Sphingolipids and Membrane Targets for Therapeutics

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Sphingolipids and membrane targets for therapeutics

1 2 3

Robbie Loewith , Howard Riezman and Nicolas Winssinger

Lipids and membranes are often strongly altered in various [1]. Therefore, membrane lipids are likely to play impor-

diseases and pathologies, but are not often targeted for tant roles in regulating protein function, including trans-

therapeutic advantage. In particular, the sphingolipids are membrane and peripheral membrane proteins, as well as

particularly sensitive to altered physiology and have been regulating membrane properties required for membrane

implicated as important players in not only several rare deformation and vesicular trafﬁcking. Because of their

hereditary diseases, but also other major pathologies, including roles in recruiting proteins to membranes and regulating

cancer. This review discusses some potential targets in the important cellular events like transcription and signal

sphingolipid pathway and describes how the initial drug transduction, lipids and the proteins they target could

compounds have been evolved to create potentially improved be prime targets for therapeutic intervention. The three

therapeutics. This reveals how lipids and their interactions with major classes of lipids are the glycerophospholipids,

proteins can be used for therapeutic advantage. We also sphingolipids, and sterols. Many studies and drugs have

discuss the possibility that modiﬁcation of the physical targeted the cholesterol synthesis pathway as hypercho-

properties of membranes could also affect intracellular lesterolemia has been associated with increased risk of

signaling and be of therapeutic interest. cardiovascular disease [2]. Among the glycerophospholi-

pids, only phosphoinositides have been extensively

Addresses addressed. The inhibition of PI-3 kinases has been devel-

Department of Molecular Biology, NCCR Chemical Biology, University

oped to treat cancer [3]. Here, we will discuss some

of Geneva, 30 quai Ernest Ansermet, CH-1205 Geneva, Switzerland

2 aspects of sphingolipid biosynthesis as targets and their

Department of Biochemistry, NCCR Chemical Biology, University of

Geneva, 30 quai Ernest Ansermet, CH-1205 Geneva, Switzerland roles in membranes, since they are modulated in a large

Department of Organic Chemistry, NCCR Chemical Biology, University number of diseases and mutations affecting their metab-

of Geneva, 30 quai Ernest Ansermet, CH-1205 Geneva, Switzerland

olism have been shown to cause disease. Sphingolipid

analogs are therapeutics because of their bioactive prop-

Corresponding authors:

erties. However, sphingolipids are also important lipids

Loewith, Robbie ([email protected]),

Riezman, Howard ([email protected]), that regulate membrane properties such as viscosity and

Winssinger, Nicolas ([email protected]) tension, which might also make them suitable as novel

targets for therapeutic intervention. Sphingolipid degra-

dation also reveals therapeutic targets [4], but this will not

Current Opinion in Chemical Biology 2019, 50:19–28

be addressed here. Finally, we will explore the

This review comes from a themed issue on Next generation

burgeoning idea that membranes per se may serve as

therapeutics

clinically relevant drug targets.

Edited by Yimon Aye and Paul J Hergenrother

For a complete overview see the Issue and the Editorial

The sphingolipid biosynthesis pathway begins with the

Available online 18th March 2019 formation of 3-ketosphinganine from serine and palmi-

https://doi.org/10.1016/j.cbpa.2019.02.015 toyl-CoA (Figure 1) by serine palmitoyltransferase (SPT).

SPT is composed of two major subunits and several small

subunits that are most likely involved in its regulation and

speciﬁcity. The enzyme is also regulated through the

action of ORM proteins, which inhibit SPT activity.

Myriocin, a natural product which was originally identi-

ﬁed with anti-fungal activity [5], has been shown to

inhibit sphingolipid biosynthesis [6], affecting intracellu-

Membrane sphingolipids as targets lar transport of glycosylphosphatidylinositol anchored

Lipids are essential for life, providing a physical mem- proteins, before SPT was identiﬁed as the target [7].

brane barrier between the interior and exterior of cells as As sphingolipids, especially ceramides and glucosylcer-

well as between intracellular compartments. Lipids are amides accumulate in a wide variety of diseases, sphin-

also used as physiological signaling molecules, called golipid biosynthesis is widely viewed as a therapeutic

bioactive lipids, which fulﬁl functions of intracellular target for many different indications [8] as a large number

signaling, as well as intercellular signaling. If the barrier of patents can be found in a simple search. However,

function of membrane lipids were their only function, a long-term myriocin treatment does not seem to be a

simple lipid composition would most likely be sufﬁcient. feasible therapeutic because of toxic side effects and

However, eukaryotic membranes have an enormous lipid because its action on SPT is at least partially irreversible

diversity, including thousands of individual lipid species [9]. Nevertheless, myriocin may be used to reduce

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20 Next generation therapeutics

