Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.     

Rapid and reliable high-performance liquid chromatographic method for analysing human plasma serotonin, 5-hydroxyindoleacetic acid, homovanillic acid and 3,4-dihydroxyphenylacetic acid

The simultaneous measurement of homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in human plasma by an ultrafiltration and microbore high-performance liquid chromatography—electrochemical detection technique is established. Conventional preparation of blood is very tedious and time-consuming, but isocratic separation of the analytes in plasma ultrafiltrates using a microbore column could be achieved within 10 min. Hence, theoretically, over 140 analyses can be performed in a working day. The detection limit (signal-to-noise ratio = 3) of this method is about 0.1–0.5 pg per injection for all analytes. The required volume of plasma samples can be less than 100 μl. Hence, blood loss is minimal, especially in repeated blood sampling. This rapid, simple and sensitive method can, therefore, be used as a routine clinical tool in the simultaneous measurement of plasma homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid.     

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response

While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Structure and function of human hemoglobin covalently labeled with periodate-oxidized adenosine triphosphate

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-A resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each beta chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P50 = 20.8 mm Hg versus 5.8 mm Hg control; 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol, pH 7.4, 0.1 M C, 20 degrees C). The subunit cooper-activity of di-ATP Hb is also reduced (nmax = 1.9 versus 2.7 control).     

Synthesis of the spin-labeled derivative of an ether-linked phospholipid possessing high antineoplastic activity.

We report here the complete synthesis of the spin-labeled derivative of an antitumor ether phospholipid, 1-O-octadecyl-2-O-(4'-doxylpentyl)-rac-glycerol-3-phosphocholine. This also represents the first time that the synthesis of a nitroxide spin-labeled diether phospholipid is described. In vitro experiments showed that at micromolar concentrations, this new analog is readily incorporated into the plasma membranes of human HL60 and mouse E8/AK.D1 leukemic cells, and subsequently kills the cells. The availability of this new probe should permit the electron spin resonance spectroscopic approach to investigate ways by which anti-tumor ether phospholipids selectively destroy the tumor cells.     

Multiple Factors from Bradykinin-Challenged Astrocytes Contribute to the Neuronal Apoptosis: Involvement of Astroglial ROS, MMP-9, and HO-1/CO System

Bradykinin (BK) has been shown to induce the expression of several inflammatory mediators, including reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), in brain astrocytes. These mediators may contribute to neuronal dysfunction and death in various neurological disorders. However, the effects of multiple inflammatory mediators released from BK-challenged astrocytes on neuronal cells remain unclear. Here, we found that multiple factors were released from brain astrocytes (RBA-1) exposed to BK in the conditioned culture media (BK-CM), including ROS, MMP-9, and heme oxygenase-1 (HO-1)/carbon monoxide (CO), leading to neuronal cell (SK-N-SH) death. Exposure of SK-N-SH cells to BK-CM or H₂O₂ reduced cell viability and induced cell apoptosis which were attenuated by N-acetyl cysteine, indicating a role of ROS in these responses. The effect of BK-CM on cell viability and cell apoptosis was also reversed by immunoprecipitation of BK-CM with anti-MMP-9 antibody (MMP-9-IP-CM) or MMP2/9 inhibitor, suggesting the involvement of MMP-9 in BK-CM-mediated responses. Astroglial HO-1/CO in BK-CM induced cell apoptosis and reduced cell viability which was reversed by hemoglobin. Consistently, the involvement of CO in these cellular responses was revealed by incubation with a CO donor CO-RM2 which was reversed by hemoglobin. The role of HO-1 in BK-CM-induced responses was confirmed by overexpression of HO-1 in SK-N-SH infected with Adv-HO-1. BK-CM-induced cell apoptosis was due to the activation of caspase-3 and cleavage of PARP. Together, we demonstrate that BK-induced several neurotoxic factors, including ROS, MMP-9, and CO released from astrocytes, may induce neuronal death through a caspase-3-dependent apoptotic pathway.     

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole‐exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer–testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell‐based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.