Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase)

Accepted Manuscript Title: Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase) Author: Pol Sanlleh´ı Jos´e Luis Abad Josefina Casas Antonio Delgado PII: DOI: Reference:

S0009-3084(15)30012-8 http://dx.doi.org/doi:10.1016/j.chemphyslip.2015.07.007 CPL 4379

To appear in:

Chemistry and Physics of Lipids

Received date: Revised date: Accepted date:

1-6-2015 14-7-2015 15-7-2015

Please cite this article as: Sanlleh´i, Pol, Abad, Jos´e Luis, Casas, Josefina, Delgado, Antonio, Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase).Chemistry and Physics of Lipids http://dx.doi.org/10.1016/j.chemphyslip.2015.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase) Pol Sanllehí,[a][b] José Luis Abad,[a] Josefina Casas[a] and Antonio Delgado*[a][b]

[a]

Research Unit on BioActive Molecules; Department of Biomedicinal Chemistry;

Institute for Advanced Chemistry of Catalonia (IQAC-CSIC); Jordi Girona 18-26; E-08034 Barcelona (Spain)

[b]

University of Barcelona (UB); Faculty of Pharmacy; Department of Pharmacology and

Medicinal Chemistry; Unit of Pharmaceutical Chemistry (Associated Unit to CSIC); Avga. Joan XXIII s/n, E-08028 Barcelona (Spain)

E-mail: [email protected] Keywords: sphingolipid, enzymes, metabolism, inhibitors, modulators

Summary Sphingolipids (SLs) are essential structural and signaling molecules of eukaryotic cells. Among them, sphingosine-1-phosphate (S1P) is a recognized promoter of cell survival, also involved, inter alia, in inflammation and tumorigenesis processes. The knowledge and modulation of the enzymes implicated in the biosynthesis and degradation of S1P are capital to control the intracellular levels of this lipid and, ultimately, to determine the cell fate. Starting with a general overview of the main metabolic pathways involved in SL metabolism, this review is mainly focused on the description of the most relevant findings concerning the development of modulators of S1P, namely inhibitors of the enzymes regulating S1P synthesis (sphingosine kinases) and degradation (sphingosine-1-phosphate phosphatase and lyase). In addition, a brief overview of the most significant agonists and antagonists at the S1P receptors is also addressed.

Abbreviations 3-kdhSo

3-ketodihydrosphingosine aSMase acid

sphingomyelinase aCDase acid ceramidase C1P ceramide-1-phosphate Cer ceramide CerK Ceramide kinase CerS ceramide synthases CerT ceramide transporter

protein cPLA2a cytosolic phospholipase

A2 Des1 dhCer desaturase dhCer dihydroceramide dhSo dihydrosphingosine

(sphinganine) ER endoplasmic reticulum FAPP2 four-phosphate adaptor

protein GC glucosyl ceramide GCS glucosyl ceramide

synthase GSL complex

glycosphingolipids HDL high-density lipoprotein LPP lipid phosphate

phosphatases nCDase neutral ceramidase nSMase neutral

sphingomyelinase PEA phosphoethanolamine

S1P sphingosine-1-phosphate S1PPase sphingosine-1-phosphate

phosphatase SK1 sphingosine kinase 1 SK2 sphingosine kinase 2 SL sphingolipids So sphingosine SM sphingomyelin SPL sphongosine-1-phosphate

lyase SPT serine palmitoyl

transferase

Introduction Sphingolipids (SL) are key components of eukaryotic cells that contribute to the structural properties of the membrane and also to the regulation and cell homeostasis. The biosynthesis of SL comprises a highly organized system that takes place in different intracellular compartments (Figure 1). Thus, the so-called biosynthesis de novo takes place in the endoplasmic reticulum (ER) and starts with the condensation of L-serine with palmitoyl-CoA to give 3-ketodihydrosphingosine (3-kdhSo) (Figure 2) in a reaction catalyzed by serine palmitoyl transferase (SPT). By the action of a reductase, the ketone group of 3-kdhSo is reduced to a hydroxyl group to afford dihydrosphingosine (dhSo), which is N-acylated to dihydroceramides (dhCer) by specific ceramide synthases (CerS) of different chain length specificities (Mullen et al., 2012). The oxidation of dhCer to ceramide (Cer) by dhCer desaturase (Des1) constitutes the last step of the biosynthesis pathway (Fabrias et al., 2012). Despite the generic term “ceramide” comprises a family of several molecular species differing in the unsaturation of the sphingoid base as well as in the nature of the N-acyl chain (Hannun

and Obeid, 2011), in this review we will refer to Cer to indicate the C18 monounsaturated species shown in Figure 2, unless otherwise stated. Due to the metabolic inter-relations between Cer and other SL metabolites (see below), Cer has also been considered as the metabolic hub of SL biosynthesis (Hannun and Obeid, 2008). Part of the Cer generated in the ER is next transported to the trans Golgi apparatus by means of the specific transporter protein CerT (Kumagai et al., 2005) for its further transformation into sphingomyelin (SM), the major SL constituent of the cell membranes. Alternatively, after vesicular transport of Cer to the cis-Golgi, biosynthesis of glucosyl ceramide (GC) by means of glucosyl ceramide synthase (GCS) takes place, prior to its subsequent transport to trans-Golgi by FAPP2 to give complex glycosphingolipids (GSL). As with SM, GSL are transported by vesicular pathways to the cell membrane where they exert several and capital functions concerning cell-cell communications and responses to external stimuli (Wennekes et al., 2009). By the action of specific cytokines or by other external stimuli, activation of neutral forms of sphingomyelinase (nSMase) and ceramidase (nCDase) can give rise to a buildup of sphingosine (So) at the membrane level. Phosphorylation of So by the action of the specific sphingosine kinase-1 (SK1) leads to sphingosine-1-phosphate (S1P), which is secreted to elicit a plentiful of extracellular actions by interaction with specific receptors (see below).

FIGURE 1

In addition to the biosynthesis de novo, which provides a flux of SL from the ER to the cell membrane, these membrane SL can also be internalized by endocytic pathways and degraded in the lysosome by acidic forms of acid sphingomyelinase (aSMase), glycosidases (GCase) and acid ceramidase (aCDase). The So thus generated can be recycled back to Cer (by the action of CerS) in what is known as the salvage pathway (Figure 1).