Figure 1

serine palmitoyl CoA

SPT

alanine mycriocin

3-ketosphinganine: R = OH (1-deoxy-3-ketosphinganine: R =H) KSR

sphinganine

C14-C28 fatty acid CerS

fumonisin B1

C14-C28 fatty acid

dehydroceramide

DH-DES Ceramidase australifunglin

sphingosine R = c14-c28 fatty acid

SphK ceramide

phosphatidylcholine UDP-glucose

SMS UGGC

sphingosine-1-phosphate sphingomyelin + diacylglycerol (S1P) glucoceramide + UDP miglustat

Current Opinion in Chemical Biology

Sphingolipid biosynthesis pathway. Only steps up until the formation of the first complex sphingolipids are shown. The sites of action of some

inhibitors of the pathway are shown.

ischemia-reperfusion injury [10] as a short-term applica- Despite a strong preference for serine, SPT can also

tion would be possible. It should be possible to ﬁnd novel accept alanine or glycine as substrates, which lead to

inhibitors of SPT that have less toxicity, especially if the production of 1-deoxysphingolipids [14]. Mutations

reversible, and these could be used for a large number of in SPT that decrease the preference for serine and

indications where ceramides and sphingolipids are accu- increase the production of 1-deoxysphingolipids are at

mulated leading to unwanted side effects. Inhibition of the origin of the rare disease, Hereditary Sensory and

SPT can be looked at as similar to inhibition of Autonomic Neuropapthy type 1 [15 ]. The exact mecha-

cholesterol production, where statins lower the amounts nism of neurotoxicity of 1-deoxysphingolipids is still

of an essential lipid by interfering at a key step early in the unknown, but understanding of the enzymology under-

pathway. Both SPT [11] and HMG-CoA reductase [12] lying the disease has led to a strategy to improve the

are important regulatory steps in cells. Another similarity quality of life in HSAN type I patients [16] by increasing

between SPT inhibition and HMG-CoA reductase inhi- serine in the diet.

bition is that downstream products, although essential,

can also be obtained through the diet [13], which can After SPT action, 3-ketosphinganine is converted to

temper the effect of inhibition of synthesis. sphinganine by 3-ketosphinganine reductase (KSR),

Current Opinion in Chemical Biology 2019, 50:19–28 www.sciencedirect.com

Sphingolipids and membrane targets for therapeutics Loewith, Riezman and Winssinger 21

which together with acyl CoA is used by multiple inhibitors for fatty hepatocellular carcinoma and per-

ceramide synthases (CerS) to form dihydroceramide. haps other lipid-driven cancers.

Mammals have 6 CerS which differ in their substrate

speciﬁcity, best described for their acyl chain length Miglustat (Zavesca, N-butyl-deoxynojirimycin: NB-DNJ,

preferences [17]. Fairly general inhibitors of ceramide Figure 2), an alkyl iminosugar which mimics the transi-

synthases, fumonisin B1 [18] and australifungin [19], tion state of the cationic intermediate in the glycosylation

have been identiﬁed, but inhibitors for speciﬁc reaction, prevents the accumulation of glucosylceramide.

ceramide synthases could be of therapeutic interest. Miglustat is a competitive inhibitor of glucosylceramide

Dihydroceramide is then desaturated by dihydrocera- synthase. It has been approved for the treatment of Type

mide desaturase (DH-DES) to form ceramide. Many I Gaucher’s disease and Niemann–Pick disease. Interest-

biological properties of ceramides, including the ability ingly, iminosugars can also bind glucocerebrosidase and

to induce apoptosis are dependent upon this desatura- act as pharmacological chaperones. Speciﬁcally, an analog

tion [20]. Therefore, DH-DES has been targeted by of miglustat (N-nonyl-deoxynojirimycin, NN-DNJ) was

small molecule inhibitors [21]. Both dihydroceramides shown to rescue lysosomal b-glucosidase activity of

and ceramides can be transported from their site of N370S, a mutant that is prevalent in Gaucher disease,

synthesis in the endoplasmic reticulum to the Golgi by stabilizing the protein and enhancing its trafﬁcking to

compartment [1] where they are converted to sphingo- the lysosome [23]. As such, it is a prominent example of a

myelin by sphingomyelin synthase (SMS) or glucosyl- corrector for metabolic disease. An alternative strategy

ceramide by glucosylceramide synthase (UCGC). In also leveraged on substrate analogs, included functionali-

most cell types sphingomyelin is more abundant than ties mimicking ceramide [24], led to the discovery of

glucosylceramide, but the latter is used to form a large eliglustat (Genz-112638) [25].

series of complex glycosphingolipids with various roles

in development, cell–cell interactions and as targets for Further drug development on this target led researchers at

virus and toxin entry. Inhibition of glucosylceramide Genzyme to the discovery of heterocyclic pharmacophore

synthesis by miglustat is used for the treatment of the deprived of conformationally ﬂexible alkyl chains

rare diseases where cholesterol and glycosphingolipids (Genz-667161 and Genz-682452). Genetic evidence of a

accumulate in endosomes and lysosomes (see below). correlation between Gaucher disease and the synucleino-

