures. Its structure was deduced as 9,11-dihydroxy-10-methylcoumarinochromone (boeravinone Y, 1) by spectroscopic data analysis and verified by chemical synthesis. The potential inhibitory effects of 1 against high mobility group box 1 (HMGB1)-mediated septic responses were investigated. Results showed that 1 effectively inhibited lipopolysaccharide-induced release of HMGB1 and suppressed HMGB1-mediated septic responses, in terms of reduction of hyperpermeability, leukocyte adhesion and migration, and cell adhesion molecule expression. In addition, 1 increased the phagocytic activity of macrophages and exhibited bacterial clearance effects in the peritoneal fluid and blood of mice with cecal ligation and puncture-induced sepsis. Collectively, these results suggested that 1 might have potential therapeutic activity against various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Lipidomic platform for structural identification of skin ceramides with α-hydroxyacyl chains.

Skin ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids via an amide bond. Typical fatty acid acyl chains are composed of α-hydroxy fatty acid (A), esterified ω-hydroxy fatty acid (EO), non-hydroxy fatty acid (N), and ω-hydroxy fatty acid (O). We recently established a lipidomic platform to identify skin ceramides with non-hydroxyacyl chains using tandem mass spectrometry. We expanded our study to establish a lipidomic platform to identify skin ceramides with α-hydroxyacyl chains. Tandem mass spectrometry analysis of A-type ceramides using chip-based direct infusion nanoelectrospray-mass spectrometry showed the characteristic fragmentation pattern of both acyl and sphingoid units, which can be applied for structural identification of ceramides. Based on the tandem mass spectrometry fragmentation patterns of A-type ceramides, comprehensive fragmentation schemes were proposed. Our results may be useful for identifying A-type ceramides in the stratum corneum of human skin.

Efficient Syntheses of 1,2,3-Triazoloamide Derivatives Using Solid- and Solution-Phase Synthetic Approaches.

Efficient synthetic routes for the preparation of secondary and tertiary 1,2,3-triazoloamide derivatives were developed. A secondary α-1,2,3-triazoloamide library was constructed and expanded by a previously developed solid-phase synthetic route and a tertiary 1,2,3-triazoloamide library was constructed by a parallel solution-phase synthetic route. The synthetic routes rely on amide formation with secondary amines and chloro-acid chlorides; SN2 reaction with sodium azide; and the selective [3 + 2] Hüisgen cycloaddition with appropriate terminal alkynes. The target secondary and tertiary 1,2,3-triazoloamide derivatives were obtained with three-diversity points in excellent overall yields and purities using the reported solid- and solution-phase synthetic routes, respectively.

Identification of metabolites of N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl)thiazol-4-yl)pivalamide including CYP3A4-mediated C-demethylation in human liver microsomes with high-resolution/high-accuracy tandem mass.

KRO-105714 [N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl)thiazol-4-yl)pivalamide] is a 2,4,5-trisubstituted 1,3-thiazole derivative that exerts anti-atopic dermatitis activity via robust suppression of the sphingosylphosphorylcholine receptor. This study used high-resolution/high-accuracy tandem mass spectroscopy (HRMS) and recombinant cDNA-expressed cytochrome P450 (P450) isoforms to identify the metabolic pathway and metabolites of KRO-105714 in human liver microsomes (HLMs) as therapeutic agents for inflammation. The incubation of KRO-105714 with pooled HLMs in the presence of NADPH generated four metabolites (M1-M4). The metabolites were identified using HRMS and confirmed using synthetic standards for M2 and M4. M1 and M2 were identified as monohydroxylated metabolites, and M3 and M4 were identified as O-demethyl KRO-105714 and C-demethyl KRO-105714, respectively. In the inhibition study with selective CYP3A4 inhibitors and incubation in recombinant cDNA-expressed P450 enzymes, all the metabolites of KRO-105714 were formed by CYP3A4 in HLMs. The CYP3A4-mediated formation of M4 from M2 was confirmed via incubation of M2 in HLMs. These results showed that the unusual C-demethylated metabolite M4 was generated from monohydroxyl metabolite M2 via a CYP3A4-mediated enzymatic reaction in HLMs.

Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine (TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimidines 6 and 30, which could be further utilized as viable lead compounds.

A derivative of imidazobenzimidazole, ML106, inhibits melanin synthesis via p38 MAPK activation.

