The discovery of sulfonylated dipeptides as potent VLA-4 antagonists.

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

Hierarchical and co-operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli.

BACKGROUND: OmpR is a transcription factor that regulates the expression of the porin genes ompF and ompC in Escherichia coli. The phosphorylation state of OmpR, directed by the osmosensor EnvZ, determines its ability to bind to the upstream regulatory regions of these genes, a total of 14 phospho-OmpR binding sites. While it has been possible to study the stoichiometry and hierarchy of the OmpR-DNA interaction in the upstream regions of ompF and ompC, their disunited location on the bacterial chromosome has made it difficult to compare the individual binding affinities of respective sites. RESULTS: Using 1,10-phenanthroline-Cu+ footprinting on a fused construct containing both the ompF and ompC upstream regulatory sequences, and gel shift experiments on oligomers corresponding to individual sites, we have established a comparative hierarchy for OmpR binding, as F1, C1 > F2, F3 > C2 > C3. In addition, the binding patterns reveal an apparent co-operative relationship between OmpR molecules bound at several upstream motifs. Densitometric analyses of the footprinted regions provide support for these observations. Mutational analysis of this construct reveals that the alteration of a conserved cytidine in the F1 motif (-86) causes a loss of OmpR affinity and disrupts hierarchical OmpR-binding in the entire ompF region. CONCLUSIONS: The present results provide a unique view of the OmpR interaction with the two respective promoters, ompF and ompC, and an insight into the question of how the expression of ompF and ompC are reciprocally regulated by medium osmolarity.

Signal transduction via the histidyl-aspartyl phosphorelay.

The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria. Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator. The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein. Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors. Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator. Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system. While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems. Here, we review the unique and conserved features of this growing family of signal transducers.

Purification and characterization of the periplasmic domain of EnvZ osmosensor in Escherichia coli.

The EnvZ-OmpR histidyl-aspartyl phosphorelay system in E. coli responds to osmolarity by differentially modulating the expression of the major outer membrane porins OmpF and OmpC. To date, the natural ligand that activates EnvZ, a transmembrane histidine kinase, has not been identified and the role of the periplasmic domain of EnvZ is unclear. We now report on the purification and characterization of the periplasmic domain of EnvZ (Lys48-Arg162) which has been expressed as a soluble protein in fusion with the maltose-binding protein. Overexpression of the fusion protein did not compete for a signal that activates EnvZ. By amylose affinity chromatography and affinity blotting, interacting proteins could not be detected. The periplasmic domain was released by factor Xa and purified to homogeneity. From circular dichroism analysis, the periplasmic domain was estimated to consist of 35% alpha-helices and 16% beta-sheets.

Nucleoside-diphosphate kinase-mediated signal transduction via histidyl-aspartyl phosphorelay systems in Escherichia coli.

Nucleoside-diphosphate kinase (NDP kinase), a key enzyme in nucleotide metabolism, is also known to be involved in growth and developmental control and tumor metastasis suppression. Interestingly, we find that coexpression of NDP kinase with Taz1, a Tar/EnvZ chimera, in the absence of its native signal, can activate a porin gene ompC-lacZ expression in Escherichia coli. Further studies show that NDP kinase can act as a protein kinase to phosphorylate histidine protein kinases such as EnvZ and CheA which are members of the His-Asp phosphorelay signal transduction systems in E. coli. Instead of ATP, the exclusive phosphodonor for histidine kinases, GTP can be utilized in vitro in the presence of NDP kinase to phosphorylate EnvZ and CheA, which then transfer the phosphoryl group to OmpR and CheY, the respective response regulators. The direct involvement of GTP for the phosphorylation of EnvZ through NDP kinase was further demonstrated by the use of a mutant EnvZ, which lost ability to be autophosphorylated with ATP. Phospho-OmpR thus formed can bind specifically to an ompF promoter sequence. These results suggest that NDP kinase may play a physiological role in signal transduction.

A synthetic inhibitor of interleukin-1 beta converting enzyme prevents endotoxin-induced interleukin-1 beta production in vitro and in vivo.