Inhibition of SPT or UCGC were also shown in a mouse pathies Parkinson disease [26] as well as Fabry disease

model to prevent appearance of hepatocellular (a glycosphingolipid storage disorder caused by the deﬁcient

carcinoma nodules in a mTORC2-driven cancer model activity of alpha-galactosidase A with cerebrovascular com-

[22 ]. In the latter model, SPT was inhibited using plication) generated an impetus to discover novel brain

myriocin treatment, whereas the UCGC was inhibited permeant glucosylceramide synthase inhibitors (miglustat

using shRNA encoded on a viral vector. This study has low brain penetration) [27]. Genz-667161 was recently

indicates a therapeutic potential for SPT and UCGC shown to reduce levels of glucosylceramide and

Figure 2

First-generation glucosylceramide synthase inhibitor Second-generation glucosylceramide synthase inhibitor

Genz-667161

AMP-DNM

miglustat (transition state mimetic for glucosyl synthase)

Genz-682452 eliglustat (competitive inhibitor with structure combining key EXEL-0346 element of ceramide and a cyclic amine in lieu of glucosyl donor)

Current Opinion in Chemical Biology

Structure of representative examples of glucoceramide synthase inhibitors.

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22 Next generation therapeutics

glucosylsphingosine in the central nervous system (CNS) of the treatment of multiple sclerosis and its applicability to

a mouse model, demonstrating target engagement. Further- other indications requiring immunomodulation prompted

more,treatment withGenz-667161 slowedthe accumulation signiﬁcant medicinal chemistry efforts to develop next gen-

of hippocampal aggregates of alpha-synuclein, ubiquitin, eration inhibitors. Notably, efforts to design compounds that

and tau, and improved the associated memory deﬁcits [26]. would not require metabolic processing (phosphorylation by

sphingosine kinase) led, for example, to the development of

In parallel, an optimized iminosugar (AMP-DNM, Figure 2), an azetidine carboxylate as a surrogate for the amino phos-

a more potent glucosylceramide synthase inhibitor than phate head group of FTY720-P. This azetidine carboxylate

structurally related miglustat, has been shown to counteract was used in many inhibitors including the clinical candidate

TNF-alpha-induced abnormalities in glycosphingolipid BAF312 (siponimod) which was recently shown to be effec-

concentrations and reverse abnormalities in insulin signal tive in a phase III trial in patients with secondary progressive

transduction [28]. A high throughput screening campaign at multiple sclerosis (SPMS) [33]. Another characteristic of

Exelixis led to the discovery of a different pharmacophore second-generation inhibitors was the replacement of the

which, following hit optimization, resulted in a low nanomolar unstructured alkyl chain of FTY720 with aryl or heteroaro-

inhibitor of glucosylceramide synthase (EXEL-0346) [29]. matic groups. Compound development wasalso aided by the

co-crystal structure of FTY720-P with S1P receptor [34 ].

Signaling sphingolipids The structure provided a detailed view of the molecular

The role of lipids as signaling molecules has long been recognition and hydrophobic volume required for activation

recognized and their role as messengers in cellular pro- of the S1P receptor. It is interesting to note that access to the

cesses, including cell proliferation, apoptosis, metabo- binding pocket from the extracellular environment is

lism, and migration is well established. Key lipid- blocked by the amino terminus and extracellular loops of

modifying enzymes respond to extracellular signals such the receptor. Access is gained by the ligand diffusing in the

as growth factors, cytokines or nutrients by changing the phospholipid bilayer and entering laterally between helices

composition of these signaling lipids in a complex within the transmembrane region of the receptor [34 ].

network harboring multiple nodes of interactions and Further medicinal chemistry optimization demonstrated

cross-regulatory mechanisms. Imbalances in this network that it was possible to obtain a direct agonist of S1P without

contribute to inﬂammation, cancer and metabolic disor- a polar acid head group such as in ozanimod which only

ders amongst other diseases [30]. Despite the apprecia- contains a polar amino alcohol head group. Notably, this

tion for their important role, the nondrug-like properties compound displayedexcellent selectivity againstS1Precep-

of signaling lipids has deterred and hampered tor type1 and S1P receptor type 5 versus S1P receptor type

pharmacological approaches to correct imbalances or ago- 3 [35]. The type 3 receptor is associated with toxicity.

nize/antagonize key signaling lipids, favoring drug

development on target classes perceived as more drug- While the majority of compounds designed against S1P

gable. The pursuit of small molecules constrained by receptor type 1 also target other subtypes of S1P recep-

some aspects of the ‘rules of ﬁve’ [31] made lipids tors, none show strong binding to S1P receptor type

somewhat outliers in the world of drug discovery. 2. Genetic evidence suggests that selectively targeting

this receptor subtype may prove beneﬁcial for the

Nonetheless, there have been notable achievements in treatment of hypertension and targeted efforts toward

the ﬁeld which should encourage more medicinal che- S1P receptor type 2 antagonists yielded the discovery of

mists to venture in the less chartered area of lipid signal- an orally available antagonist [36].