In an alternative degradation process, S1P can be generated at the ER to be further transformed

by

sphongosine-1-phosphate

lyase

(SPL)

into

2-hexadecenal

and

phosphoethanolamine (PEA), which is ultimately incorporated into the biosynthesis of glycerolipids.

FIGURE 2

Phosphorylated sphingolipids An important group of SL metabolites are those showing a phosphate group at the terminal hydroxyl group. In this context, ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) are nowadays recognized as signaling molecules that regulate cell differentiation, survival, inflammation, angiogenesis, calcium homeostasis and immunity, among other functions(Kihara et al., 2007). In general, the roles of phosphorylated SL are opposed to those of ceramides, which are potent inducers of cell cycle arrest and apoptosis (Arana et al., 2010). Since phosphorylation and dephosphorylation are closely interconnected metabolic pathways in the cell, the balance between these two types of metabolites is crucial for cell fate and homeostasis (see below). In this context, ceramide kinase (CerK)(Bajjalieh and Batchelor, 2000; Bornancin, 2011) catalyzes the biosynthesis of C1P in the trans-Golgi, despite other localizations for this kinase have been shown (Carre et al., 2004; Van Overloop et al., 2006). Further transport by the specific transporter CPTP (Yamaji and Hanada, 2015) is involved in the transfer or C1P to the plasma membrane, where it is probably rapidly degraded to Cer by specific phosphatases. Nevertheless, C1P, together with S1P, have been implicated in the regulation of inflammatory responses (Gomez-Munoz et al., 2013) and, particularly, C1P has been shown to be a direct activator of cytosolic phospholipase A2 (cPLA2a) (Pettus et al., 2004). In addition, the CerK/C1P pathway is required for PLA2 activation in response to

cytokines (Pettus et al., 2003). The finding that bone marrow-derived macrophages from CerK deficient mice still have significant levels of C1P, suggests that other metabolic pathways, such as acyl transfer of long chain fatty acyl chains to Cer by acyl transferases or the cleavage of SM by a specific SMase D may be implicated in the formation of C1P (Boath et al., 2008). The ability of C1P to induce macrophages migration when administered exogenously, has led to hypothesize on the existence of a specific extracellular, low affinity Gi-coupled C1P receptor, which has emerged as a potential drug target for the treatment of illnesses associated to macrophage migration as the main cause of the pathology, such as chronic inflammation or metastasis of malignant tumors (Granado et al., 2009).

Sphingosine-1-phosphate: cellular roles and its modulation by synthetic compounds Probably the most studied actions of S1P are those derived from its high levels in plasm (ranging from 100 to 400 nM, depending, on the population, and reaching up to 1 µM depending on the method used to isolate plasma) (Hänel et al., 2007; Książek et al., 2015) arising from its generation in the plasma membrane from the high pools of SM and the successive action of nSMase, nCDase and SK1 (Figure 1). The S1P thus generated is transported, via the Spinster homologue 2 (SPNS2) transporter, across the membrane to exert its extracellular roles associated to the binding to specific lipid G-protein coupled S1P1-5 receptors in an autocrine (also known as “inside-out” signaling) or paracrine manner to give rise to a series of downstream signaling pathways that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and immunity responses including, inter alia, lymphocyte trafficking and differentiation, cell growth, cell survival, and cytokine and chemokine production (Table 1) (Adada et al., 2013; Maceyka and Spiegel, 2014; Pulkoski-Gross et al., 2015; Rosen et al., 2013; Sanchez and Hla, 2004; Spiegel and Milstien, 2011).

TABLE 1

Erythrocytes and platelets constitute a buffer system for S1P in blood. They efficiently incorporate and store S1P, and protect it from cellular degradation. Despite they are not able to biosynthesize S1P, they can phosphorylate So as an additional pathway to account for the high levels of intracellular S1P than can be reached (up to 5 µM in plasma-free medium supplemented with the same concentration of S1P)(Hänel et al., 2007). Another reason for the high levels of S1P found in these types of cells has been attributed to their lack of both SPL and the specific S1P transporter SPNS2 (Santos and Lynch, 2015). Nevertheless, the S1P produced in erythrocytes can also be exported to the extracellular space by the ATP-binding cassette ABCC1 (Mitra et al., 2006) to reach S1P receptors after binding to albumin and highdensity lipoprotein (HDL) (Spiegel and Milstien, 2011). Interestingly, the structure of the S1P1 receptor, in complex with the selective antagonist ML056 (Figure 4) (also known as (R)-W156, (Rosen et al., 2013)) has been solved at 2.8 Å resolution (Hanson et al., 2012). Remarkably, the family of G-protein coupled S1P receptors share around 40% sequence identity (Sanchez and Hla, 2004) and have been validated as pharmacological targets. This has boosted the development of specific S1P modulators, some of which have gained relevance in clinical practice, as, for example, FTY720 (Fingolimod, Gilenya®) (Figure 3). Detailed pharmacological studies carried out on this compound showed that it is a pro-drug that requires an enantioselective (S)-phosphorylation by SK2 for activation as S1P agonist.(Albert et al., 2005). Fingolimod was the first orally active drug approved for the treatment of relapsing-remitting multiple sclerosis for its ability to act as a S1P1,3-5 agonist at nM concentrations (Brinkmann et al., 2010; Strader et al., 2011). Despite it is out of the scope of this review, the design of FTY720 analogs with improved receptor selectivity has been an active field of research. In addition, several selective agonists and antagonists on the different

S1P receptors have been reported (Cahalan, 2014; Roberts et al., 2013). Some representative examples are listed in Tables 2 and 3 and Figures 3 and 4.

TABLE 2

FIGURE 3

TABLE 3

FIGURE 4

While the GPCRs S1P1-5 receptors are well-characterized and universally accepted as mediating S1P extracellular signaling, there is evidence that S1P can also act inside the cell as a second messenger during inflammation (Spiegel and Milstien, 2011). In addition, S1P can be biosynthesized in the nucleus of endothelial cells and T cells by the action of SK2 (see below), where it exerts important immune functions as a result of the activation of gene transcription by inhibition of histone deacetylases 1 and 2 (HDAC 1/2) (Fyrst and Saba, 2010; Spiegel and Milstien, 2011).