We investigated the effects of ML106 on melanogenesis in B16F10 melanoma cells. Our results showed that ML106 decreased melanin content and tyrosinase activity in a dose-dependent manner. Interestingly, ML106 did not inhibit microphthalmia-associated transcription factor (MITF) expression, but did decrease tyrosinase expression. Thus, we further investigated the expression and degradation of tyrosinase and related signal transduction pathways. Although ML106 increased glycogen synthase kinase 3beta (GSK3beta) activation, the level of beta-catenin level was not affected. Thus, we excluded the involvement of GSK3beta and beta-catenin in ML106-induced hypopigmentation. However, ML106 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), causing down-regulation of tyrosinase. Thus, we next investigated whether tyrosinase down-regulation was due to proteasomal degradation by p38 MAPK activation. We found that ML106-induced tyrosinase down-regulation was restored by MG132, a proteasome inhibitor. Thus, we propose that ML106 has hypopigmentary activity through tyrosinase degradation via p38 MAPK phosphorylation.

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 µl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 µl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Synthesis and biological evaluation of triazolothienopyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of triazolothienopyrimidine (TTPM) compounds as potent HIV-1 replication inhibitors during a high-throughput screening campaign that evaluated more than 200,000 compounds using a cell-based full replication assay. Herein, we report the optimization of the antiviral activity in a cell-based assay system leading to the discovery of aryl-substituted TTPM derivatives (38, 44, and 45), which exhibited significant inhibition of HIV-1 replication with acceptable safety margins. These novel and potent TTPMs could serve as leads for further development.

In vitro metabolism of obovatol and its effect on cytochrome P450 enzyme activities in human liver microsomes.

Obovatol, a major constituent of the leaves of Magnolia obovata Thunb, is known to inhibit nuclear factor-κB activity and arachidonic acid-induced platelet aggregation. This study was performed to identify the metabolites of obovatol in human liver microsomes. Human liver microsomes incubated with obovatol in the presence of NADPH and/or UDPGA resulted in the formation of six metabolites, M1-M6. M1 and M2 were identified as hydroxyobovatol, on the basis of liquid chromatography/tandem mass spectrometric (LC-MS/MS) analysis. M1, M2 and obovatol were further metabolized to their glucuronide conjugates, obovatol-glucuronide (M3), obovatol-diglucuronide (M4) and hydroxyobovatol-glucuronide (M5 and M6). The inhibitory potency of obovatol on eight major human P450s was also investigated in human liver microsomes. In these experiments, obovatol strongly inhibited CYP2C19-mediated S-mephenytoin hydroxylase activity with an IC(50) value of 0.8 µM, which could have implications for drug-drug interactions.

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability.

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.

Solid-phase synthesis of thiazolo[4,5-b]pyridine derivatives using Friedländer reaction.

Traceless solid-phase synthesis of 2,5,6,7-tetrasubstituted thiazolo[4,5-b]pyridine derivatives is described. Thorpe-Ziegler type cyclization of solid supported cyanocarbonimidodithioate with alpha-halo ketones afforded thiazole resin, which were converted to the desired thiazolopyridine resin by the Friedländer protocol under microwave irradiation conditions. After oxidation of sulfides to sulfones, nucleophilic desulfonative substitution with amines gave the target thiazolo[4,5-b]pyridine derivatives in good overall yields.

Dosimetric comparison of four different external beam partial breast irradiation techniques: three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, helical tomotherapy, and proton beam therapy.

BACKGROUND AND PURPOSE: As an alternative to whole breast irradiation in early breast cancer, a variety of accelerated partial breast irradiation (APBI) techniques have been investigated. The purpose of our study is to compare the dosimetry of four different external beam APBI (EB-APBI) plans: three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), helical tomotherapy (TOMO), and proton beam therapy (PBT). METHODS AND MATERIALS: Thirty patients were included in the study, and plans for four techniques were developed for each patient. A total dose of 30Gy in 6Gy fractions once daily was prescribed in all treatment plans. RESULTS: In the analysis of the non-PTV breast volume that was delivered 50% of the prescribed dose (PD), PBT (mean: 16.5%) was superior to TOMO (mean: 22.8%), IMRT (mean: 33.3%), and 3D-CRT (mean: 40.9%) (p<0.001). The average ipsilateral lung volume percentage receiving 20% of the PD was significantly lower in PBT (0.4%) and IMRT (2.3%) compared with 3D-CRT (6.0%) and TOMO (14.2%) (p<0.001). The average heart volume percentage receiving 20% and 10% of the PD in left-sided breast cancer (N=19) was significantly larger with TOMO (8.0%, 19.4%) compared to 3D-CRT (1.5%, 3.1%), IMRT (1.2%, 4.0%), and PBT (0%, 0%) (p<0.001). CONCLUSIONS: All four EB-APBI techniques showed acceptable coverage of the PTV. However, effective non-PTV breast sparing was achieved at the cost of considerable dose exposure to the lung and heart in TOMO.

Stimulatory Effects of KPR-A148 on Osteoblast Differentiation and Bone Regeneration.

Background: Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation in vitro and bone regeneration in vivo. Methods: The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on in vivo bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining. Results: KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10 µM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model. Conclusion: These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.