ing. A common thread through these efforts is that the

ﬁrst-generation inhibitors are generally based on a sub- As an alternative approach to targeting S1P receptors,

strate mimetic with relatively poor potency and selectiv- inhibition of sphingosine kinase has also been pursued.

ity. However, these inhibitors play a crucial role in S1P is produced by sphingosine kinase (two subtypes in

establishing the drugability of the target. Accrued efforts mammals: SphK1 and SphK2). Furthermore, in addition

on a given target yields more drug-like small molecules, to the well characterized interaction of S1P with cell

which are chemically more distinct from the substrate and surface S1P receptors, there is mounting evidence that

provide better efﬁcacy. S1P also has intracellular targets [37]. SphK is a key

regulator of S1P levels and the S1P:Sph/ceramide ratio.

The fortuitous discovery of FTY720 (ﬁngolimod, Figure 3) Also, ceramide synthase2 (CerS2) is inhibited by S1P [38].

as an immunosuppressant and the latter discovery that its S1P has also been reported to regulate the histone acety-

phosphorylated metabolite (FTY720-P) binds to S1P recep- lation in the nucleus [39]. Increases in S1P levels have

tor, thus promoting receptor internalization and degradation, been linked to diseases including sickle cell disease,

instigated tremendous research in the area [32]. The immu- cancer, and ﬁbrosis. The development of SphK inhibitors

nosuppressive effect stems from the depletion of S1P recep- parallels those of S1P receptor agonists with the ﬁrst

tor on lymphocytes resulting in their sequestration in generation inhibitors (such as N,N-dimethylsphingosine:

lymphoid organs [19]. The clinical success of FTY720 for DMS [40] and SK1-I [41]) closely mimicking the

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Sphingolipids and membrane targets for therapeutics Loewith, Riezman and Winssinger 23

Figure 3

O HO H2N OH C H 6 13 O OH O aliphatic region head group P O OH

myriocin (natrual product, lead OH

OH H N

SPK2 2 compound with off target activity) H N

2 OH

simplified analog OH

FTY720-p: active metabolite FTY720 (fingolimod) S1P ligand (prodrug) medicinal chemistry

O N O N O N F3C N OH N O more rigidmimic of the aliphatic region OH N azetine carboxylate

H BAF312 (siponimod) as a surrogate of the

RPC1063 (ozanimod) amino phosphate

S1P1 and S1P5 selective agonist headgroup HO Selective S1P2 antagonist

F H N N O

O F O

CO2H

co-crystal structure of FTY720 with S1P11 receptor

OH screening of S OH synthetic compound NH OH libraries N OH HN

N Cl

SK1-I (selective inhibitor) DMS (off target inhibition of PKC) OH SK1-II competitive inhibitor of SphK1 medicinal chemistry optimization

N H N Cl O

ABC294640: selective SphK1 inhibitor Second generation SphK inhibitor H OH N S

N N Amgen 82: potent dual HO SphK1-2 inhibitor

CF conformationally constained 3

mimics of S1P head group O O OH S O

N PF543: potent and selective SphK1 inhibitor co-crystal structure of PF543 with SphK1 co-crystal structure of SP with SphK1

Current Opinion in Chemical Biology

Representative examples of S1P modulators (top) and SphK inhibitors (bottom).

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24 Next generation therapeutics

À5 À4

structure of sphingosine and acting as competitive inhi- conﬁnes, between 10 N/m to 5 Â 10 N/m, with cell

À2

bitors. Screening of synthetic compound libraries led to rupture occurring at tensions >10 N/m [50–53]. Con-

the discovery of SK1-II with a drug-like heterocyclic core sistently, all cell types analyzed were found to actively

[42]. Further optimization of the compound yielded counteract perturbations in plasma membrane tension

ABC294640 [43], a ﬁrst-in-class orally available inhibitor demonstrating that this is indeed a tightly controlled

of SK2, which is currently in clinical trials (Phase I, biophysical parameter [54–56]. Tension per se can serve

patients with solid tumors) [44]. The publication of co- to integrate and transfer information within cells and

crystal structure of SphK1 with S1P [45] and ﬁrst genera- tissues [57–59], although recent studies have unexpect-

tion inhibitor SK1-II enabled structure-based design of edly suggested that membrane tension may, in fact, only

potent second generation inhibitors such as Amgen 82 be transmitted over very short distances in vivo [60,61].