Modulation of S1P metabolism The levels of S1P depend on the balance of three different processes: a) phosphorylation of So at the 1-position by specific kinases, which use ATP as phosphate source and present different cellular localizations, b) dephosphorylation of S1P by ubiquitous general lipid phosphatases or by specific S1P phosphatases located at the ER, and c) degradation of S1P by the action of the specific lyase SPL, also located at the ER. Together with C1P (see above),

the dynamic balance between phosphorylated and non-phosphorylated SL, as well as their subcellular localization, turns out to be crucial for cell fate. Thus, phosphorylated SLs exert mitogenic effects while Cer and other sphingoid bases are considered pro-apoptotic. Based on these premises, the notion that the ratio S1P/Cer can act as a “sphingolipid rheostat” (Newton et al., 2015; Spiegel and Milstien, 2003) emerged as an important concept to understand cellular homeostasis (see SPL section, below). This concept can be better understood by also considering CerK for its ability to regulate the intracellular levels of C1P, another important phosphorylated SL.

Sphingosine kinases Sphingosine kinases (SK) catalyze the transfer of phosphate from ATP to So to generate S1P. Two SK isoforms (SK1 and SK2) have been identified, showing more than 50% sequence identity (Liu et al., 2000). Concerning substrate specificity, both enzymes can also phosphorylate dhSo. However, SK2 is less restrictive and can accept a wider variety of lipidic structures as, for example, phytosphingosine, L-threo-dhSo (also known as DHS or safingol, Figure 5) (Liu et al., 2000), and also FTY720, which is converted into the active (S)-phosphorylated form (see above) (Paugh et al., 2003). Isoform SK1 is mostly localized in the cytosol, in close proximity to the plasma membrane. It participates in the biosynthesis of S1P, which is subsequently transported to the extracellular space for autocrine or paracrine signaling to give rise, inter alia, to cell proliferation and inhibition of de novo Cer synthtesis. Another localization of SK1 is near the lysosomes, where it traps protonated So (as ammonium salt) to form zwitterionic S1P for its subsequent degradation by SPL (see below and Figure 1) or its recycling, as part of the salvage pathway (Hannun and Obeid, 2008), after dephosphorylation to So by a specific phosphatase (see below). On the other hand, the isoform SK2 is mostly nuclear and the resulting S1P has been

reported to inhibit HDAC 1/2 to control gene expression, as indicated above. Other reported localizations for SK2 are mitochondria, where S1P plays an important role in the cytochrome c-oxidase assembling required for mitochondrial respiration (Strub et al., 2011) and in the ER, where So is phosphorylated as the first step of the salvage pathway, in concert with sphingosine-1-phosphate phosphatase (S1PPase), leading to Cer and triggering of apoptosis (Maceyka et al., 2005) (see Figure 1). From a functional standpoint, both SK are quite similar and partly redundant, since the absence of any of the isoforms does not give rise to severely altered phenotypes. However, the simultaneous lack of both SK1 and SK2 in a double knockout mouse is lethal due to an incomplete maturation of the vascular system and brain (Mizugishi et al., 2005). The importance of SK as potential targets for drug discovery is linked to the above indicated role of phosphorylated SL, in particular S1P, in cell proliferation and its implication in several diseases as, for example, sickle cell disease, cancer, and fibrosis.(Santos and Lynch, 2015). Quite recently, the structures of SK1 (expressed as N- and C-truncated versions of the native enzyme) in its apo- form and in complexes with ADP, and the inhibitors SKI-II (Figure 6) (Wang et al., 2013) and PF-543 (Figure 7) (Wang et al., 2014) at 2.0–2.3 Å and 1.8 Å resolution, respectively, have been disclosed.

SK inhibitors The interest of SK inhibitors as potential drugs (Pitman and Pitson, 2010) stems from the fact that SK1 is usually up-regulated in several forms of cancer and that its genetic ablation leads to sensitization of cancer cells to chemotherapeutic agents. Concerning SK2, its involvement in cancer development is still controversial (Neubauer and Pitson, 2013). The field of SK inhibitors has been reviewed in recent works where the interested reader will find a thorough account of the progress made in this area in the last years (Gangoiti et al.,

2010; Orr Gandy and Obeid, 2013; Patwardhan et al., 2015; Plano et al., 2014; Pyne et al., 2011; Santos and Lynch, 2015). In general, the requirements for an ideal SK inhibitor do not differ much from those of any ideal drug, namely high potency and selectivity for a particular isoform, lack of toxicity, predictable pharmacokinetics, metabolic stability, and suitable properties for oral administration. However, we apparently are still far from an inhibitor meeting all of the above criteria. It is expected that the structural information obtained from the above mentioned X-ray enzyme-inhibitor co-crystals will help along this way.

Sphingolipid analogs as SK inhibitors The first SK inhibitors reported in the literature were found among natural products and SL analogs, generally showing low potency and selectivity. Since many of them are losing relevance, we address the interested reader to previous review articles on the topic (Gangoiti et al., 2010; Plano et al., 2014). Nevertheless, some of these inhibitors deserve special attention. This is the case of the SL analog safingol (Figure 5), which has been extensively used as pharmacological tool. Despite being a moderate SK1 inhibitor (Ki ~ 3-6 µM)(Buehrer and Bell, 1992), safingol behaves as a SK2 substrate with several off-target effects (Coward et al., 2009). Nevertheless, in combination with cisplatin, a phase I study of safingol in patients with locally advanced or metastatic solid tumors has been completed (ClinicalTrials.gov ID: NCT00084812). Another SL analogue is N,N-dimethylsphingosine (DMS, Figure 5). It was initially described as a PKC inhibitor (Igarashi et al., 1989; Merrill Jr. et al., 1989), but later reported as a dual SK inhibitor, competitive for SK1 and uncompetitive for SK2, with Ki in the low µM range for both enzymes (Cuvillier et al., 1996; Liu et al., 2000; Yatomi et al., 1996). Jaspine B (pachastrissamine) (Figure 5), isolated as a cytotoxic component of the sponge Pachastrissa sp (Kuroda et al., 2002), is a natural marine product structurally related to SL.

