Functional Activity of Etrasimod and a Diverse Panel of Sphingosine-1-Phosphate Receptor (S1PR) Modulators at S1P4 Receptors John W. Adams1, Steve Chang2, Xiaohua Chen2, John Frazer2, Ibragim Gaidarov2, Minh Le2, and David J Unett2 1 Arena Pharmaceuticals and 2 Beacon Discovery, San Diego, CA Abstract ID: 6545
No Recovery 5h Recovery
Results I 1 0 0
Introduction I 1 0 0 Figure 6 shows label-free, dynamic mass redistribution
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responses obtained using the same S1PR4 expressing d 7 5
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All compounds produced robust arrestin recruitment 5 0 a 5 0 m
The S1P receptor modulator FTY720 (Fingolimod) m
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r o with a range of efficacies. S1P was quite potent in this 2 5 o With the exception of etrasimod, all compounds elicited
N 2 5 exerts numerous anti-inflammatory effects at least in N assay (4.5 nM) (Fig. 1). DMR responses. The potencies of test compounds in all part by producing sustained S1PR1 receptor 0
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internalization, resulting in loss of cellular 1 2 0 0
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responsiveness to S1P. FTY720 and a number of next- r
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generation S1PR modulators have varying degrees of f
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Etrasimod is a next-generation small molecule S1PR a
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modulator currently in clinical development for e 2 5
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ulcerative colitis and additional indications. Like other s
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S1PR1 modulators, etrasimod induces persistent o
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p s R Figure 3 S1PR4 internalization and receptor recovery 4 0 internalization of S1PR1 on T-cells, preventing their e
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at the cell surface 5h and 20h after compound wash-
egress from lymph nodes, producing lymphopenia and R
out. M immunosuppression. 0 D 2 0 - 1 2 - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 There is increasing awareness that, the S1PR4 lo g [ c m p d ] , M receptor, which is expressed largely in immune cells In GTPgS assays using CHO-S1PR4 membranes, the 0 (e.g. dendritic cells), may provide additional Fig. 1 Dose-responses of S1PR modulator-induced potency of S1P was 215 nM, consistent with a number - 1 2 - 1 0 - 8 - 6 - 4 recruitment of b-Arrestin to S1PR4. opportunities for immunomodulation. However, of literature reports (Fig. 4). While most compounds lo g [ c m p d ] , M signaling via S1PR4 has so far been poorly produced G-protein activation, etrasimod was unique characterized. in exhibiting inverse agonist activity. Figure 6. S1PR4 mediated DMR responses. All of the compounds induced robust receptor We therefore sought to characterize the functional internalization (Fig. 2). For most compounds, 3 0 0 0 activity of etrasimod and a panel of S1P receptor E t r a s im o d internalization potencies matched those observed in Arrestin Internaliz’n DMR GTPgS cAMP O z a n im o d modulators in a range of S1PR4 functional assays arrestin recruitment. The most notable exception was 2 5 0 0 F T Y 7 2 0 (P ) designed to interrogate signaling pathways and S1P, which was 100-fold less potent in producing Etrasimod 147 206 NR 680* NR receptor internalization. S 1 P internalization. 2 0 0 0
M Ozanimod 482 549 5234 3310 NR P 1 2 0 C
E t r a s im o d 1 5 0 0 FTY720(P) 2.3 4.4 1.7 1.3 16 )
P 1 0 0 O z a n im o d Methods 1
S 1 0 0 0
S1P 4.5 453 6.0 215 5397
f F T Y 7 2 0 (P ) o 8 0
% S 1 P ( 5 0 0 Table 1 Summary of potencies (nM) of S1PR n - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4
S1P and a panel of S1P receptor modulators were o
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t modulators in all S1PR4 functional assays. lo g [ c m p d ] , M
evaluated in the following panel of functional assays: a
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a 4 0 b-Arrestin recruitment: Performed in a clonal n
r Figure 4 S1PR4 mediated GTPgS responses. Conclusions e
S1PR4/HEK293 cell line using the PathHunter t
n 2 0 I technology from DiscoveRx. • S1PR4 was confirmed to be Gi-coupled but high Receptor internalization: Performed using HEK293 0 In cAMP assays using forskolin-stimulated S1PR4 CHO receptor expression was required to detect this cells stably expressing N-terminally HA-tagged S1PR4. - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 cells, S1P produced a low-potency response (Fig. 5). activity in cAMP assays. lo g [ c m p d ] , M Cells were exposed to test compounds at 37C and FTY720(P) elicited reductions in cAMP consistent with • The reported low potency of S1P induced G-protein then fixed. Cell surface receptor was quantified using its activity in the GTPgS assay. Etrasimod and activation at S1PR4 was confirmed. However, S1P- Figure 2 Dose-responses of S1PR modulator-induced a fluorescently labelled anti-HA antibody by flow ozanimod were inactive in cAMP assays. mediated b-arrestin recruitment was potent and internalization of S1PR4. cytometry. robust, perhaps suggesting S1PR4 signals primarily
GTPgS binding: G-protein activation was evaluated in 1 . 7 5 through the arrestin pathway. GTPgS assays using membranes prepared from CHO Receptor recycling was evaluated in a similar manner. • Etrasimod exhibits clear, biased signaling at S1PR4,
cells stably expressing S1PR4 at high levels. ) l 1 . 5 0 E t r a s im o d Cells were exposed to S1PR modulators (10 mM) for l inducing robust arrestin recruitment and internalizing
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1.5h at 37C. Compounds were then removed and the / the receptor with no detectable G-protein signaling cAMP Modulation: Gai-mediated reductions of l
o F T Y 7 2 0 (P ) forskolin-stimulated intracellular cAMP were cells thoroughly washed. Receptor expression at the m 1 . 2 5 in cAMP or DMR assays, and clear inverse agonist
p S 1 P ( activity in GTPgS assays.
measured in an S1PR4-CHO cell line (HTRF, Cisbio) cell surface was determined either immediately or P M
after a 5h or 20h recovery period at 37C (Fig. 3). A Dynamic Mass Redistribution assays: Compounds c 1 . 0 0 • Etrasimod induces persistent internalization of S1PR4 were evaluated in label-free dynamic mass Etrasimod, like FTY720(P), maintained robust receptor that is maintained >20 h in recombinant systems. internalization for 20h. In contrast, normal cell redistribution assays (Corning Epic®) using the same 0 . 7 5 • Etrasimod may act as a robust, functional antagonist - 1 2 - 1 0 - 8 - 6 - 4 S1PR4 expressing CHO cells, to capture a more holistic surface S1PR4 expression was observed at the 5h at S1P4 receptors in the immune system, inducing lo g [ c m p d ] , M view of functional responses. timepoint for S1P, ozanimod and S1P. prolonged receptor internalization without G-protein activation. Studies in primary immune cells are Figure 5. S1PR4 mediated cAMP responses. underway.
Funded by Arena Pharmaceuticals, San Diego, CA