Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System*.

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.

EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures.

EGFR signaling plays important roles in the development of eccrine sweat glands. We previously demonstrate that Matrigel induces eccrine sweat gland cells to reconstruct the three-dimensional (3D) structures of eccrine sweat glands, but the mechanisms are still unknown. In the study, eccrine sweat gland cells were cultured within a 3D Matrigel, and EGFR inhibitor AG1478, or MEK1/2 inhibitor U0126, were added to the medium respectively. The morphology of the 3D-reconstructed eccrine sweat gland-like structures was observed, the localization of phospho-EGFR was detected, and protein levels of EGFR, phospho-EGFR, phospho-JAK, phospho-AKT and phospho-ERK were examined. The results showed that cells treatment with AG1478 from Day 0 of 3D cultures blocked formation of spheroid-like structures. AG1478 administration caused reduced phospho-EGFR, concomitant with downregulation of phospho-ERK1/2, but not phospho-JAK or phospho-AKT. Phospho-EGFR and phospho-ERK were reduced, and only a small number of 3D-structures were formed following treatment with U0126. We conclude that EGFR plays important roles in Matrigel-induced 3D structures of eccrine sweat gland-like structures, and ERK1/2 signaling is responsible, at least in part, for the effect of EGFR.

Ultrasensitive amplification-free detection of protein kinase based on catalyzed assembly and enumeration of gold nanoparticles.

A single-particle enumeration method based on phosphorylation-directed in situ assembly of gold nanoparticles is developed for the ultrasensitive sensing of cellular protein kinase A activity. In comparison to existing strategies, the proposed new method demonstrates five orders of linear range and improves the detection limit up to 10-to-1000 fold without the involvement of target amplification.

Synthesis and Evaluation of the Anticancer and Trypanocidal Activities of Boronic Tyrphostins.

Molecules containing an (cyanovinyl)arene moiety are known as tyrphostins because of their ability to inhibit proteins from the tyrosine kinase family, an interesting target for the development of anticancer and trypanocidal drugs. In the present work, (E)-(cyanovinyl)benzeneboronic acids were synthesized by Knoevenagel condensations without the use of any catalysts in water through a simple protocol that completely avoided the use of organic solvents in the synthesis and workup process. The in vitro anticancer and trypanocidal activities of the synthesized boronic acids were also evaluated, and it was discovered that the introduction of the boronic acid functionality improved the activity of the boronic tyrphostins. In silico target fishing with the use of a chemogenomic approach suggested that tyrosine-phosphorylation-regulated kinase 1a (DYRK1A) was a potential target for some of the designed compounds.

WP1066, a small molecule inhibitor of the JAK/STAT3 pathway, inhibits ceramide glucosyltransferase activity.

WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a ceramide glucosyltransferase inhibitor. Ceramide glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of ceramide glucosyltransferase with an IC50 of 7.2 µM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of ceramide glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.

Screening of the 'Stasis Box' identifies two kinase inhibitors under pharmaceutical development with activity against Haemonchus contortus.

BACKGROUND: In partnership with the Medicines for Malaria Venture (MMV), we screened a collection ('Stasis Box') of 400 compounds (which have been in clinical development but have not been approved for illnesses other than neglected infectious diseases) for inhibitory activity against Haemonchus contortus, in order to attempt to repurpose some of the compounds to parasitic nematodes. METHODS: We assessed the inhibition of compounds on the motility and/or development of exsheathed third-stage (xL3s) and fourth-stage (L4) larvae of H. contortus using a whole-organism screening assay. RESULTS: In the primary screen, we identified compound MMV690767 (also known as SNS-032) that inhibited xL3 motility by ~70% at a concentration of 20 µM after 72 h as well as compound MMV079840 (also known as AG-1295), which induced a coiled xL3 phenotype, with ~50% inhibition on xL3 motility. Subsequently, we showed that SNS-032 (IC50 = 12.4 µM) and AG-1295 (IC50 = 9.92 ± 1.86 µM) had a similar potency to inhibit xL3 motility. Although neither SNS-032 nor AG-1295 had a detectable inhibitory activity on L4 motility, both compounds inhibited L4 development (IC50 values = 41.24 µM and 7.75 ± 0.94 µM for SNS-032 and AG-1295, respectively). The assessment of the two compounds for toxic effects on normal human breast epithelial (MCF10A) cells revealed that AG-1295 had limited cytotoxicity (IC50 > 100 µM), whereas SNS-032 was quite toxic to the epithelial cells (IC50 = 1.27 µM). CONCLUSIONS: Although the two kinase inhibitors, SNS-032 and AG-1295, had moderate inhibitory activity on the motility or development of xL3s or L4s of H. contortus in vitro, further work needs to be undertaken to chemically alter these entities to achieve the potency and selectivity required for them to become nematocidal or nematostatic candidates.