(dual SphK1-2 inhibitor) wherein the hydroxylated piper- How plasma membrane tension is sensed and regulated

idine head group mimics the head group of S1P. remains poorly understood, due, in large part, to a lack of

Researchers at Pﬁzer identiﬁed the most potent SphK1 appropriate tools and to the inherent challenges incurred

inhibitor (PF-543) by combining fragments from two by the interdisciplinary approaches needed to address

different screening hits [46 ]. The co-crystal structure these questions. More egregious is our lack of under-

of this inhibitor with SphK1 clearly show a good overlap standing of tension in endo-membranes, which are, of

between the positioning of the aryl groups of PF-543 and course, even less experimentally accessible.

the alkyl chain of S1P as well as the respective head

groups [47]. As shown by caveolinopathies, loss of membrane tension

homeostasis seems to lead to disease. Caveolae are small

Membranes as drug targets pits found in the plasma membrane of many mammalian

Thus far we have highlighted the roles of sphingolipids cells. Their core structural protein components are the

species in signaling and disease. Using small molecules to caveolins and cavins while the Eps15 homology domain

target the biosynthetic enzymes that generate these lipids (EHD) proteins and pacsin/Syndapin proteins are addi-

or the protein interfaces that interact with these lipids is tionally required for efﬁcient caveolae formation/stability.

an obvious, but under developed, avenue of future drug Adipocytes, endothelial cells and muscle cells show a

design. Indeed, targeting proteins is presently common particularly high density of caveolae which may sequester

practice and of the 1578 FDA-approved drugs, 96% target up to 50% of total membrane surface area. Consistent

proteins [37]. However, there is no a priori reason to limit with this observation, caveolae are now believed to play a

drug-target-space to proteins. Lipids, like amino acids, major role in mechanoprotection, although other tissue

also assemble into macromolecular structures such as lipid speciﬁc functions have also been proposed [62 ]. Direct

droplets and membranes. In this ﬁnal section we ask: measurements demonstrated that caveolae-ﬂattening

might these lipid-based macromolecular structures also helps buffer plasma membrane tension increases in cells

be directly targetable for therapeutic gain? experiencing hypo-osmotic shock [53]. Furthermore, rel-

ative to controls, cells and tissues lacking caveolae present

As elaborated above, cells dynamically adjust both the extensive membrane damage upon exposure to physical

chemical composition as well as biophysical aspects of stress. Patients harboring mutations that affect caveolae

their membranes. Membrane lipid composition varies formation present various diseases (caveolinopathies)

between organisms, cell types, organelles, leaﬂets and including muscular dystrophies, cardiomyopathies, pul-

even within a leaﬂet by partitioning into subdomains and monary arterial hypertension and glaucoma — diseases

how these speciﬁc chemical compositions affect the func- linked by the fact that cells in affected tissues are highly

tion of membrane proteins is only now starting to be susceptibility to plasma membrane rupture [62 ,63]. Col-

addressed. This is an important avenue of research as loss lectively, these observations demonstrate a potentially

of membrane lipid composition homeostasis is correlated causal link between loss of plasma membrane tension

with various diseases [1]. Furthermore, the discrete bio- regulation and human disease and thus provoke the

physical properties of different membranes could, in question: can membrane tension be targeted for thera-

principle, present privileged nodes for therapeutic peutic gain?

intervention.

The Target Of Rapamycin (TOR) is an atypical

Membrane tension is deﬁned as the in-plane counter- eukaryote protein kinase that assembles into two, distinct

acting force to surface expansion. In some experimental multiprotein complexes known as TORC1 and TORC2

settings tension can be measured, often by the tedious [64]. In 2012, it was reported that blocking sphingolipid

process of determining the force required to extract a biosynthesis in yeast strongly activates TORC2 but not

membrane tube from a membrane surface. In vitro, mem- TORC1 [65 ]. Furthermore, hypo-osmotic shock and

À8

brane tension is highly variable ranging from 10 N/m to physical ‘pulling’ of the plasma membrane of yeast spher-

À2

10 N/m [48,49], whereas in vivo, when measurable, oplasts also hyperactivated TORC2. Although it could

plasma membrane tension is held to much narrowing not be measured at that time, all three of these otherwise

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Sphingolipids and membrane targets for therapeutics Loewith, Riezman and Winssinger 25

orthogonal manipulations presumably caused an increase These results with PalmC, albeit in the model eukaryote

in the tension of the plasma membrane (by respectively Saccharomyces cerevisiae, lend validation to the concept that

reducing the amount of lipid that can contribute to the the biophysical properties of membranes serve as eligible

membrane, increasing turgor pressure, and, physically drug targets (in this case as a means to inhibit TORC2

stretching the membrane). TORC2 activation by these signaling). Attempting to identify drugs that target mem-

manipulations requires the translocation of the paralogous branes might sound rather unorthodox; however, we note

