Characterization of a benzyl-phenoxy-ethanamine binding protein in Trypanosoma equiperdum and the possible relation between binding affinity and trypanocidal activity.

A new family of benzyl-phenoxy-ethanamine derivatives has been assayed for trypanocidal activity. Using tritiated morpholino-benzyl-phenoxy-ethanamine as a probe, it is shown that this ligand is able to bind specifically to a protein contained in extracts of Trypanosoma equiperdum. The binding is saturable and of high affinity (KD = 4 nM: Bmax = 200 fmol (mg protein)-1). The in vitro activities of the investigated compounds against this parasite correlate with their affinities to the putative binding site. Moreover, using an azido functionalized morpholino-benzyl-phenoxyethanamine as photoprobe a major M(r) = 40,000 protein was specifically revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This molecular weight corresponds with the previously observed value determined for the antioestrogen binding site protein of rat liver which has been shown to specifically bind antioestrogens of the triphenylethylene family and phenoxyethanamine derivatives

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS).

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds