ABSTRACT
Edelfosine is an inhibitor of SK3 channel mediated cell migration. However, this compound bears adverse in vivo side effects. Using cell SK3 dependent cell-migration assay, patch-clamp, (125)I-apamin binding, and in vivo experiments we tested the ability of 15 lipid derivatives with chemical structures inspired from edelfosine to inhibit SK3 channels. Using a structure-activity relationship approach we identified an edelfosine analog named Ohmline (1-O-hexadecyl- 2-O-methyl-sn-glycero-3-lactose) with potent inhibitory effects on the SK3 channel. Its potency was greater for SK3 channels than for SK1 channels; it did not affect IKCa channels and only slightly but not significantly affected SK2 channels. This is the first SKCa channel blocker that can be used to discriminate between SK2 and SK1/SK3 channels and represents a useful tool to investigate the functional role of SK3 channels in peripheral tissues (that do not express SK1 channels). This compound, which acts with an IC(50) of 300 nM, did not displace apamin from SKCa channels and had no effect on non-specific edelfosine targets such as protein kinase C (PKC), receptors for platelet activating factor (PAF) and lysophosphatidic acid (LPA), as well as non-cancerous cells. This is promising because the pitfalls associated with the use of edelfosine-like compounds have been that their effective and high concentrations are often cytotoxic due to their detergent-like character causing normal cell lysis. Finally, Ohmline reduced metastasis development in a mice model of tumor indicating that this compound could become a lead compound for the first class of lipid-antimetastatic agent.
Subject(s)
Cell Movement/drug effects , Glycolipids/pharmacology , Potassium Channel Blockers/pharmacology , Small-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Migration Assays , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/pathology , Female , Glycolipids/chemistry , Humans , Mice , Mice, Nude , Molecular Structure , Phospholipid Ethers/chemistry , Phospholipid Ethers/pharmacology , Platelet Membrane Glycoproteins/agonists , Protein Kinase C/antagonists & inhibitors , Receptors, G-Protein-Coupled/agonists , Receptors, Lysophosphatidic Acid/agonists , Structure-Activity RelationshipABSTRACT
Two competitive immunoassays, a laboratory assay based on microwell plates and a field test based on the use of polystyrene tubes, have been developed for the quantification of thiram in lettuces. Concerning the laboratory assay, the calibration curve for thiram had a linear range of 11 to 90 ng/mL and a detection limit of 5 ng/mL. Precision of the assay presented coefficient of variation values <9% and the recovery of thiram from lettuce averaged 89% across the range of the immunoassay method using 30 min extraction with water/acetone (50:50, v/v). The tube-based method was developed in order that an extract of lettuce, containing thiram at the MRL (8 ppm), would be found on the linear part of the standard curve. The calibration curve for thiram has a linear range of 100 to 800 ng/mL (1.39 to 11.1 ppm in lettuce) and a detection limit of 40 ng/mL.
Subject(s)
Fungicides, Industrial/analysis , Lactuca/chemistry , Pesticide Residues/analysis , Thiram/analysis , Enzyme-Linked Immunosorbent Assay/methods , Reproducibility of Results , Sensitivity and Specificity , Thiram/immunology , Time FactorsABSTRACT
An enzyme-linked immunosorbent assay (ELISA) was developed for the fungicide thiram. Two types of haptens were synthesized. The first type exhibits the two symmetrical N-alkyl dithiocarbamate patterns of thiram with a spacer arm linked to one of the N-methyl terminal group. The second type exhibits one of the two symmetrical N-alkyl dithiocarbamate patterns of thiram with a variable-length spacer arm linked to one sulfur atom. Polyclonal antibodies suitable for thiram detection were obtained from immunization with an hapten of the first type, while haptens of the second type were used as coating antigens to develop a competitive ELISA against thiram. The IC(50) value for thiram was estimated to be 0.24 microg/mL, with a detection limit of 0.03 microg/mL. The assay seems to be thiram-specific since no or little cross-reaction with other dithiocarbamates were observed.
Subject(s)
Fungicides, Industrial/analysis , Haptens/chemistry , Thiram/analysis , Binding, Competitive , Enzyme-Linked Immunosorbent AssayABSTRACT
A number of N-4 (4-dialkylamino-2-butynyl) phosphoramide derivatives related to the oxotremorine structure were prepared and evaluated for their muscarinic activity on isolated perfused rat hearts. A structure-activity relationship study revealed that among other factors, muscarinic activity is directly related to the environment of the phosphorus atom.