Structural insights into sphingosine-1-phosphate receptor activation.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

Deconstructing the Pharmacological Contribution of Sphingosine-1 Phosphate Receptors to Mouse Models of Multiple Sclerosis Using the Species Selectivity of Ozanimod, a Dual Modulator of Human Sphingosine 1-Phosphate Receptor Subtypes 1 and 5.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator that binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), is approved for the treatment of relapsing multiple sclerosis (MS) in multiple countries. Ozanimod profiling revealed a species difference in its potency for S1P5 in mouse, rat, and canine compared with that for human and monkey. Site-directed mutagenesis identified amino acid alanine at position 120 to be responsible for loss of activity for mouse, rat, and canine S1P5, and mutation back to threonine as in human/monkey S1P5 restored activity. Radioligand binding analysis performed with mouse S1P5 confirmed the potency loss is a consequence of a loss of affinity of ozanimod for mouse S1P5 and was restored with mutation of alanine 120 to threonine. Study of ozanimod in preclinical mouse models of MS can now determine the S1P receptor(s) responsible for observed efficacies with receptor engagement as measured using pharmacokinetic exposures of free drug. Hence, in the experimental autoimmune encephalomyelitis model, ozanimod exposures sufficient to engage S1P1, but not S1P5, resulted in reduced circulating lymphocytes, disease scores, and body weight loss; reduced inflammation, demyelination, and apoptotic cell counts in the spinal cord; and reduced circulating levels of the neuronal degeneration marker, neurofilament light. In the demyelinating cuprizone model, ozanimod prevented axonal degradation and myelin loss during toxin challenge but did not facilitate enhanced remyelination after intoxication. Since free drug levels in this model only engaged S1P1, we concluded that S1P1 activation is neuroprotective but does not appear to affect remyelination. SIGNIFICANCE STATEMENT: Ozanimod, a selective modulator of human sphingisone 1-phosphate receptor subtypes 1 and 5 (S1P1/5), displays reduced potency for rodent and dog S1P5 compared with human, which results from mutation of threonine to alanine at position 120. Ozanimod can thus be used as a selective S1P1 agonist in mouse models of multiple sclerosis to define efficacies driven by S1P1 but not S1P5. Based on readouts for experimental autoimmune encephalomyelitis and cuprizone intoxication, S1P1 modulation is neuroprotective, but S1P5 activity may be required for remyelination.

Sphingosine-1-phosphate: From insipid lipid to a key regulator.

It is a great honor to be asked to write a "Reflections" article by one of the true icons of biochemistry, Herb Tabor. I felt humbled, especially since it follows many written by biochemists I admire and whose contributions have shaped major advances in biochemistry and molecular biology in the last century. Here I present my personal reflections on my adventure with the bioactive sphingolipid metabolite sphingosine-1-phosphate intertwined with those of my family life as a wife, mother, and grandmother. These reflections brought back many memories of events in my early career that played significant roles in determining the path I have taken for more than 40 years and that brought much fun and satisfaction into my life. It has been an exciting journey so far, with many surprises along the way, that still continues.

Acute Rodent Tolerability, Toxicity, and Radiation Dosimetry Estimates of the S1P1-Specific Radioligand [11C]CS1P1.

PURPOSE: In preclinical studies with rodent models of inflammatory diseases, [11C]CS1P1 has been identified as a promising imaging agent targeting sphingosine-1-phosphate receptor 1 (S1P1) in the central nervous system and other tissues. In preparation for USA Food and Drug Administration (FDA) approval of [11C]CS1P1 for human use, an acute biodistribution study in mice and an acute tolerability and toxicity evaluation in rats were conducted. PROCEDURES: Acute organ biodistribution and excretion data was obtained using male and female Swiss Webster mice intravenously (IV) injected with 4.8-10 MBq of [11C]CS1P1. The organ residence times for each harvested organ were calculated using the animal biodistribution data, and were entered in the program OLINDA/EXM for C-11 to obtain human radiation dosimetry estimates. Acute tolerability and toxicity studies were conducted in male and female Sprague Dawley rats. Rats were administered an IV bolus of either the vehicle control or 0.3 mg/kg CS1P1. Blood samples were collected and a gross post-mortem examination was conducted at day 2 or day 15 post-injection. RESULTS: The extrapolated human radiation dose estimates revealed that the highest organ dose was received by the liver with 24.05 µGy/MBq in males and 32.70 µGy/MBq in females. The effective dose (ED) estimates of [11C]CS1P1 were calculated at 3.5 µSv/MBq in males and 5.9 µSv/MBq in females. The acute tolerability and toxicity study identified 0.3 mg/kg as a no observable adverse effect level (NOAEL) dose, which is a ~ 300-fold dose multiple of the human equivalent dose of the mass to be injected for positron emission tomography (PET) imaging studies in humans as a no-observable-effect limit. CONCLUSIONS: The toxicity study in rats suggested that injection dose of radiotracer [11C]CS1P1 with mass amount < 10 µg is safe for performing a human PET study. The dosimetry data supported an injection of 0.74 GBq (20 mCi) dose for human studies would be acceptable.