Inhibitory Effect of KP-A038 on Osteoclastogenesis and Inflammatory Bone Loss Is Associated With Downregulation of Blimp1.

Excessive osteoclastic activity results in pathological bone resorptive diseases, such as osteoporosis, periodontitis, and rheumatoid arthritis. As imidazole-containing compounds possess extensive therapeutic potential for the management of diverse diseases, we synthesized a series of imidazole derivatives and investigated their effects on osteoclast differentiation and function. In the present study, we found that a novel imidazole derivative, KP-A038, suppressed receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and bone-resorbing activity in vitro and attenuated lipopolysaccharide (LPS)-induced bone destruction in vivo. KP-A038 significantly inhibited the induction of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and the expression of its target genes, including tartrate-resistant acid phosphatase (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), and matrix metallopeptidase 9 (Mmp9). KP-A038 upregulated the expression of negative regulators of osteoclast differentiation, such as interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6). Consistently, KP-A038 downregulated the expression of B lymphocyte-induced maturation protein-1 (Blimp1 encoded by Prdm1), a repressor for Irf8 and Bcl6. Moreover, administration of KP-A038 reduced LPS-induced bone erosion by suppressing osteoclast formation in vivo. Thus, our findings suggest that KP-A038 may serve as an effective therapeutic agent for the treatment and/or prevention of bone loss in pathological bone diseases, including osteoporosis and periodontitis.

Monoglycerides from the brown alga Sargassum sagamianum: Isolation, synthesis, and biological activity.

Polyunsaturated fatty acid-derived monoglycerides were characterized from the marine brown alga Sargassum sagamianum, collected from Jeju Island, Korea. A new compound of this structural class was isolated and determined to be 1-octadecatetraenoyl glycerol, by combined spectroscopic methods. Based on the structures and bioactivity of these compounds, a series of monoglycerides were synthesized using glycerol and various fatty acids. Several compounds exhibited moderate to significant inhibition of phospholipase A(2) and cyclooxygenase-2.

Performance evaluation of field-in-field technique for tangential breast irradiation.

Conventional hard or dynamic wedge systems are commonly applied to reduce the dose inhomogeneity associated with whole breast irradiation. We evaluated the dosimetric benefits of the field-in-field (FIF) technique by comparing it with the electronic compensator (EC), Varian enhanced dynamic wedge (EW) and conventional hard wedge (HW) techniques. Data were obtained from 12 patients who had undergone breast-conserving surgery (six left-sided and six right-sided). For these patients, the average breast planning target volume (PTV) was 447.4 cm(3) (range, 211.6-711.8 cm(3)). For the experiments, a 6 MV photon beam from a Varian 21 EX was used, the HW and EW angles were applied from 15 to 45 degrees, while 40-50% isodose values were chosen to achieve the best dose distribution for electronic compensation. In applying the FIF technique, we used two or three subfields for each portal. To evaluate the performance for each planning technique, we analysed a dose-volume histogram (DVH) for the PTV and organs-at-risk (OARs). To evaluate the effects of these techniques on dose inhomogeneity, we defined the PTV Dose Improvement (PDI) index, which was derived from a PTV volume between 97-103% of the differential DVHs. In addition, we compared the average monitor units (MUs) for each technique. The average PDI index with FIF is 76.4%, while the PDI indices for other treatments were 65.8, 41.8 and 50.9% for EC, EW and HW, respectively. This study demonstrated an improved performance using the FIF technique compared with the conventional HW/EW system, as well as a new modality for EC. We demonstrated that FIF is a very useful technique for improving PTV conformity, while protecting the OARs from breast tangential irradiation.

Characterization of CYPs and UGTs Involved in Human Liver Microsomal Metabolism of Osthenol.

Osthenol is a prenylated coumarin isolated from the root of Angelica koreana and Angelica dahurica, and is an O-demethylated metabolite of osthole in vivo. Its various pharmacological effects have been reported previously. The metabolic pathway of osthenol was partially confirmed in rat osthole studies, and 11 metabolic products were identified in rat urine. However, the metabolic pathway of osthenol in human liver microsomes (HLM) has not been reported. In this study, we elucidated the structure of generated metabolites using a high-resolution quadrupole-orbitrap mass spectrometer (HR-MS/MS) and characterized the major human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes involved in osthenol metabolism in human liver microsomes (HLMs). We identified seven metabolites (M1-M7) in HLMs after incubation in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and uridine 5'-diphosphoglucuronic acid (UDPGA). As a result, we demonstrated that osthenol is metabolized to five mono-hydroxyl metabolites (M1-M5) by CYP2D6, 1A2, and 3A4, respectively, a 7-O-glucuronide conjugate (M6) by UGT1A9, and a hydroxyl-glucuronide (M7) from M5 by UGT1A3 in HLMs. We also found that glucuronidation is the dominant metabolic pathway of osthenol in HLMs.