Tyrphostin AG-related compounds attenuate H2O2-induced TRPM2-dependent and -independent cellular responses.

PURPOSE: TRPM2 is a Ca2+-permeable channel that is activated by H2O2. TRPM2-mediated Ca2+ signaling has been implicated in the aggravation of inflammatory diseases. Therefore, the development of TRPM2 inhibitors to prevent the aggravation of these diseases is expected. We recently reported that some Tyrphostin AG-related compounds inhibited the H2O2-induced activation of TRPM2 by scavenging the intracellular hydroxyl radical. In the present study, we examined the effects of AG-related compounds on H2O2-induced cellular responses in human monocytic U937 cells, which functionally express TRPM2. METHODS: The effects of AG-related compounds on H2O2-induced changes in intracellular Ca2+ concentrations, extracellular signal-regulated kinase (ERK) activation, and CXCL8 secretion were assessed using U937 cells. RESULTS: Ca2+ influxes via TRPM2 in response to H2O2 were blocked by AG-related compounds. AG-related compounds also inhibited the H2O2-induced activation of ERK, and subsequent secretion of CXCL8 mediated by TRPM2-dependent and -independent mechanisms. CONCLUSION: Our results show that AG-related compounds inhibit H2O2-induced CXCL8 secretion following ERK activation, which is mediated by TRPM2-dependent and -independent mechanisms in U937 cells. We previously reported that AG-related compounds blocked H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. The inhibitory effects of AG-related compounds on TRPM2-independent responses may be due to scavenging of the hydroxyl radical.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

BACKGROUND AND PURPOSE: The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACH: Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTS: Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30 µM genistein or 10 µM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 µM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONS: The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .

[Lipid derivative of benzylidene malononitrile AG490 attenuates airway inflammation of mice with neutrophilic asthma].

Objective To observe the effect of lipid derivative of benzylidene malononitrile AG490 on the airway inflammation in a mouse model of neutrophilic asthma (NA). Methods Fifty-four specific pathogen-free (SPF) female C57BL/6 mice were randomly divided into 3 groups: NA group, AG490-treated NA (NAAG) group, and normal control (NC) group, 18 mice in each group. The NA group and the NAAG group were sensitized by airway instillation of ovalbumin (OVA) and lipopolysaccharide (LPS) on day 0, 6 and 13. The NAAG group was injected with AG490 (500 µg/mouse, i.p.) three times a week, from day 0 after the first sensitization, for 3 weeks. Mice were challenged on day 21, 22 for 1 hour/time with an aerosol of 10 g/L OVA. At 24 hours after the final challenge, bronchoalveolar lavage fluid (BALF) was collected. The total number and differential counts of nucleated cells and the percentage of each type were determined. HE staining and PAS staining was employed for observing the lung pathological changes. The percentages of Th17 cells and regulatory T cells (Treg) in the lung issue were determined by flow cytometry. The level of interleukin-17 (IL-17) in BALF was measured using ELISA. Results Compared with the NA group, the total number of nucleated cells, the percentage of neutrophils and the percentage of eosinophils in BALF in the NAAG group were obviously reduced; lung tissue pathologic changes were improved in the NAAG group; goblet cell hyperplasia and the level of IL-17 in BALF in the NAAG group were significantly down-regulated; the proportion of Treg in the lung increased and the proportion of Th17 cells in the lung decreased in the NAAG group. Conclusion After NA mice are treated with AG490 during the sensitization phase, the proportion of Treg in the lung would increase and the proportion of Th17 cells in the lung would decrease. AG490 could attenuate the airway inflammation in the mouse model of NA.