The cytotoxicity found for this natural product in several cell lines (Kuroda et al., 2002; Ledroit et al., 2003) has been associated to its activity as sphingomyelin synthase inhibitor (Salma et al., 2012, 2011, 2009). However, Jaspine B, its diasteroisomers (Yoshimitsu et al., 2011) and some derivatives thereof have also been evaluated as SK inhibitors showing a moderate inhibition of both isoforms in the low µM range (Byun et al., 2013). The fact that some toxic effects have been associated to increased levels of dhCer (Canals et al., 2009), makes conceivable to assume a complex mode of action for this interesting natural product. Another SL related SK inhibitor is compound SK1-I (BML258) (Figure 5), a N-methyl sphingosine analogue that incorporates a phenyl group as part of the sphingoid chain (Paugh et al., 2008). Despite being a low µM SK1 inhibitor (Ki of 10 µM), it shows around 10 times lower affinity as SK2 inhibitor and also shows little activity against a panel of kinases. Recently, some dhSo derivatives have been reported as non-selective SK inhibitors (Byun et al., 2013). Thus, the thiourea dhSo analogue F-02 (Figure 5) inhibited SK2 with an IC50 of around 20 µM and SK1 with an IC50 of around 70 µM (Byun et al., 2013). In the same work, 1-deoxydihydrosphingosine, referred as compound 55-21 (Figure 5), albeit identical to the marine natural product spisulosine (Cuadros et al., 2000a, 2000b) is described as a selective SK1 inhibitor, with an IC50 of 7.1 µM, around 100 times lower than that found as SK2 inhibitor. Another 1-deoxysphingolipid analog (compound 77-7, Figure 5) has also been reported as a moderately selective SK1 inhibitor with IC50 around 30 µM, 10 times lower than as SK2 inhibitor.

FIGURE 5

More recently, the SL analog SG12 (Figure 5), initially described as selective human SK2 inhibitor (IC50 = 22 µM), together with a series of structurally related analogs (Kim et al.,

2005), has also been reported to be an apoptotic agent after phosphorylation by SK2 (HaraYokoyama et al., 2013). Another SL analog that merits consideration is compound 5c (Figure 5), a synthetic intermediate of a series of SL analogues designed as SK inhibitors. Although its potency as SK1 inhibitor is similar to that of DMS, compound 5c shows an outstanding SK1 selectivity (Wong et al., 2009). As a result of the chemical modifications of the S1P agonist FTY-720 (see above), several interesting SK antagonists have emerged. Thus, the corresponding (S)-FTY-720 vinylphosphonate (Figure 4) inhibits SK1 (Ki = 14.5 µM) in an uncompetitive manner (with respect to sphingosine) (Lim et al., 2011b). At 50 µM, the compound has also been reported to inhibit SK2 (around 70% from the maximal activity of the assay on purified SK2 at 10 µM So) (Lim et al., 2011a). Structural modifications of (S)-FTY720 vinylphosphonate revealed the need of the phosphate group for inhibition and the surprising finding that the replacement of the amino group with an azido group turns the compound into a modest SK1 activator (Z. Liu et al., 2013). Interestingly, examples of SK1 activators, as observed for the Cer analog K6PC5 (N-[(1-hydroxymethyl-2-hydroxy)ethyl]-2-(n-hexyl)-3-oxodecanamide) are scarce in the literature (Bernacchioni et al., 2011; Ji et al., 2015; Kwon et al., 2007; Park et al., 2008).

Non lipidic, non-selective SK inhibitors Many of the most recent SK inhibitors have been discovered as a result of HTS campaigns that have led to a vast array of new and structurally unrelated lead compounds. One of the first inhibitors reported as a result of this strategy was compound SKI-II (Figure 6) (French et al., 2006, 2003), a mixed SK inhibitor with a competitive Ki of 17 µM and an uncompetitive Ki of 48 µM on SK1 (Lim et al., 2011b). Among the several modes of action revealed by this compound (Santos and Lynch, 2015), a recent finding has allowed its identification as a non-competitive inhibitor of Des1 (Ki = 0.3 µM), the last enzyme

implicated in the de novo biosynthesis of Cer (Cingolani et al., 2014). This interesting finding should be taken into consideration when using SKI-II as pharmacological tool. Compound VPC96091 is a representative example of the first generation of amidino derivatives that have been developed as SK inhibitors on the last years. In particular, this compound is a high affinity and selective SK1 inhibitor with a Ki of around 100 nM, but suffers from a short half-life (Kharel et al., 2011). Along this line, the structurally related guanidino compound SLR080811 showed an improved half-life, albeit with a 100 times lower affinity (Ki around 10 µM) as SK1 inhibitor and 10 times more selective as SK2 inhibitor (Ki around 1 µM)(Kharel et al., 2012). Compound 82, also reported in the literature as Amgen 82, is the most representative compound of a large focused library of SK inhibitors arising from a structure-guided design. Amgen 82 is characterized by its dual SK1 and SK2 inhibition in the nM range, with Ki values around 20 and 100 nM, respectively. In addition to its favorable pharmacokinetic and physico-chemical

properties,

the

docking

studies

show

that

the

(2R,4S)-2-(hydroxymethyl)piperidin-4-ol moiety is crucial for the establishment of key hydrogen bonding interactions with Asp81 and Asp178 of human SK1 (Gustin et al., 2013). Compound trans-12b has also been reported as a SK inhibitor with modest SK2 selectivity (Raje et al., 2012). From a structural standpoint, the quaternary ammonium salt is singular, albeit bio-equivalent to the guanidine moiety present in other SK inhibitors of this type (Figure 6). Finally, in a recent report, compound MPA08 has been disclosed as the first small molecule SK inhibitor (SK1: 27 µM; SK2: 6.9 µM) acting at the ATP-binding site (Pitman et al., 2015).

