Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.
ABSTRACT: FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.
Project description:The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.
Project description:FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720's anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity.
Project description:FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P(1), and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.
Project description:FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and the leukotriene C(4) transporter Abcc1 (Mrp1). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S1P also require 5-lipoxygenase-mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL19, CCL21, VLA-4alpha, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb1, and Abcc1, and comparison of the effects of FTY720 with those of S1P, suggest a model of sequential engagement of Abcb1, SP1 receptors, 5-lipoxygenase, and Abcc1 to enhance T cell migration and homing.
Project description:FTY720 (Fingolimod), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P(1) receptor internalization and targeting to the proteasomal degradative pathway, and thus functions as an antagonist of S1P(1) by depleting the functional S1P(1) receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)- and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P(1) (EC(50) 20+/-3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P(1,3,4) (K(i) 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P(2), and S1P(5). Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K(i) values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)- and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P(2) transcripts and traces of S1P(5). (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. The biological effects of the (R)- and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets.
Project description:In the search for bioactive sphingosine 1-phosphate (S1P) receptor ligands, a series of 2-amino-2-heterocyclic-propanols were synthesized. These molecules were discovered to be substrates of human-sphingosine kinases 1 and 2 (SPHK1 and SPHK2). When phosphorylated, the resultant phosphates showed varied activities at the five sphingosine-1-phosphate (S1P) receptors (S1P(1-5)). Agonism at S1P(1) was displayed in vivo by induction of lymphopenia. A stereochemical preference of the quaternary carbon was crucial for phosphorylation by the kinases and alters binding affinities at the S1P receptors. Oxazole and oxadiazole compounds are superior kinase substrates to FTY720, the prototypical prodrug immunomodulator, fingolimod (FTY720). The oxazole-derived structure was the most active for human SPHK2. Imidazole analogues were less active substrates for SPHKs, but more potent and selective agonists of the S1P(1) receptor; additionally, the imidazole class of compounds rendered mice lymphopenic.
Project description:Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S1P1-5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.
Project description:Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.
Project description:Rationale:Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. Objective:To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. Methods:Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. Results:FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. Conclusions:Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.
Project description:Sphingosine 1-phosphate (S1P), a lysophospholipid mediator, is generated from sphingosine by sphingosine kinases and binds five known cell surface receptors. S1P receptor 1 (S1P1) plays an essential role in lymphocyte egress from secondary lymphoid organs (SLO), as evinced by the inability of lymphocytes to exit from the SLO in mice lacking lymphocytic S1P1. Fingolimod hydrochloride (FTY720) is a first-in-class, orally active, S1P receptor modulator with a structure closely related to sphingosine. FTY720 was first synthesized by chemical modification of a natural product, myriocin. FTY720 is effectively converted to an active metabolite, FTY720 phosphate (FTY720-P) by sphingosine kinases. FTY720-P shows high affinity to 4 of the S1P receptors (S1P1, S1P3, S1P4, and S1P5). In particular, FTY720-P strongly induces internalization and degradation of S1P1, inhibits S1P responsiveness of lymphocytes in the SLO, and acts as a functional antagonist at lymphocytic S1P1. Consequently, FTY720 inhibits S1P1-dependent lymphocyte egress from the SLO to decrease circulation of lymphocytes including autoreactive Th17 cells and is highly effective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Because FTY720 shows a superior efficacy in relapsing remitting MS patients compared to intramuscular interferon-?-1a (Avonex®), S1P1 is presumed to be a useful target for the therapy of MS.