Molecular Mechanism of S1P Binding and Activation of the S1P1 Receptor.

Sphingosine-1-phosphate (S1P) is a lipidic mediator in mammals that functions either as a second messenger or as a ligand. In the latter case, it is transported by its HDL-associated apoM carrier and circulated in blood where it binds to specific S1P receptors on cell membranes and induces downstream reactions. Although S1P signaling pathways are essential for many biological processes, they are poorly understood at the molecular level. Here, the solved crystal structures of the S1P1 receptor were used to evaluate molecular dynamics (MD) simulations to generate greater detailed molecular insights into the mechanism of S1P signaling. The MD simulations provided observations at the coarse-grained and atomic levels indicating that S1P may access the receptor binding pocket directly from solvents. Lifting of the bulky N-terminal cap region of the receptor precedes initial S1P binding. Glu1213.29 guides S1P penetration, and together with Arg2927.34 is responsible for the stabilization of S1P in the binding pocket, which is consistent with experimental predictions. The complete binding of S1P is followed by receptor activation, wherein Trp2696.48 moves toward the transmembrane helix (TM) 7, resulting in the formation of an enhanced hydrogen bond network in the lower region of TM7. The distance between TM3 and TM6 is subsequently increased, resulting in the opening of the intracellular binding pocket that enables G protein binding. Further analysis of the force distribution network in the receptor yielded a detailed molecular understanding of the signal transmission network that is activated upon agonist binding.

Automated production of a sphingosine-1 phosphate receptor 1 (S1P1) PET radiopharmaceutical [11C]CS1P1 for human use.

Automated synthesis of a radiopharmaceutical 3-((2-fluoro-4-(5-(2'-methyl-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1,2,4-oxadiazol-3-yl)benzyl) (methyl-11C)amino)propanoic acid ([11C]CS1P1) for PET imaging sphingosine-1 phosphate receptor 1 (S1P1) was accomplished by a two-step-one-pot procedure in a Siemens CTI methylation automated module using TR-19 cyclotron. The synthesis of [11C]CS1P1 was successfully validated under current Good Manufacturing Practices (cGMP) conditions, resulting in a consistent average radiochemical yield of ∼15%, molar activity of ∼3129 GBq/µmol (decay corrected to end of bombardment, EOB), and radiochemical purity > 95%. The radiopharmaceutical product meets all quality control criteria for human use for an Investigational New Drug (IND) application to permit human studies.

Design, synthesis, and in vitro bioactivity evaluation of fluorine-containing analogues for sphingosine-1-phosphate 2 receptor.

Twenty eight new aryloxybenzene analogues were synthesized and their in vitro binding potencies toward S1PR2 were determined using a [32P]S1P competitive binding assay. Out of these new analogues, three compounds, 28c (IC50 = 29.9 ±â€¯3.9 nM), 28e (IC50 = 14.6 ±â€¯1.5 nM), and 28g (IC50 = 38.5 ±â€¯6.3 nM) exhibited high binding potency toward S1PR2 and high selectivity over the other four receptor subtypes (S1PR1, 3, 4, and 5; IC50 > 1000 nM). Each of the three potent compounds 28c, 28e, and 28g contains a fluorine atom that will allow to develop F-18 labeled PET radiotracers for imaging S1PR2.

