C - Chemistry – Metallurgy – 07 – K
Patent
C - Chemistry, Metallurgy
07
K
C07K 14/705 (2006.01) C07K 5/00 (2006.01) C07K 5/06 (2006.01) C07K 5/072 (2006.01) G01N 33/534 (2006.01) G01N 33/567 (2006.01)
Patent
CA 2092733
The leukotriene (LT)D4 receptor has been defined by radioligand binding studies as a member of the family of G-protein coupled receptors. In order to characterize this receptor a radioiodinated, photoactivable azido derivative of LTD4 ([125l]Azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of [125l]Azido-LTD4 specific binding to guinea-pig lung membranes were directly comparable with those of [s H]LTD4 specific binding to this tissue, in agreement with [125l]Azido-LTD4 binding to a G-protein coupled LTD4 receptor. [125l]Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. [125l]Azido-LTD4 specific binding was also strongly inhibited by the non-hydrolysable GTP analogue GTP.gamma.S with ATP.gamma.S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G- protein. The peptidyl leukotrienes competed for [125l]Azido-LTD4 specific binding with the following rank order of potency: LTD4 > LTE4 >> LTC4, while the non-peptidyl LTB4 was virtually inactive. Two structurally different LTD4-receptor antagonists, MK- 571 and ICI 204,219, also competed for [1251]Azido-LTD4 specific binding to guinea-pig lung membranes, with nanomolar potency, whereas the leukotriene biosynthesis inhibitor MK-886 was 10000- fold less active. Photoactivation of [125l]Azido-LTD4 under equilibrium binding conditions revealed the selective radiolabeling of a 45 kDa protein in guinea-pig lung membranes, as visualized by SDS-PAGE and autoradiography. The photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the [125l]Azido-LTD4 binding assays. In addition, the radiolabeling of this 45 kDa protein was saturable, modulated by cations, and inhibited by nucleotide analogues in an analogous way to [125l]Azido-LTD4 specific binding to guinea-pig lung membranes. It is therefore proposed that this novel 45 kDa protein represents the LTD4 receptor.
Metters Kathleen M.
Zamboni Robert
Goudreau Gage Dubuc
Merck Frosst Canada Inc.
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