A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP relative to GMP, FdUMP, and dUMP was determined in the presence and absence of U89 by ultrafiltration analysis, which revealed that although the binding of GMP and dGMP could not be detected in the absence of U89 both were bound in its presence. The Kd for dGMP was about the same as that for dUMP and FdUMP, with binding of the latter two nucleotides being increased by two orders of magnitude by U89. An explanation for the binding of dGMP was provided by x-ray diffraction studies that revealed an extensive stacking interaction between the guanine of dGMP and the benzoquinazoline ring of U89 and hydrogen bonds similar to those involved in dUMP binding. In addition, binding energy was provided through a water molecule that formed hydrogen bonds to both N7 of dGMP and the hydroxyl of Tyr-94. Accommodation of the larger dGMP molecule was accomplished through a distortion of the active site and a shift of the deoxyribose moiety to a new position. These rearrangements also enabled the binding of GMP to occur by creating a pocket for the ribose 2' hydroxyl group, overcoming the normal TS discrimination against nucleotides containing the 2' hydroxyl.

Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. We evaluated rolipram, a type IV phosphodiesterase inhibitor, for its efficacy in preventing EAE in the common marmoset Callithrix jacchus. In a blinded experimental design, clinical signs of EAE developed within 17 days of immunization with human white matter in two placebo-treated animals but in none of three monkeys that received rolipram (10 mg/kg s.c. every other day) beginning 1 week after immunization. In controls, signs of EAE were associated with development of cerebrospinal fluid pleocytosis and cerebral MRI abnormalities. In the treatment group, there was sustained protection from clinical EAE, transient cerebrospinal fluid pleocytosis in only one of three animals, no MRI abnormality, and marked reduction in histopathologic findings. Rolipram-treated and control animals equally developed circulating antibodies to myelin basic protein. Thus, inhibition of type IV phosphodiesterase, initiated after sensitization to central nervous system antigens, protected against autoimmune demyelinating disease.

ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans.

Examination of the structural basis for antiviral activity, oral pharmacokinetics, and hepatic metabolism among a series of symmetry-based inhibitors of the human immunodeficiency virus (HIV) protease led to the discovery of ABT-538, a promising experimental drug for the therapeutic intervention in acquired immunodeficiency syndrome (AIDS). ABT-538 exhibited potent in vitro activity against laboratory and clinical strains of HIV-1 [50% effective concentration (EC50) = 0.022-0.13 microM] and HIV-2 (EC50 = 0.16 microM). Following a single 10-mg/kg oral dose, plasma concentrations in rat, dog, and monkey exceeded the in vitro antiviral EC50 for > 12 h. In human trials, a single 400-mg dose of ABT-538 displayed a prolonged absorption profile and achieved a peak plasma concentration in excess of 5 micrograms/ml. These findings demonstrate that high oral bioavailability can be achieved in humans with peptidomimetic inhibitors of HIV protease.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2nd generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

The Structure of theCyprinid herpesvirus 3ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

A simple sustained release device for the ethylene binding inhibitor 1-methylcyclopropene

The efficacy of 1-methylcyclopropene (1-MCP) gas to prevent the adverse effects of ethylene is limited by its short-term residual activity in some plants. Development of a simple 1-MCP sustained release device that prolongs 1-MCP exposure is reported herein. Sustained release devices comprised of polyvinylchloride tubes containing 0.1 g SmartFresh(TM) powder (a.i. 3.3% 1-MCP) and 1.25 ml deionised water were used to release 1-MCP into fibreboard cartons containing cut Geraldton waxflower (Chamelaucium uncinatum Schauer) cv. CWA Pink bunches during export shipment by air (107 h) from Australia to the UK. The devices protected flowers against abscission induced by subsequent test exposures to ethylene (1011,mul l(-1), 12 h, 20 degreesC) for 3-5 days after arrival. In contrast, pre-shipment treatments with either a single application of 790 nl l(-1) 1-MCP for 14 h at 2 degreesC or a 0.2 mM Ag+ (as silver thiosulphate; STS) pulse for 14 h at 2 degreesC protected flowers against exogenous ethylene for only 1-2 days of post-export life. However, pre-shipment 1-MCP fumigation was up to about three-fold more effective than either sustained 1-MCP release or pre-shipment STS treatments in reducing floral organ and leaf abscission from bunches during export. Thus, it is suggested that a combination of pre-shipment 1-MCP fumigation before export with sustained 1-MCP release during shipment should maximise efficacy against ethylene-induced waxflower flower abscission. (C) 2004 Elsevier B. V. All rights reserved.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.