S1PR1 as a Novel Promising Therapeutic Target in Cancer Therapy.

Sphingosine-1-phosphate (S1P) can regulate several physiological and pathological processes. S1P signaling via its cell surface receptor S1PR1 has been shown to enhance tumorigenesis and stimulate growth, expansion, angiogenesis, metastasis, and survival of cancer cells. S1PR1-mediated tumorigenesis is supported and amplified by activation of downstream effectors including STAT3, interleukin-6, and NF-κB networks. S1PR1 signaling can also trigger various other signaling pathways involved in carcinogenesis including activation of PI3K/AKT, MAPK/ERK1/2, Rac, and PKC/Ca, as well as suppression of cyclic adenosine monophosphate (cAMP). It also induces immunological tolerance in the tumor microenvironment, while the immunosuppressive function of S1PR1 can also lead to the generation of pre-metastatic niches. Some tumor cells upregulate S1PR1 signaling pathways, which leads to drug resistant cancer cells, mainly through activation of STAT3. This signaling pathway is also implicated in some inflammatory conditions leading to the instigation of inflammation-driven cancers. Furthermore, it can also increase survival via induction of anti-apoptotic pathways, for instance, in breast cancer cells. Therefore, S1PR1 and its signaling pathways can be considered as potential anti-tumor therapeutic targets, alone or in combination therapies. Given the oncogenic nature of S1PR1 and its distribution in a variety of cancer cell types along with its targeting advantages over other molecules of this family, S1PR1 should be considered a favorable target in therapeutic approaches to cancer. This review describes the role of S1PR1 in cancer development and progression, specifically addressing breast cancer, glioma, and hematopoietic malignancies. We also discuss the potential use of S1P signaling modulators as therapeutic targets in cancer therapy.

A Computational Approach to the Study of the Binding Mode of S1P1R Agonists Based on the Active-Like Receptor Model.

Sphingosine-1-phosphate receptor 1 (S1P1R), a member of the G protein-coupled receptor (GPCR) family, is an attractive protein target for the treatment of autoimmune diseases, and a diverse array of S1P1R agonists have been developed. Rational drug design based on S1P1R remains challenging due to the limited information available on the binding mode between S1P1R and its agonists. In this work, the active-like state of S1P1R was modeled via Gaussian accelerated molecular dynamics (GaMD) based on its inactive form, which was further validated by docking studies with two representative S1P1R agonists. Moreover, with the usage of the induced active-like state, the binding mode between S1P1R and its agonists was studied through molecular dynamics simulations and MM-GBSA calculations. The results of those studies indicated that four groups of binding site residues were the major contributors to the ligand and receptor interactions. In addition, this model was verified by five chemically similar compounds synthesized in-house and 1145 known S1P1R agonists collected from the BindingDB database. The elucidation of the key binding characteristics will further complete the cognition of S1P1R, which can guide the rational design of novel S1P1R agonists.

Constraints on GPCR Heterodimerization Revealed by the Type-4 Induced-Association BRET Assay.

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically important family of cell-surface receptors encoded by the human genome. In many instances, the distinct signaling behavior of certain GPCRs has been explained in terms of the formation of heteromers with, for example, distinct signaling properties and allosteric cross-regulation. Confirmation of this has, however, been limited by the paucity of reliable methods for probing heteromeric GPCR interactions in situ. The most widely used assays for GPCR stoichiometry, based on resonance energy transfer, are unsuited to reporting heteromeric interactions. Here, we describe a targeted bioluminescence resonance energy transfer (BRET) assay, called type-4 BRET, which detects both homo- and heteromeric interactions using induced multimerization of protomers within such complexes, at constant expression. Using type-4 BRET assays, we investigate heterodimerization among known GPCR homodimers: the CXC chemokine receptor 4 and sphingosine-1-phosphate receptors. We observe that CXC chemokine receptor 4 and sphingosine-1-phosphate receptors can form heterodimers with GPCRs from their immediate subfamilies but not with more distantly related receptors. We also show that heterodimerization appears to disrupt homodimeric interactions, suggesting the sharing of interfaces. Broadly, these observations indicate that heterodimerization results from the divergence of homodimeric receptors and will therefore likely be restricted to closely related homodimeric GPCRs.