FIGURE 6

Selective SK1 inhibitors Among the recent SK1 inhibitors reported in the literature, some highly selective compounds deserve special attention. This is the case of the modest inhibitor SKI-178, with a Ki value of 1.33 µM, which inhibits SK1 in a non-competitive manner with respect to ATP binding (Hengst et al., 2010). Another modest but highly selective SK1 inhibitor is RB-005, with an IC50 of 3.6 µM (Baek et al., 2013). Nowadays, the so-called “second generation” of SK inhibitors has arisen as a result of intensive screening programs of many of the lead pharmaceutical companies. The most promising compounds are active at the nM level and some of them are totally selective towards one of the SK isoforms. Some of the most outstanding leads within this category are indicated next. Thus, PF-543 (Figure 7) is claimed as one of the most potent (Ki = 4 nM) and SK1 selective (around 100 fold compared with SK2) inhibitor reported so far. Despite its poor pharmacokinetics (Schnute et al., 2012), it has shown a promising profile for the reversion of sickle cell disease (Zhang et al., 2014). As also reported for the SK1/SK2 inhibitor Amgen 82 (Figure 6)(Rex et al., 2013), SK1 inhibition by PF-543 showed no effect on the proliferation and survival of cancer cells, despite the observed alteration of S1P levels (Schnute et al., 2012). Compound SLP7111228 has been reported as a potent (Ki = 48 nM) and selective (around 200 fold) SK1 inhibitor with a favorable in vivo stability (Patwardhan et al., 2015). Interestingly, this compound resulted from the chemical manipulation of the initially discovered SK2 inhibitor SLP120701 (see below). Another new generation selective SK1 inhibitor is Genzyme 51, with an IC50 around 60 nM, an acceptable pharmacokinetic profile and SK2 inhibition at concentrations close to 10 µM. (Xiang et al., 2010, 2009).

FIGURE 7

Finally, it is expected that the development of selective SK1 inhibitors will benefit from the structural knowledge of the SK1 receptor and its co-crystallization with selective ligands (Wang et al., 2014, 2013), which represents a breakthrough for the rational design of new structure-based inhibitors. Selective SK2 inhibitors Contrary to SK1, the repertoire of selective SK2 inhibitors is much more limited, which can be attributed, to some extent, to the partial knowledge of the role of this enzyme in cell homeostasis and its implication in pathological processes (Neubauer and Pitson, 2013). In addition to the mixed SK1/SK2 inhibitors mentioned in the above sections, some well-recognized selective SK2 inhibitors have emerged. One of them is (R)-FTY720-OMe (Figure 8), rationally designed to block the phosphorylation site of the known SK2 substrate FTY720 (see above). The compound is a So-competitive SK2 inhibitor, with a Ki = 16 µM and devoid of SK1 inhibition (Pyne et al., 2011). The compound is also able to reduce the proliferation of MCF-7 breast cancer cells and to prevent cell migration, which suggests a more complex and interesting mode of action (Lim et al., 2011a). Another interesting SK2 inhibitor is ABC294640 (Figure 8), nowadays considered as the main representative of the “first generation” of selective SK2 inhibitors and one of the most interesting and promising candidate to enlarge the therapeutic arsenal of potential new drugs (French et al., 2010). ABC294640 is a selective SK2 inhibitor (Ki = 9.8 µM), being also active against a panel of different kinases and also able to inhibit the proliferation of different cancer cell lines (French et al., 2006). The compound showed an excellent oral bioavailability in vivo and moderate toxicity, which makes ABC294640 an excellent drug candidate for the treatment of Crohn’s disease, as evidenced by the promising preliminary results (Maines et al., 2010). This compound is also currently under phase I clinical trials for pancreatic cancer and solid tumors (ClinicalTrials.gov ID: NCT01488513) and for diffuse large B cell lymphoma

(ClinicalTrials.gov ID: NCT02229981). The thiazolidine-2,4-dione derivative K145 (Figure 8) has been reported as an another selective SK2 inhibitor in the low µM range (Ki = 6.4 µM). Moreover, it does not affect CerK at concentrations up to 10 µM. Due to its growth inhibition and apoptotic effects in U937 cells, the compound has emerged as a novel lead candidate for further development (K. Liu et al., 2013). An interesting group of new generation SK2 inhibitors is found among some guanidine-based analogs bearing an oxazole ring as part of their structure. Among them, compound SLP120701 (Figure 8) has emerged as a representative SK2 selective inhibitor (Ki =1 µM), as evidenced by the decrease of S1P levels in histiocytic lymphoma (U937) cells (Patwardhan et al., 2015). This compound is a result of the structural modification of the closely related guanidine oxazole SLR080811 (Figure 8), another SK2 inhibitor (Ki =1.3 µM) with 10 fold lower affinity towards SK1 receptors (Neubauer and Pitson, 2013).

FIGURE 8

Sphingosine-1-phosphate phosphatases (S1PPase) The phosphatase super-family comprises a heterogeneous group of enzymes characterized by the presence of conserved residues essential for their catalytic activity. The mammalian lipid phosphate phosphatases (LPP) and the S1PPase are members of this superfamily. LPPs are integral membrane proteins that dephosphorylate a variety of phosphorylated lipid substrates. Three mammalian LPP isoforms have been cloned, termed LPP1, LPP2 and LPP3, together with another member named PRG-1 (plasticity-related gene-1). They differ in tissue distribution but, overall, they are widely expressed. All three LPPs are capable of dephosphorylating LPA, S1P, PA and C1P in vitro, thereby producing monoacylglycerol, So, diacylglycerol (DAG) and Cer, respectively. Concerning S1PPase, there are two ER-localized

isoforms (named SPP1 and SPP2) that catalyze the dephosphorylation of S1P. They differ from LPP in the substrate specificity, which is limited to S1P, dhS1P, and phytosphingosine 1-phosphate, whereas LPA and PA are not substrates. Concerning tissue distribution, S1PPase1 is mainly expressed in kidney and placenta whereas S1PPase2 is predominant in the heart and kidney. To date, there are no specific inhibitors for these enzymes, and only N-ethylmaleimide and propranolol have been reported as inhibitors of use as pharmacological tools. (Brindley, 2004; Gomez-Munoz et al., 2013; Pyne et al., 2004).

