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CELLULAR AND MOLECULAR

Desensitization of the Human Motilin Receptor by Motilides

Gut Hormone Lab, Center for Gastroenterological Research, Department of Pathophysiology, Katholieke Universiteit Leuven, Leuven, Belgium (L.T., I.D., T.T., T.L.P.); Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium (J.P., P.R.); Euroscreen, Brussels, Belgium (E.B.); and Kosan Biosciences, Hayward, California (Y.L., C.C.)

Tachyphylaxis may have contributed to the failure of the motilide ABT-229 [N-ethyl, N-methyl 4'' deoxy erythromycin (EM)-B enolether] in clinical trials. We compared the desensitizing potency of structurally related motilides [EM-A, EM-A enolether (ME4), N-ethyl, N-methyl EM-A (ME36), EM-B enolether (ME67), N-ethyl, N-methyl EM-A enolether (EM523), ABT-229 and 4'' deoxy EM-A enolether (KOS1326)] in a Chinese hamster ovary (CHO)-K1 cell line expressing the human motilin receptor (MTLR) and in rabbit duodenal segments. CHO-MTLR cells were preincubated with motilides prior to stimulation with motilin. The negative logarithm of the preincubation concentration reducing the maximal motilin-induced Ca²⁺ flux to 50% was calculated (pDC₅₀). Internalization was visualized in CHO-K1 cells containing an enhanced green fluorescent protein (EGFP)-tagged MTLR and quantified in binding experiments. The contractile response of repeated stimulations was measured in duodenal segments. In CHO-MTLR cells, the pDC₅₀ was ABT-229 (8.78) > motilin (7.77) > EM-A (4.78), different from their order of potency to induce Ca²⁺ release (pEC₅₀): motilin (9.39) > ABT-229 (8.46) > EM-A (7.11). In cells with the EGFP-tagged MTLR, ABT-229 decreased membrane fluorescence by 25 ± 2% compared with 16 ± 2% for motilin and 8 ± 2% for EM-A. Binding studies confirmed that EM-A did not induce MTLR internalization (residual binding 96 ± 4% compared with motilin, 31 ± 3% and ABT-229, 21 ± 1%). Comparison of the pDC₅₀ and pEC₅₀ values of the other motilides ME4 (5.90; 8.08), ME67 (6.03; 8.12), ME36 (3.32; 6.62), EM-523 (6.02; 8.22), and KOS1326 (7.32; 8.14) suggested that the strong desensitizing properties of ABT-229 are mostly related to the removal of the 4''-OH of the cladinose sugar. The decline of the contractile response in duodenal segments correlated with the pDC₅₀. The ability to desensitize and internalize the MTLR is not only determined by potency. This may be an important criterion for the development of a clinically useful compound.

Address correspondence to: T. L. Peeters, Gut Hormone Lab, Gasthuisberg O & N, B-3000 Leuven, Belgium. E-mail: theo.peeters{at}med.kuleuven.ac.be

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