BAR-domain proteins Slm1 and Slm2 from caveolae-like that several clinically used drugs are thought to function

membrane invaginations known as eisosomes into plasma by altering membrane ‘ﬂuidity’. These include the

membrane domains containing TORC2 [65 ]. Con- volatile anesthetics isoﬂurane, halothane, enﬂurane, sevo-

versely, the loss of membrane tension triggered by ﬂurane, and methoxyﬂurane [68], and the antipsychotic

hyper-osmotic shock leads to TORC2 inactivation. drug chlorpromazine [69] (Figure 4 top). We note, how-

ever, that the precise mode of action of these compounds

As a means to characterize the regulatory events remains controversial [70,71]. While there is a broad array

upstream of TORC2, a high throughput screen to iden- of biophysical methods to study the effect of small

tify small molecules that antagonize TORC2 signaling molecules on proteins (afﬁnity, promotion or inhibition

was performed. This screen led to the identiﬁcation of of protein–protein interactions), it is not the case for the

palmitoyl carnitine (PalmC, Figure 4 top) [66 ]. Given impact of small molecules on membranes. The develop-

its detergent-like structure and the fact that TORC2 is ment of probes, such as Flipper-TR, that can speciﬁcally

regulated downstream of plasma membrane tension, it measure changes in the biophysical properties of mem-

was tested whether the primary target of PalmC was the branes in situ will certainly help in this direction.

yeast plasma membrane. Indeed, this appears to be the Including membranes (plasma membranes as well as

case: addition of PalmC to giant unilamellar vesicles in endomembranes) as viable targets would open new

vitro provokes an immediate increase in membrane opportunities in drug-discovery and potentially provide

surface area, and, similarly to hyper-osmotic shock, addi- an inroad to address hitherto unmet medical needs. It is

tion of PalmC to yeast cells triggers an immediate clear that some drugs partition in the membrane and, as

decrease in plasma membrane tension as readout by discussed for FTY720, gain access to their target protein

the ﬂuorescent membrane tension probe Flipper-TR through lateral diffusion within a membrane. Membrane

(ﬂuorescent lipid tension reporter) [67 ] and inhibition composition and properties may affect the partitioning

of TORC2 signaling (Figure 4 bottom). and diffusion. While these questions are difﬁcult to

Figure 4

Palm C

isoflurane enflurane chlorpromazine

PalmC Addition

Twisted Flipper-TR Planar Flipper-TR PIP - ENRICHED 2 STRUCTURE Outside MCT Outside

Inside PIP Inside 2 Sim1/2 Active Inactive TORC2 TORC2 PIP 2 PM Tension Phase Separation Decresase

Current Opinion in Chemical Biology

Membranes can be targeted by small molecules. (Top) Structure of palmitoylcarnitine (PalmC) and other small molecules that potentially interact

with membranes. (Bottom) Cartoon illustrating that PalmC intercalates into the plasma membrane of yeast cells, triggering a massive drop in

membrane tension as visualized by changes in Flipper-TR fluorescence lifetime imaging (insets). Drop in membrane tension leads to phase

separation of Phosphatidylinositol 4,5-bisphosphate (PIP2) and TORC2 inhibition. See Ref. [66 ] for details.

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26 Next generation therapeutics

sphingosine-like immunosuppressant, Isp-1/myriocin.

address with current technologies, this challenge is an

Biochem Biophys Res Commun 1995, 211:396-403.

opportunity to innovate.

8. Fox TE, Finnegan CM, Blumenthal R, Kester M: The clinical

potential of sphingolipid-based therapeutics. Cell Mol Life Sci

Conclusions 2006, 63:1017-1023.

It is abundantly clear that dysregulation of lipid biosyn- 9. Wadsworth JM, Clarke DJ, McMahon SA, Lowther JP, Beattie AE,

Langridge-Smith PR, Broughton HB, Dunn TM, Naismith JH,

thesis and the loss of membrane homeostasis lead to dire

Campopiano DJ: The chemical basis of serine

medical consequences. To better understand the molec- palmitoyltransferase inhibition by myriocin. J Am Chem Soc

2013, 135:14276-14285.

ular roots behind these lipid/membrane-related diseases

new tools to study the unique composition and 10. Reforgiato MR, Milano G, Fabrias G, Casas J, Gasco P, Paroni R,

Samaja M, Ghidoni R, Caretti A, Signorelli P: Inhibition of

biophysical properties of speciﬁc membranes are being

ceramide de novo synthesis as a postischemic strategy to

developed, such as Secondary ion mass spectrometry reduce myocardial reperfusion injury. Basic Res Cardiol 2016,

111:12.