Sphingosine-1-phosphate lyase (SPL) Sphingosine-1-phosphate lyase (SPL) is a key enzyme of SL catabolism that catalyzes the irreversible degradation of S1P into phosphoethanolamine (PEA) and hexadecenal (Bandhuvula and Saba, 2007). In combination with S1PPase, the levels of S1P can be regulated by the so-called “sphingolipid rheostat”, a system that controls cell fate based on the ratio of intracellular proliferative S1P and the apoptogenic So and Cer (Newton et al., 2015; Spiegel and Milstien, 2003). From a mechanistic standpoint, SPL belongs to the superfamily of PLP (pyridoxal 5’-phosphate)-dependent enzymes. The enzyme is located in the ER and possesses an N-terminal lumenal domain, a transmembrane segment and a soluble PLP-binding domain, responsible for the catalytic activity (Ikeda et al., 2004). Recombinant human SPL has been expressed in E.coli (Van Veldhoven et al., 2000) and, more efficiently, in Sf9 insect cells (Weiler et al., 2014). A co-crystal of the human recombinant enzyme with the selective inhibitor C (see Figure 11, below) was also reported in the same work (Weiler et al., 2014). Alternatively, the crystal structures of yeast SPL and that of a putative prokaryotic homolog from Symbiobacterium thermophilum (StSPL), expressed in E.coli, have also been reported. These structures have been used as reliable homology models of the human enzyme (Bourquin et al., 2010). Based on the structural, biochemical and the mutagenesis studies

available, a plausible reaction mechanism for SPL can be postulated. As shown in Figure 9, the base-promoted retro-aldol cleavage, initiated by proton abstraction of the hydroxyl moiety of the intermediate aldimine A, is expected to be crucial for enzyme functionality.

FIGURE 9

Sphingolipid analogs as SPL inhibitors The development of selective SPL inhibitors has attracted the scientific community as an indirect way to increase the levels of S1P and the opportunities that S1P modulation offers for the treatment of multiple sclerosis (Matloubian et al., 2004; Schwab et al., 2005). Attempts to design selective SPL inhibitors have been accomplished with varied results. The first inhibitor reported in the literature was 1-desoxydihydrosphingosine-1-phosphonate (Figure 10) (Stoffel and Grol, 1974). It was described as a competitive inhibitor with a Ki = 5 µM. The compound behaves also as a substrate, but it is cleaved at reduced rate in comparison with S1P. Due to its hemolytic properties, this phosphonate was proved to be highly toxic when administered intravenously. The SL analogue 2-vinylsphinganine-1-phosphate (2VS1P) (described as a mixture of stereoisomers) (Figure 10) has also been reported as a potent SPL inhibitor with a IC50 value of 2.4 µM. Given the reactivity of the vinyl group at C2, it is presumed that this compound could interact in a covalent manner with the enzyme and, therefore, act as an irreversible inhibitor.(Boumendjel and Miller, 1994). Compound FTY720, initially reported as an immunomodulatory agent by its direct activity as S1P receptors 1,3-5 agonists after phosphorylation by SK2 (see above), was also reported as SPL antagonist in vitro (Bandhuvula et al., 2005). However, this effect is only observed at concentrations around 100-fold higher than those required as S1P agonist (Brinkmann et al., 2010). Using a protocol for (2E)-hexadecenal, quantitation as a semicarbazone derivative and mouse liver homogenate

as the enzymatic source, Berdyshev et al. reported that FTY720 inhibited SPL in vitro with an IC50 of 52.4 µM.(Berdyshev et al., 2011) In contrast, (S)-FTY720P, the active form of fingolimod on S1P receptors, was determined to be inactive against SPL.(Bandhuvula et al., 2005; Berdyshev et al., 2011).

FIGURE 10

Non lipidic SPL inhibitors A series of inhibitors structurally unrelated to S1P have been reported on the last years as a result of extensive HTS screening programs. This is the case of the oxopyridylpyrimidine A (Figure 11), which was reported as a direct inhibitor of SPL (IC50 = 2.1 µM) and able to increase S1P levels 275% over controls in HepG2 cells, despite it failed to elicit and in-vivo lymphopenic effect, probably because of tight protein binding (Bagdanoff et al., 2009). References in the literature mentioning this compound are very scarce. Gatfield et al. used this compound alongside the Des1 inhibitor GT11 and the functional S1P1 receptor antagonist ponesimod in order to study the role of the enzyme in the physiological S1P1 receptor recycling process.(Gatfield et al., 2014). Very recently, as a result of a massive screening campaign carried out by Novartis using a corporate chemical library of around 250,000 compounds, the piperazinophatlazine derivative B (Figure 11) was identified as an interesting SPL inhibitor in the low micromolar range.(IC50 = 2.4 µM) (Billich et al., 2013; Loetscher et al., 2013). As a result of a lead optimization process, compounds C (Loetscher et al., 2013) and D (Weiler et al., 2014) (Figure 11) were later reported as improved inhibitors acting in the submicromolar and nanomolar range (IC50 values of 0.21 µM and 0.024 µM for C and D, respectively). The reported crystal structure of human SPL in complex with C demonstrated that the inhibitor is hosted in the substrate binding site, specifically at the branching point of a

Y-shaped channel that links the buried active site to the exterior.(Cosconati et al., 2014; Weiler et al., 2014). Interestingly, compound C is able to induce lymphopenia and confers protection in animal models of multiple sclerosis (Weiler et al., 2014). Very recently, kidney toxicity, probably associated to an on-target effect of S1P has been reported (Schumann et al., 2015).

FIGURE 11

Functional SPL antagonists Some compounds have been reported as functional SPL antagonists by their ability to reproduce a phenotypic SPL inhibition in vivo, despite their lack of activity on the isolated enzyme. In this context, the vitamin B6 antagonist 4-deoxypyridoxine (DOP) (Figure 12) has been reported as a SPL inhibitor and also as a non-selective inhibitor of PLP-dependent enzymes (Bassi et al., 2006). It has been suggested that DOP, either by itself of after metabolization, is able to compete with PLP as an essential co-factor of the enzyme. Although it does not inhibit SPL in biochemical and cellular assays (Bagdanoff et al., 2009; Billich et al., 2013; Loetscher et al., 2013), it causes reduction of SPL activity when administered in vivo (Bagdanoff et al., 2009; Schwab et al., 2005). A similar scenario is observed for THI and structurally

related

analogs

(Figure

12).