Inhibitory effects of Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential channel activation.

Some transient receptor potential (TRP) proteins including TRPA1, TPRM2 and TRPV1 are oxidative stress-sensitive Ca(2+)-permeable channels. Ca(2+) signaling via these TRP channels activated by oxidative stress has been implicated in the aggravation of various inflammatory diseases and pain sensation. We recently reported that Tyrphostin AG490 exerted inhibitory effects on H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. In order to identify stronger inhibitors of oxidative stress-sensitive TRP channels than AG490, we examined the inhibitory effects of Tyrphostin AG-related compounds on H2O2-induced TRP channel activation in human embryonic kidney 293 cells expressing TRP channels. AG555 and AG556 blocked the activation of TRPM2 by H2O2 more strongly than AG490. Regarding TRPV1 and TRPA1, none of the three compounds tested affected H2O2-induced TRPV1 activation; however, AG555 and AG556 reduced H2O2-induced TRPA1 activation more than AG490. Thus, we herein identified AG555 and AG556 as new compounds that exert stronger inhibitory effects on H2O2-induced TRPM2 and TRPA1 activation than AG490. Edaravone, a hydroxyl radical scavenger used in the treatment of cerebral hemorrhage and cerebral infarction, did not affect H2O2-induced TRPM2 or TRPA1 activation. AG555 and AG556 may be useful seed compounds as therapeutic agents for several TRP-related diseases associated with oxidative stress.

UV-Vis spectroscopy and solvatochromism of the tyrosine kinase inhibitor AG-1478.

The effect of twenty-one solvents on the UV-Vis spectrum of the tyrosine kinase inhibitor AG-1478 was investigated. The absorption spectrum in the range 300-360nm consisted of two partially overlapping bands at approximately 340nm and 330nm. The higher energy absorption band was more sensitive to solvent and exhibited a peak position that varied from 327nm to 336nm, while the lower energy absorption band demonstrated a change in peak position from 340nm to 346nm in non-chlorinated solvents. The fluorescence spectrum of AG-1478 was particularly sensitive to solvent. The wavelength of peak intensity varied from 409nm to 495nm with the corresponding Stokes shift in the range of 64nm to 155nm (4536cm(-1) to 9210cm(-1)). We used a number of methods to assess the relationship between spectroscopic properties and solvent properties. The detailed analysis revealed that for aprotic solvents, the peak position of the emission spectrum in wavenumber scale correlated with the polarity (dielectric constant or ET(30)) of the solvent. In protic solvents, a better correlation was observed between the hydrogen bonding power of the solvent and the position of the emission spectrum. Moreover, the fluorescence quantum yields were larger in aprotic solvents as compared to protic solvents. This analysis underscores the importance of polarity and hydrogen-bonding environment on the spectroscopic properties of AG-1478. These studies will assume relevance in understanding the interaction of AG-1478 in vitro and in vivo.

Inhibition of Janus kinases by tyrosine phosphorylation inhibitor, Tyrphostin AG-490.

Janus kinases (JAKs) belong to a crucial family of tyrosine kinases, implicated in the patho-physiology of multiple cancer types, and serve as striking therapeutic targets. To date, many potent, either ATP-competitive (PTK domain) or non-ATP-competitive JAK inhibitors have been identified. Among them, Tyrphostin AG-490 (2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide) is a well-known ATP-competitive inhibitor. However, its mode of action, details of interacting residues, and induced conformational changes in JAK-specific binding sites remain elusive. Here, through comparative structure analysis, molecular docking, and molecular dynamics simulation assays, we explored comparative binding patterns of AG-490 against JAK1, JAK2, and JAK3. Our results entail noteworthy observations about the binding affinity of AG-490 by illustrating distinctive amino acid residues lying at the conserved ATP-binding domains of JAK family members. By subsequent assessment of their structural homology and conserved structural folds, we highlight intriguing prospects to design more specific and potent inhibitors for selective targeting of JAK family members. Our comparative study provides a platform for the rational design of precise and potent inhibitor for selective targeting of JAK family members.