A potent, reversible, tetrapeptide inhibitor of interleukin-1 beta converting enzyme (ICE), L-709,049, has been shown to suppress the in vitro production of mature IL-1 beta. We now report that this inhibitor also effectively suppresses the production of mature IL-1 beta in a murine model of endotoxic shock. Intraperitoneal administration of L-709,049 reduced the elevations of IL-1 beta in the plasma and peritoneal fluid of mice treated with LPS in a dose-related manner (ED50 = 2 +/- 0.9 mg/kg). LPS-induced elevations in IL-1 alpha and IL-6 in these mice were unaffected, indicating that the inhibitor specifically affected IL-1 beta production. Immunoblot analysis of plasma and peritoneal fluid indicated that L-709,049 suppressed the formation of mature IL-1 beta production in vivo. When mouse blood was incubated in vitro with LPS, IL-1 beta was released into the plasma. This assay was used to determine ex vivo the activity of an ICE inhibitor in the blood following its administration to mice. Blood obtained 15 minutes after ip administration of 10 mg/kg of L-709,049 to mice produced 80% less IL-1 beta than control blood, and IL-1 beta production returned to control levels in blood obtained 30 minutes after injection of this inhibitor. In addition, the capacity of the blood plasma obtained from these animals to prevent the cleavage of a synthetic substrate by ICE disappeared within 1 h of ip administration of 50 mg/kg of inhibitor.

IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor.

The major form of IL-1 beta-converting enzyme (ICE) identified in THP.1 monocytic cells and human monocytes is the 45-kDa precursor protein (p45), which is found in the cytoplasm. Cytoplasmic extracts of these cells show no pIL-1 beta cleavage activity, indicating that the p45 has no detectable catalytic activity. pIL-1 beta cleavage activity can only be observed after incubation in vitro when p45 breaks down to the active p20 form of the enzyme. LPS stimulation of human monocytes or THP.1 monocytic cells results in no change in the amount of p45 or its activity and no detectable appearance of p20 ICE. Immunoprecipitation of [35S]Met-labeled LPS-stimulated monocyte extracts revealed only p45 with no other co-precipitating protein. The inability to identify active ICE in stimulated monocytic cells was probably a reflection of the very low levels of active ICE present.

Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells.

Interleukin-1 beta-converting enzyme (ICE) was purified from dialyzed cytoplasmic extracts of THP.1 human monocytic cells by a combination of DEAE-5PW and SP-5PW ion exchange and C4 reverse phase high performance liquid chromatography. Sequence information from tryptic and Asp.N peptides on the isolated 20-kDa (p20) and a 10-kDa (p10) proteins enabled the subsequent cloning of ICE (Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774) and localized the active site Cys. Immunoblots with ICE specific antibodies and NH2-terminal sequencing indicated that ICE active column fractions contained in addition to p20 and p10 an alternatively processed form of the p20 protein (p22) containing an extra 16 amino acids NH2-terminal to the p20. Furthermore, immunoblot analysis of the ion exchange column effluent showed that p20 and p22 were found together in three separate fractions distinguished by differences in p10: an intact p10 with complete ICE activity, a COOH-terminally truncated form of p10 with decreased ICE activity, and an absence of p10 with no ICE activity. These results indicate that the p10 protein is essential for ICE activity and that the ICE holoenzyme contains an intact p10 subunit paired with a p20 or p22 catalytic subunit.

Health behaviours in a Canadian community college sample: prevalence of drug use and interrelationships among behaviours.

This study investigated the prevalence of drug use among a Canadian college sample and the covariation of drug taking and other health-related behaviours. A representative sample of students at a community college in Alberta were interviewed using telephone surveys, mail-in questionnaires and face-to-face interviews. Data was collected on drug, alcohol and caffeine use, cigarette smoking, eating habits, sleep habits and exercise. While use of illicit drugs did not appear to be widespread, alcohol appeared to be a primary substance abuse problem for a minority of subjects. Factor analysis indicated that the various health habits did not form one dimension of health-related behaviours. Four separate factors emerged: abusive drinking, eating habits, a drug use factor (caffeine intake, smoking, cannabis and hallucinogen use), and exercise levels. Findings are discussed in terms of their implications for future research, treatment and intervention.