(nanoSIMS) [72] and the Flipper-TR probe discussed

above [67 ], but more are needed. Imaging technologies, 11. Breslow DK, Weissman JS: Membranes in balance:

mechanisms of sphingolipid homeostasis. Mol Cell 2010,

like the ﬂuorsescent membrane tension probe [67 ], that

40:267-279.

can report on membrane changes would empower our

12. Brown MS, Goldstein JL: Multivalent feedback regulation of

ability to link such changes to disease states. Ultimately,

HMG CoA reductase, a control mechanism coordinating

these tools will help guide the design, characterization, isoprenoid synthesis and cell growth. J Lipid Res 1980,

21:505-517.

and, ideally clinical validation of novel therapeutics that

correct lipid imbalances and/or loss of homeostatic control 13. Vesper H, Schmelz EM, Nikolova-Karakashian MN, Dillehay DL,

Lynch DV, Merrill AH Jr: Sphingolipids in food and the

of the biophysical set points of various membranes. The

emerging importance of sphingolipids to nutrition. J Nutr

progression into the clinic and the approval of several 1999, 129:1239-1250.

small molecules targeting sphingolipid processing

14. Zitomer NC, Mitchell T, Voss KA, Bondy GS, Pruett ST, Garnier-

enzymes and receptors clearly highlight the advances Amblard EC, Liebeskind LS, Park H, Wang E, Sullards MC et al.:

Ceramide synthase inhibition by fumonisin B1 causes

that have been made. To date, there is still a lack of

accumulation of 1-deoxysphinganine: a novel category of

small molecules that target lipid transport proteins. bioactive 1-deoxysphingoid bases and 1-

deoxydihydroceramides biosynthesized by mammalian cell

lines and animals. J Biol Chem 2009, 284:4786-4795.

Conﬂict of interest statement

15. Penno A, Reilly MM, Houlden H, Laura M, Rentsch K,

Nothing declared. Niederkoﬂer V, Stoeckli ET, Nicholson G, Eichler F, Brown RH Jr

et al.: Hereditary sensory neuropathy type 1 is caused by the

accumulation of two neurotoxic sphingolipids. J Biol Chem

Acknowledgements 2010, 285:11178-11187.

We thank Margo Riggi for help with Figure 4. This work was supported by This article identiﬁes two neurotoxic sphingolipids, 1-deoxysphingolipids,

the Swiss National Centre of Competence in Research (NCCR) Chemical that accumulate in the rare disease HSAN type I due to modiﬁcation of the

enzymatic speciﬁcity of serine palmitoyltransferase.

Biology, the Swiss National Science Foundation, the European Research

Council.

16. Garofalo K, Penno A, Schmidt BP, Lee HJ, Frosch MP, von

Eckardstein A, Brown RH, Hornemann T, Eichler FS: Oral L-serine

supplementation reduces production of neurotoxic

References and recommended reading

deoxysphingolipids in mice and humans with hereditary

Papers of particular interest, published within the period of review,

sensory autonomic neuropathy type 1. J Clin Invest 2011,

have been highlighted as: 121:4735-4745.

of special interest 17. Levy M, Futerman AH: Mammalian ceramide synthases. IUBMB

of outstanding interest Life 2010, 62:347-356.

18. Merrill AH Jr, Sullards MC, Wang E, Voss KA, Riley RT:

1. Harayama T, Riezman H: Understanding the diversity of

Sphingolipid metabolism: roles in signal transduction and

membrane lipid composition. Nat Rev Mol Cell Biol 2018,

disruption by fumonisins. Environ Health Perspect 2001,

19:281-296. 109:283-289.

2. Nair P: Brown and Goldstein: the cholesterol chronicles. Proc

19. Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J,

Natl Acad Sci U S A 2013, 110:14829-14832.

Milligan J, Thornton R, Shei GJ, Card D, Keohane C et al.:

Alteration of lymphocyte trafﬁcking by sphingosine-1-

3. Pons-Tostivint E, Thibault B, Guillermet-Guibert J: Targeting PI3K

phosphate receptor agonists. Science 2002, 296:346-349.

signaling in combination cancer therapy. Trends Cancer 2017,

3:454-469.

20. Hannun YA, Obeid LM: Sphingolipids and their metabolism in

physiology and disease. Nat Rev Mol Cell Biol 2018, 19:175-191.

4. Canals D, Perry DM, Jenkins RW, Hannun YA: Drug targeting of

sphingolipid metabolism: sphingomyelinases and

21. Casasampere M, Ordonez YF, Pou A, Casas J: Inhibitors of

ceramidases. Br J Pharmacol 2011, 163:694-712.

dihydroceramide desaturase 1: therapeutic agents and

pharmacological tools to decipher the role of

5. Kluepfel D, Bagli J, Baker H, Charest MP, Kudelski A: Myriocin, a

dihydroceramides in cell biology. Chem Phys Lipids 2016,

new antifungal antibiotic from Myriococcum albomyces. J

197:33-44.

Antibiot (Tokyo) 1972, 25:109-115.

22. Guri Y, Colombi M, Dazert E, Hindupur SK, Roszik J, Moes S,

6. Horvath A, Sutterlin C, Manningkrieg U, Movva NR, Riezman H:

Jenoe P, Heim MH, Riezman I, Riezman H et al.: mTORC2

Ceramide synthesis enhances transport of Gpi-anchored

promotes tumorigenesis via lipid synthesis. Cancer Cell 2017,

proteins to the Golgi-apparatus in yeast. EMBO J 1994,

13:3687-3695. 32:807-823 e812.

