Predictability of Peripheral Lymphocyte Reduction of Novel S1P1 Agonists by In Vitro GPCR Signaling Profile

Abstract

Surrogate readouts of G-protein-coupled receptor signaling pathways using highly engineered systems are often employed in the drug discovery process. However, accumulating data have demonstrated the importance of selecting relevant biological activity rather than technically facile assays to support high-throughout screening and subsequent structureactivity relationship studies. Here we report a case study using sphingosine-1-phosphate receptor 1 (S1P 1 ) as the model system to compare compound activity in six different in vitro assays with their ability to predict in vivo efficacy. S1P 1 has long been validated as a therapeutic target for autoimmune diseases. In this article, in vivo and in vitro studies on 19 S1P 1 agonists are reported. In vitro activities of these S1P 1 agonists, together with S1P and FTY720p, on Ca 2+ mobilization, adenylyl cyclase inhibition, extracellular signal-related kinase (ERK) phosphorylation, β-arrestin recruitment, and receptor internalization, were determined. The in vitro potency of these compounds was correlated with their ability to induce peripheral lymphocyte reduction. The results revealed that inhibition of adenylyl cyclase and induction of β-arrestin recruitment and receptor internalization are good indicators to predict in vivo efficacy, whereas induction of Ca 2+ mobilization through G qi/5coupling and ERK phosphorylation is irrelevant. This study demonstrated the importance of identifying an appropriate in vitro assay to predict in vivo activity based on the biological relevance in the drug discovery setting.

Parallel Fourier ptychographic microscopy for high-throughput screening with 96 cameras (96 Eyes)

“…( a ) Montage of all 96 brightfield images captured by the 96 Eyes imager; ( b ) Magnified view of well A8 on the 96-well plate; ( c ) Low-resolution, out-of-focus raw image captured by the consumer-grade CMOS sensor; ( d ) Restored intensity of the object; ( e ) Restored phase; ( f ) Fluorescence response from eGFP; ( g ) Composite image of phase channel in gray color and eGFP channel in green color. The cells are S1P 1 -eGFP-Human bone osteosarcoma epithelial cells 32 (U2OS; ATCC number HTB-96), seeded and fixed in the 96-well culture plate. Refer to Materials and methods for the detailed cell preparation protocol.…”

“…S1P 1 -eGFP-Human bone osteosarcoma epithelial cells 32 (U2OS; ATCC number HTB-96) were maintained in Dulbecco’s Modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, and 1% Penicillin Streptomycin (Life Technologies). Cells are seeded at a density of 8,000 cells per well in the 96-well culture plate (Grenier UV-Star or Cell Star) and incubated overnight at 37 °C and 5% carbon dioxide.…”

Structural Biology of the S1P1 Receptor

“…Recently, it has been established that the S1P 1 receptor agonists act, at least partially, through a functional antagonist mechanism. This finding introduces the possibility of designing agonist compounds that preferentially induce internalization of the receptor over other signaling pathways, a phenomenon known as biased ligand signaling (Xu et al 2013;Healy et al 2013). This concept could be an important mechanism for further improvements to the safety profile of immunomodulatory S1P 1 receptor agonists, and indeed, many other classes of small molecule therapeutics designed to modulate G protein-coupled receptor pharmacology.…”

S1P1 Modulator-Induced Gαi Signaling and β-Arrestin Recruitment Are Both Necessary to Induce Rapid and Efficient Reduction of Blood Lymphocyte Count In Vivo

“…This was previously proposed to explain SEW2871-and S1P-induced lymphopenia (Jo et al, 2005). The requirement for potent and effective G ai protein signaling has been also indirectly suggested by Xu et al (2013), who analyzed the correlation between in vitro potency testing of S1P 1 receptor modulators and peripheral lymphocyte count reduction. Interestingly, cAMP signaling, b-arrestin recruitment, and S1P 1 receptor internalization were all found to be good predictors of in vivo lymphopenia, but inhibition of cAMP accumulation, which is caused by G ai protein signaling, clearly showed the highest correlation (Xu et al, 2013).…”

“…The requirement for potent and effective G ai protein signaling has been also indirectly suggested by Xu et al (2013), who analyzed the correlation between in vitro potency testing of S1P 1 receptor modulators and peripheral lymphocyte count reduction. Interestingly, cAMP signaling, b-arrestin recruitment, and S1P 1 receptor internalization were all found to be good predictors of in vivo lymphopenia, but inhibition of cAMP accumulation, which is caused by G ai protein signaling, clearly showed the highest correlation (Xu et al, 2013). Similarly, BMS-986104, a S1P 1 receptor modulator that is potent and fully efficacious in G ai protein signaling, but which induces only partial receptor internalization, was recently reported to be fully efficacious in inhibiting lymphocyte egress in mice (Dhar et al, 2016).…”