Target Class Profiling of Small-Molecule Methyltransferases.

Target class profiling (TCP) is a chemical biology approach to investigate understudied biological target classes. TCP is achieved by developing a generalizable assay platform and screening curated compound libraries to interrogate the chemical biological space of members of an enzyme family. In this work, we took a TCP approach to investigate inhibitory activity across a set of small-molecule methyltransferases (SMMTases), a subclass of methyltransferase enzymes, with the goal of creating a launchpad to explore this largely understudied target class. Using the representative enzymes nicotinamide N-methyltransferase (NNMT), phenylethanolamine N-methyltransferase (PNMT), histamine N-methyltransferase (HNMT), glycine N-methyltransferase (GNMT), catechol O-methyltransferase (COMT), and guanidinoacetate N-methyltransferase (GAMT), we optimized high-throughput screening (HTS)-amenable assays to screen 27,574 unique small molecules against all targets. From this data set, we identified a novel inhibitor which selectively inhibits the SMMTase HNMT and demonstrated how this platform approach can be leveraged for a targeted drug discovery campaign using the example of HNMT.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Differential anti-inflammatory and analgesic effects by enantiomers of zaltoprofen in rodents.

In the present study, we investigated the effect of zaltoprofen enantiomers on inflammation and pain and compared their effect with racemic zaltoprofen. S(+)-zaltoprofen potently inhibited the inflammatory response in carrageenan-induced paw edema model, whereas R(-)-zaltoprofen did not. Moreover, the anti-inflammatory effect of S(+)-zaltoprofen was stronger than that of racemic zaltoprofen, suggesting that S(+)-zaltoprofen is an active component of racemic zaltoprofen in terms of anti-inflammatory activity. In contrast, the results of acetic acid-induced writhing model demonstrated that no significant analgesic effect was observed by racemic zaltoprofen and zaltoprofen enantiomers at doses used in carrageenan-induced paw edema model. However, racemic zaltoprofen and zaltoprofen enantiomers all exerted an analgesic effect at higher doses, which is inconsistent with the result of carrageenan-induced paw edema model. Gastric ulcers induced by racemic zaltoprofen and zaltoprofen enantiomers were minimal. Taken together, these results suggest that S(+)-zaltoprofen is a potent and active anti-inflammatory component of racemic zaltoprofen, but both S(+)-zaltoprofen and R(-)-zaltoprofen might seem to contribute to the analgesic effect of racemic zaltoprofen.

Widdrol induces apoptosis via activation of AMP-activated protein kinase in colon cancer cells.

Widdrol, a natural sesquiterpene present in Juniperus sp., has been shown to exert anticancer and antifungal effects. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is a potential therapeutic target for human cancers. In this study, we found that AMPK mediates the anticancer effects of widdrol through induction of apoptosis in HT-29 colon cancer cells. We showed that widdrol induced the phosphorylation of AMPK in a dose- and time-dependent manner. The selective AMPK inhibitor compound C abrogated the inhibitory effect of widdrol on HT-29 cell growth. In addition, we demonstrated that widdrol induced apoptosis and this was associated with the activation of caspases, including caspase­3/7 and caspase-9, in HT-29 cells. We also demonstrated that transfection of HT-29 cells with AMPK siRNAs significantly suppressed the widdrol-mediated apoptosis and the activation of caspases. However, cell cycle arrest induced by widdrol was not affected by transfection of HT-29 cells with AMPK siRNAs. Furthermore, widdrol inhibited HT-29 tumor growth in a human tumor xenograft model. Taken together, our results suggest that the anticancer effect of widdrol may be mediated, at least in part, by induction of apoptosis via AMPK activation.

DBM1285 suppresses tumor necrosis factor alpha production by blocking p38 mitogen-activated protein kinase/mitogen-activated protein kinase-activated protein kinase 2 signaling pathway.

Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory cytokine that plays an important role in the development of various inflammatory diseases. TNF-alpha has been considered as a potential therapeutic target for the treatment of chronic inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In this study, we report that cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (DBM1285) is a novel inhibitor of TNF-alpha production. DBM1285 concentration-dependently inhibited lipopolysaccharide (LPS)-induced TNF-alpha secretion in various cells of macrophage/monocyte lineage, including mouse bone marrow macrophages, THP-1 cells, and RAW 264.7 cells. However, LPS-induced mRNA expression of TNF-alpha was not affected by DBM1285 in these cells. Further studies demonstrated that the inhibitory effect of DBM1285 on TNF-alpha production might be mediated by post-transcriptional regulation through the modulation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) signaling pathway. We also confirmed that DBM1285 directly inhibits p38 MAPK enzymatic activity. In vivo administration of DBM1285 inhibited LPS-induced increase in the plasma level of TNF-alpha in mice. Whole-blood in vivo target inhibition assay also revealed that DBM1285 attenuates p38 MAPK activity after oral administration in mice. Moreover, DBM1285 suppressed zymosan-induced inflammation and adjuvant-induced arthritis in murine models. Collectively, these results suggest that DBM1285 inhibits TNF-alpha production, at least in part, by blocking the p38 MAPK/MK2 pathway. Furthermore, in vivo results suggest that DBM1285 might be a possible therapeutic candidate for the treatment of TNF-alpha-related chronic inflammatory diseases.

Glabridin, an isoflavan from licorice root, inhibits inducible nitric-oxide synthase expression and improves survival of mice in experimental model of septic shock.

(R)-4-(3,4-Dihydro-8,8-dimethyl)-2H,8H-benzo[1,2-b:3,4-b']dipyran-3yl)-1,3-benzenediol (glabridin), a flavonoid present in licorice extract, is known to have antimicrobial, anti-inflammatory, and cardiovascular protective activities. In the present study, we report the inhibitory effect of glabridin on nitric oxide (NO) production and inducible nitric oxide (iNOS) gene expression in murine macrophages. Glabridin attenuated lipopolysaccharide (LPS)-induced NO production in isolated mouse peritoneal macrophages and RAW 264.7 cells, a mouse macrophage-like cell line. Moreover, iNOS mRNA expression was also blocked by glabridin treatment in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced nuclear factor (NF)-kappaB/Rel DNA binding activity and NF-kappaB/Rel-dependent reporter gene activity were significantly inhibited by glabridin in RAW 264.7 cells and that this effect was mediated through the inhibition of inhibitory factor-kappaB degradation and p65 nuclear translocation. Moreover, reactive oxygen species generation was also suppressed by glabridin treatment in RAW 264.7 cells. In contrast, the activity of mitogen-activated protein kinases was unaffected by glabridin treatment. In animal model, in vivo administration of glabridin increased the rate of survival of LPS-treated mice and inhibited LPS-induced increase in plasma concentrations of nitrite/nitrate and tumor necrosis factor-alpha. Collectively, these data suggest that glabridin inhibits NO production and iNOS gene expression by blocking NF-kappaB/Rel activation and that this effect was mediated, at least in part, by inhibiting reactive oxygen species generation. Furthermore, in vivo anti-inflammatory effect of glabridin suggests a possible therapeutic application of this agent in inflammatory diseases.