Showing papers by "Dennis C. Dean published in 2003"

A Small Molecule α4β1/α4β7 Antagonist Differentiates between the Low-Affinity States of α4β1 and α4β7: Characterization of Divalent Cation Dependence

Abstract: An α4β1/α4β7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of α4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both α4β1 and α4β7, but 2 S -[(1-benzenesulfonyl-pyrrolidine-2 S -carbonyl)-amino]-4-[4-methyl-2 S -(methyl-{2-[4-(3- o -tolyl-ureido)-phenyl]-acetyl}-amino) pentanoylamino]-butyric acid (BIO7662), a specific α4β1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that α4β1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly α4β7 with a K D of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated α4β7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily α4β1 with a K D of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four α4β1/α4β7 antagonists to block binding of activated α4β1 or α4β7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to α4β1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that α4β1 and α4β7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.

Synthesis of high specific activity 35S‐labelled N‐methanesulfonyl farnesylcysteine and a photoactive analog

Abstract: Prenylated cysteine analogs, which mimic the prenylated cysteine residue of prenylated GTP-binding proteins (G-proteins), have been used in a variety of contexts for the study of prenylated G-protein behavior. In earlier work in this area, we prepared the photoactive analog [35S]4 and showed that it labelled RhoGDI upon photolysis; those results were consistent with the idea that GDI contains an isoprenoid binding site. Here, we describe the preparation of [35S]N-methanesulfonyl labelled analogs (1a and 2a) of N-acetyl farnesylcysteine and its methyl ester together with an improved synthetic procedure for photoactive analogs 3 and 4; specific activities of ∼1100 Ci/mmol were achieved. Compounds 1a and 2a in unlabelled form were used as competitors in photolysis reactions to show that the methanesulfonamido group is a reasonable acetamide substitution. Additional experiments show that the photoactive ester [35S]3 can cross-link GDI in both purified form and crude bacterial extract. However, the extent of cross-linking obtained with the ester ([35S]3) is significantly less than that observed with the free acid ([35S]4) despite the fact that the esterified form probably more closely reflects the structure of the C-terminus of a prenylated protein; using the GDI·Cdc42 co-crystal structure, the structural basis for these results is discussed. Copyright © 2002 John Wiley & Sons, Ltd.

Synthesis of two non-peptidyl GnRH receptor antagonists via [14C]carbonylation

Abstract: In support of a program to develop a new gonadotropin releasing hormone (GnRH) receptor antagonist, two 14C labelled candidate tracers, 14C-1 and 14C-2, were synthesized for utilization in metabolism studies. A slight modification of the Medicinal Chemistry route for the synthesis of the antagonists provided iodide 4. Palladium (0) catalyzed [14C]carbonylation of 4 proceeded in good chemical yield to afford acid 14C-3 which served as a common precursor to 14C-1 and 14C-2. Copyright © 2003 John Wiley & Sons, Ltd.

The changing environment of graduate and postdoctoral training in drug metabolism: viewpoints from academia, industry, and government.

Abstract: This article is an invited report of a symposium sponsored by the Drug Metabolism Division of the American Society for Pharmacology and Experimental Therapeutics held at Experimental Biology 2002 in New Orleans. The impetus for the symposium was a perceived shortage in the supply of graduate students qualified for drug metabolism research positions in industry, academia, and government. For industry, recent hiring stems largely from the expansion of drug metabolism departments in an effort to keep pace with the demands of drug discovery and new technologies. In turn, regulatory scientists are needed to review and verify the results of the increased number and volume of studies required for drug development and approval. Thus the initial source of training, academia, has been forced to recognize these external hiring pressures while trying to attract and retain the faculty, postdoctoral scientists, and students necessary for active teaching and research programs. The trend of the expansion of the interdisciplinary nature of traditional drug metabolism to include emerging technologies such as pharmacogenetics, transporters, and proteomics and the implications for future needs in training and funding were acknowledged. There was also consensus on the value of partnerships between academia and industry for increasing student interest and providing training in disciplines directly applicable to industrial drug metabolism research. Factors affecting the sources of these trainees, such as federal funding, the number of trainees per institution, and recent issues with immigration restrictions that have limited the flow of scientists were also discussed.

Derives d'alcyne utilises comme marqueurs pour la liaison au recepteur du glutamate metabotropique

Abstract: La presente invention concerne des composes derivees d'alcyne marques isotopiquement, en particulier des composes marques au 11C, 13C, 14C, 18F, 15O, 13N, 35S, 2H, et au 3H. La presente invention concerne plus particulierement des alcynes heterocycliques marques au 11C, 13C, 14C, 18F, 15O, 13N, 35S, 2H, et au 3H et leurs procedes de preparation. La presente invention concerne egalement un procede d'utilisation des composes d'alcyne heterocycliques marques au 11C, 18F, 15O, ou au 13N comme marqueurs en imagerie tomographique par emission de positions (TEP), en particulier dans l'etude de troubles metaboliques chez les mammiferes, et plus precisement de troubles modules par les recepteurs du glutamate metabotropique de type 5 (mGluR5).