Oral

administration

of

2-acetyl-4-tetrahydroxybutylimidazole (THI), originally identified as a minor constituent of caramel color III, caused immunosuppression in rodents (Gugasyan et al., 1998), leading to a phenotype similar to that observed in mice expressing reduced levels of SPL (Bagdanoff et al., 2009). As for DOP (see above), these compounds only inhibit SPL in vivo and its effect is reverted by dietary vitamin B6.(Schwab et al., 2005) Very recently, Ohtoyo et al. investigated the mode of action of THI by focusing in particular on the vitamin B6 concentration in the

assay system. Using a vitamin B6-deficient culture medium in a cell-based assay, they reported the first successful detection of SPL inhibition by THI in in vitro experiments (Ohtoyo et al., 2015). In addition, derivatives of THI, such as LX2931 and LX2932, have also been described.(Bagdanoff et al., 2010, 2009) LX2931, developed for the treatment of rheumatoid arthritis, was the first clinically studied inhibitor of SPL. After evaluation in phase I clinical trial in healthy human subjects it failed at phase II, apparently due to sub therapeutic dosing.(Bigaud et al., 2014)

FIGURE 12

Concluding remarks The importance of phosphorylated SLs as signaling molecules that finely regulate key cellular events is nowadays well recognized and constitutes a field of active research. In this context, S1P is illustrative of the role of this type of SLs at both extra- and intracellular levels and how its proper modulation can be judiciously used in several therapeutic areas. However, regardless the acquired knowledge of the metabolic pathways involved in the biosynthesis and degradation of S1P, the need for pharmacological tools to selectively control these processes is still required. Specific kinases (SK1 and SK2), with different intracellular locations and functions, have been characterized and efforts towards the development of selective inhibitors have been noteworthy. In this context, several “brute force” HTS campaigns have led to the discovery of some promising hits as SK inhibitors. However, despite a much limited progress has been made on SPL inhibitors, it is expected that rational design strategies based on the X-ray structures of the target enzymes, either free or co-crystallized with high affinity ligands, will gain relevance in the development of more efficient structure-based inhibitors.

Acknowledgments Partial financial support from the “Ministerio de Ciencia e Innovación”, Spain (Projects SAF2011-22444 and CTQ2014-54743-R are acknowledged). The authors also want to thank “Fundació La Marató de TV3” for financial support through Projects 20112130 and 20112132.

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Graphical Abstract CN

S1P

N

N

N N

CN N

So

S1P SK1

SPL PEA+ hexadecenal

S1P

Sph

Cer SK2

O

O S

O

Cl N OH

This file, together with Figure 1, have been uploaded in tiff versión.

O HN

N

S1P

Cytokine SPNS2 transporter

S1P receptor

So

S1P

Cer SM nCDase nSMase

SK1

endocytosis

CPTP intracellular ef f ects SM

Cer1P

GSL

GSL CERK

GCase aSMase Cer SM aCDase

Lysosome

Cer

SM

Golgi

So

FAPP2

GC apoptosis

So

GC

GSL

GCS

CERT Recycling (*)

So

Cer SM

SK1 (*)

S1P

Mitochondria Ca2+

1: SPT 3-kdhSo dhSo 2: r eductase dhCer 3: CerS Cer 4: Des1

Cer

SK2

(*) (*) S1PPase SPL CerS PEA+ hexadecenal S1P Sph Cer Degradation aCDase SK2

De novo

Serine + palmitoyl CoA

Palmitoyl CoA

ER SK2 SM

Glycerolipids

Cer

Sph

S1P

Nucleus

Figure 1. Compartmentalization of SL biosynthesis. For the structures, see Figure 2. Adapted from (Maceyka and Spiegel, 2014); (*): salvage pathway; For abbreviations, see above. O HO

C13H27

HO

NH2

C13H27

HO

Dihydrosphingosine (dhSo)

OH C13H27 NH2

Sphingosine (So)

H3 C H3 C

CH3 N

OH C13H27

HO

NHCOR

NH2

3-Ketodihydrosphingosine (3kdhSo)

HO

OH

OH

O O

Dihydroceramides (dhCer) OH

O P

O

Sphingomyelins (SM)

Ceramides (Cer) O

C13H27 NHCOR

C13H27 NHCOR

HO

O P

OH

O

C13H27 NHR

R=H: Sphingosine-1-phosphate (S1P) R=acyl: Ceramide-1-phosphate (CerP)

Figure 2. Chemical structures of the most relevant SL (see also Figure 1)

O OH R

Cl

S

O

CF3

HO

O

KRP203

S

F3C

OH

N H

EtO

N

CYM5541

Compound 9f

N S

COOH

O

F MeO

F

N N

R

HO

N

F COOH

BAF312

O

O

ML248

COOH

Compound 31a

N

N

N

N O

S N

Cl

N

NH

O

S

N

O

N Cl

OH F3C

O N

O

COOH

N

CS2100

Cl ASP4058

N O

RP001

O N

N

N

F3C

N OH

N

O N

O

NH

N

N H

N

CYM5442

O N

O

O N

O

O O

XAX162

ML178

NC

O N SEW2871

F3C

Br O

N CF3

NO2

S

Cl

AUY954 O N

EtO N

S N

·HCl

HO

R = H: FTY720 R = PO3H2: (S)-FTY720P

Br O

O

S

HO

NH3

Cl

N H

NH2

N

S

N

F Ponesimod

O

COOH

R= CF3: SID 46371153 R= CN: CYM5520

AMG369

Figure 3. Structures of the S1P receptor agonists listed in Table 2

NH2

H N O

O HN S O

N

O

N

O

Cl

N N O P OH HO X

Cl Cl

TASP0277308 NH2

O

O P HO HO

OH P OH O

NIBR0213

HO

N N

NH2

N

N H

H N

H N

Cl

O JTE013

(S)-FTY720 vinylphosphonate

W146 (ML056)

N Cl

Cl OH O

O B

O

Cl

NH

H N

N

N

NH2 HO

O

Cl

OH P O OH

O

Cl

O

S

N H

Cl

TY52156

O

HO

H N

F

Compound 22

O

N

X=O: VPC23019 X=CH2 VPC44116 H N

OH

N H

H N

SPM242

ML131

N

Figure 4. Structures of the S1P receptor antagonists listed in Table 3 OH HO

OH

C15H21

HO

NH2

CH3

HN

CH3

C15H31

CH3

NH

OH

C15H31 NH2

F-02

55-21

OH

SK1-I (BML258) OH

O

CH3 CH3

NHMe

Jaspine B

OH

CF3

HO H2N

DMS

OH

OH

C14H29

C13H27 N

DHS (Safingol)

