S1P1 Signalling Maintained Despite Receptor Internalisation

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The immunomodulator, FTY720 (fingolimod) is currently undergoing phase III clinical trials for the treatment of relapsing-remitting multiple sclerosis. FTY720 is a prodrug that, once phosphorylated to FTY720-P, is believed to act primarily by targeting sphingosine-1-phosphate (S1P) receptors on lymphocytes and endothelial cells. This leads to retention of lymphocytes in lymph nodes that, in turn, prevents attacks on myelin sheaths.

FTY720-PAlthough FTY720-P is a potent agonist of several S1P receptors, its beneficial effects in multiple sclerosis are believed to be mediated primarily through the S1P1 receptor. Because specific knockout of the S1P1 receptor on haematopoietic cells in mice and treatment with FTY720 show similar effects on lymphocyte recirculation, the efficacy of FTY720-P has been attributed to ‘functional antagonism’ leading to complete internalisation and desensitisation of receptors. However, writing in the journal Nature Chemical Biology, scientists at Novartis now provide evidence that, despite internalisation, signalling by S1P1 receptors bound to FTY720-P persists for hours. Although calcium signalling – which depends on cell surface localisation of the receptor – was inhibited by treatment with FTY720, other signalling pathways remained activated. In both stably transfected and primary cell lines, persistent activation of S1P1 receptors by FTY720 led to prolonged inhibition of adenylate cyclase and increased ERK phosphorylation. Similar effects were not observed with the endogenous agonist, S1P, and by exploring analogues of FTY-720, the length of the aliphatic side chain was found to be crucially important for persistent signalling and receptor internalisation.

Shortening the lipophilic side chain by one methylene group decreased the effect and shortening by two methylene groups abolished the effect completely, despite all three compounds having similar intrinsic potencies. The ability of the S1P1 antagonist, WN146 – which does not itself induce lymphocyte sequestration – to inhibit prolonged S1P1 signalling caused by treatment with FTY720-P suggests that direct agonism rather than functional antagonism may be the predominant mechanism of action of FTY720-P.


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