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.

Fluorescence-based screening assays for the NAD⁺-dependent histone deacetylase smSirt2 from Schistosoma mansoni.

Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.

Therapeutic targets in subependymoma.

Subependymomas are usually treated with surgical resection; however, no standard, defined alternative medical therapy is recommended for patients who are not surgical candidates, owing to a paucity of molecular, immunological, and genetic characterization. To address this, an ex vivo functional analysis of the immune microenvironment in subependymoma was conducted, a subependymoma cytokine/chemokine microarray was constructed for the evaluation of operational immune and molecular pathways, and a subependymoma cell line was derived and used to test a variety of cytotoxic agents that target operational pathways identified in subependymoma. We found that immune effectors are detectable within the microenvironment of subependymoma; however, marked immune suppression is not observed. The subependymoma tissue microarrays demonstrated tumor expression of p53, MDM2, HIF-1α, topoisomerase II-ß, p-STAT3, and nucleolin, but not EGFRvIII, EphA2, IL-13RA2, CMV, CTLA-4, FoxP3, PD-1, PD-L1, EGFR, PDGF-α, PDGF-ß, PDGFR-α, PDGFR-ß, PTEN, IGFBP2, PI3K, MDM4, IDH1, mTOR, or Jak2. A topoisomerase inhibitor (WP744, IC50=0.83 µM) and a p-STAT3/HIF-1α inhibitor (WP1066, IC50=3.15 µM) demonstrated a growth inhibition of the subependymoma cell proliferation. Cumulatively, these data suggest that those agents that interfere with oncogenes operational in subependymoma may have clinical impact.

Endothelin-1 stimulates resistin gene expression.

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Intriguing roles of reactive intermediates in dissociation chemistry of N-phenylcinnamides.

In mass spectrometry of protonated N-phenylcinnamides, the carbonyl oxygen is the thermodynamically most favorable protonation site and the added proton is initially localized on it. Upon collisional activation, the proton transfers from the carbonyl oxygen to the dissociative protonation site at the amide nitrogen atom or the α-carbon atom, leading to the formation of important reactive intermediates. When the amide nitrogen atom is protonated, the amide bond is facile to rupture to form ion/neutral complex 1, [RC(6)H(4)CH=CHCO(+)/aniline]. Besides the dissociation of the complex, proton transfer reaction from the α-carbon atom to the nitrogen atom within the complex takes place, leading to the formation of protonated aniline. The presence of electron-withdrawing groups favored the proton transfer reaction, whereas electron-donating groups strongly favored the dissociation (aniline loss). When the proton transfers from the carbonyl oxygen to the α-carbon atom, the cleavage of the C(α)-CONHPh bond results in another ion/neutral complex 2, [PhNHCO(+)/RC(6)H(4)CH=CH(2)]. However, in this case, electron-donating groups expedited the proton transfer reaction from the charged to the neutral partner to eliminate phenyl isocyanate. Besides the cleavage of the C(α)-CONHPh bond, intramolecular nucleophilic substitution (a nucleophilic attack of the nitrogen atom at the ß-carbon) and stepwise proton transfer reactions (two 1,2-H shifts) also take place when the α-carbon atom is protonated, resulting in the loss of ketene and RC(6)H(5), respectively. In addition, the H/D exchanges between the external deuterium and the amide hydrogen, vinyl hydrogens and the hydrogens of the phenyl rings were discovered by D-labeling experiments. Density functional theory-based (DFT) calculations were performed to shed light on the mechanisms for these reactions.

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells.

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 µM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

Quinazoline-tyrphostin as a new class of antitumor agents, molecular properties prediction, synthesis and biological testing.

A new series of substituted quinazolin-4-(3H)-one-tyrphostin derivatives was prepared and screened for their cytotoxic activity against three tumor cell lines, namely human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human hepatocellular liver carcinoma cell line (HepG2) using the colorimetric MTT assay. Among the current series, 10 compounds exhibited remarkable in vitro antiproliferative activity against the three tested cell lines with the IC(50) values ranging from 0.009 to 0.015 mM. All the compounds showed suitable drug like characteristics according to Lipinski's rule.