HO S

O

N

CH3

77-7

C14H29

O

C8H17 O

HO

N

NH2 SG-12

Figure 5. SL-related SK inhibitors

O OtBu

C13H27 5c

OH O

S

·HCl

C12H25 Cl

NH H2N ·HCl N O N

NH2

N

NH

N

HN

N

C8H17

Me

I

C8H17

VPC96091

SKI-II

Me Me N

trans-12b

SLR080811 OH

S F3C

N

O S NH O

N N H

OH

O HN S O

N

Amgen 82

MPA08

Figure 6. Non-lipidic and non-selective SK inhibitors MeO O

OMe

O S

HN N

O

OMe

N N H

N

O

OH

PF-543

SKI-178 O

OH

OH N

N H

N

RB-005

N

N

HN

HN

O N

C8H17

N O

NH2 ·HCl

C8H17 Genzyme 51

SLP7111228

Figure 7. Selective SK1 inhibitors

MeO

O

NH2 OH

Cl

O

C8H17

N S

BuO

HN (R)-FTY720-OMe

NH2

N

O K145

ABC294640 N O N H2N

C8H17

NH

H2N N O

N

N

·HCl

N

·HCl

NH C8H17

SLP120701

SLR080811

Figure 8. Selective SK2 inhibitors

SPL 2-hexadecenal H O

Lys 353 H SPL

O HO P O O

S1P

H

O *

O

HN

PLP

H C13H27 CH3

O O P O O

C13H27 N

O

O P O

O

hidrolysis CH3 NH2

NH

Aldimine A

O

O O P O O

O O P O O

PEA

NH2

Figure 9. Base-promoted retro-aldol cleavage of the intermediate internal aldimine in SPL.

O

OH

OH P O

OH

HO O P O O

C13H27

C13H27 NH3

NH3

1-desoxysphinganine-1-phosphonate

2VS1P

Figure 10. Sphingolipid analogs reported as SPL inhibitors

NH2 O

O

N

N H N

N

N

N

N N

N

N N

CN N

O A

B

Cl

CN

N

N

N N

CN

N

N

N

CN

N N

N

C

D

Figure 11. Non lipidic SPL inhibitors

OH

HN

HO

OH

O

N

N

HO

DOP

THI

OH

OH

HN

OH HO

N

N

N HO

OH

OH

HN

OH O

LX2931

OH N HO

OH

LX2932

Figure 12. Functional SPL antagonists

Table 1. Distribution and functions of sphingosine-1-phosphate receptors; modified from (Cuvillier, 2012) Receptor

Tissue expression

Functional expression

S1P1

S1P2

S1P3

S1P4

S1P5

Endothelial cells Smooth muscle cells Cardiomyocytes Immune cells (dendritic cells, macrophages, eosinophils, mastocytes, lymphocytes T and B, NK, NKT) Neuronal cells

Smooth muscle cells Cardiomyocytes Immune cells (dendritic cells, macrophages, eosinophils, mastocytes, NKT)

Endothelial cells Smooth muscle cells Cardiomyocytes Immune cells (dendritic cells, eosinophils, lymphocytes T) Neuronal cells Immune cells (dendritic cells, lymphocytes T, NKT) Immune cells (dendritic cells, NK) Neuronal cells (oligodendrocytes)

Angiogenesis Endothelial integrity Cardiomyocytes survival in ischemia/reperfusion Vascular tone regulation (relaxation) Lymphocyte circulation Migration Neurogenesis Cardiomyocytes survival in ischemia/reperfusion Vascular system and audition Vascular tone regulation (contraction) Neuronal excitability Inhibition of migration Mastocytes degranulation Cardiomyocytes survival in ischemia/reperfusion Vascular tone regulation (relaxation) Heart rate regulation

Maintenance of dendritic cells functions Inhibition of T cell proliferation NK circulation Oligodendrocytes survival

Table 2. S1P receptor agonists (see Figure 3 for structures) Compound

Receptor

Reference

FTY720 (Fingolimod)

S1P1 S1P3 S1P5

(Albert et al., 2005)

BAF312

S1P1 S1P3 S1P5

(Fryer et al., 2012)

ASP4058

S1P1 S1P5

(Yamamoto et al., 2014)

Compound 9f

S1P1 S1P3

(Meng et al., 2012)

CS2100

S1P1 S1P3

(Nakamura et al., 2012)

Ponesimod

S1P1

(Bigaud et al., 2014)

KRP203

S1P1

(Song et al., 2008)

AUY954

S1P1

(Pan et al., 2006)

SEW2871

S1P1

(Jo et al., 2005)

CYM5442

S1P1

(Gonzalez-Cabrera et al., 2008)

RP001

S1P1

(Cahalan et al., 2011)

AMG369

S1P1

(Cahalan, 2014).

Compound 31a

S1P1

(Skidmore et al., 2014)

SID46371153

S1P2

(Cahalan, 2014).

CYM5541

S1P3

(Guerrero et al., 2013)

ML178

S1P4

(Guerrero et al., 2012)

CYM5038 (ML248)

S1P4

(Urbano et al., 2011)

XAX162

S1P2

(Satsu et al., 2013)

CYM5520

S1P2

(Satsu et al., 2013)

Table 3. S1P receptor antagonists (see Figure 4 for structures) Compound

Receptor

Reference

VPC23019

S1P1 S1P3

(Davis et al., 2005)

VPC44116

S1P1

(Foss Jr. et al., 2007)

TASP0277308

S1P1

(Fujii et al., 2011)

NIBR0213

S1P1

(Quancard et al., 2012)

ML056 (R)-W146

S1P1

(Rosen et al., 2013; Tarrason et al., 2011)

(S)-FTY720 vinylphosphonate

S1P1 S1P3 S1P4

(Valentine et al., 2010)

JTE013

S1P2

(Osada et al., 2002); in combination with SK1-I, see (Chen et al., 2015)

Compound 22

S1P2

(Kusumi et al., 2015)

TY52156

S1P3

(Murakami et al., 2010)

SPM242

S1P3

(Jo et al., 2012)

ML131

S1P4

(Oldstone et al., 2010)