A mouse hepatocellular carcinoma model system showed that mTORC2-

7. Miyake Y, Kozutsumi Y, Nakamura S, Fujita T, Kawasaki T: dependent increases in lipid synthesis proceeds and is required for

Serine palmitoyltransferase is the primary target of a development of tumors.

Current Opinion in Chemical Biology 2019, 50:19–28 www.sciencedirect.com

Sphingolipids and membrane targets for therapeutics Loewith, Riezman and Winssinger 27

23. Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW: 39. Hait NC, Allegood J, Maceyka M, Strub GM, Harikumar KB,

Chemical chaperones increase the cellular activity of N370S Singh SK, Luo C, Marmorstein R, Kordula T, Milstien S et al.:

beta-glucosidase: a therapeutic strategy for Gaucher disease. Regulation of histone acetylation in the nucleus by

Proc Natl Acad Sci U S A 2002, 99:15428-15433. sphingosine-1-phosphate. Science 2009, 325:1254-1257.

24. Lee L, Abe A, Shayman JA: Improved inhibitors of 40. Yatomi Y, Ruan FQ, Megidish T, Toyokuni T, Hakomori SI,

glucosylceramide synthase. J Biol Chem 1999, 274:14662-14669. Igarashi Y: N,N-dimethylsphingosine inhibition of sphingosine

kinase and sphingosine 1-phosphate activity in human

25. McEachern KA, Fung J, Komarnitsky S, Siegel CS, Chuang WL, platelets. Biochemistry 1996, 35:626-633.

Hutto E, Shayman JA, Grabowski GA, Aerts JMFG, Cheng SH

et al.: A speciﬁc and potent inhibitor of glucosylceramide 41. Paugh SW, Paugh BS, Rahmani M, Kapitonov D, Almenara JA,

synthase for substrate inhibition therapy of Gaucher disease. Kordula T, Milstien S, Adams JK, Zipkin RE, Grant S et al.: A

Mol Genet Metab 2007, 91:259-267. selective sphingosine kinase 1 inhibitor integrates multiple

molecular therapeutic targets in human leukemia. Blood 2008,

26. Sardi SP, Clarke J, Kinnecom C, Tamsett TJ, Li LY, Stanek LM, 112:1382-1391.

Passini MA, Grabowski GA, Schlossmacher MG, Sidman RL et al.:

CNS expression of glucocerebrosidase corrects alpha- 42. French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN,

synuclein pathology and memory in a mouse model of Eberly JL, Yun JK, Smith CD: Discovery and evaluation of

Gaucher-related synucleinopathy. Proc Natl Acad Sci U S A inhibitors of human sphingosine kinase. Cancer Res 2003,

2011, 108:12101-12106. 63:5962-5969.

27. Ashe KM, Budman E, Bangari DS, Siegel CS, Nietupski JB, 43. French KJ, Zhuang Y, Maines LW, Gao P, Wang WX, Beljanski V,

Wang B, Desnick RJ, Scheule RK, Leonard JP, Cheng SH et al.: Upson JJ, Green CL, Keller SN, Smith CD: Pharmacology and

Efﬁcacy of enzyme and substrate reduction therapy with a antitumor activity of ABC294640, a selective inhibitor of

novel antagonist of glucosylceramide synthase for Fabry sphingosine kinase-2. J Pharmacol Exp Ther 2010, 333:129-139.

disease. Mol Med 2015, 21:389-399.

44. Britten CD, Garrett-Mayer E, Chin SH, Shirai K, Ogretmen B,

28. Aerts JM, Ottenhoff R, Powlson AS, Grefhorst A, van Eijk M, Bentz TA, Brisendine A, Anderton K, Cusack SL, Maines LW et al.:

Dubbelhuis PF, Aten J, Kuipers F, Serlie MJ, Wennekes T et al.: A phase I study of ABC294640, a ﬁrst-in-class sphingosine

Pharmacological inhibition of glucosylceramide synthase kinase-2 inhibitor, in patients with advanced solid tumors. Clin

enhances insulin sensitivity. Diabetes 2007, 56:1341-1349. Cancer Res 2017, 23:4642-4650.

29. Richards S, Larson CJ, Koltun ES, Hanel A, Chan V, Nachtigall J, 45. Wang Z, Min X, Xiao SH, Johnstone S, Romanow W, Meininger D,

Harrison A, Aay N, Du HW, Arcalas A et al.: Discovery and Xu HD, Liu JS, Dai J, An SZ et al.: Molecular basis of sphingosine

characterization of an inhibitor of glucosylceramide synthase. kinase 1 substrate recognition and catalysis. Structure 2013,

J Med Chem 2012, 55:4322-4335. 21:798-809.

30. Wymann MP, Schneiter R: Lipid signalling in disease. Nat Rev 46. Schnute ME, McReynolds MD, Kasten T, Yates M, Jerome G,

Mol Cell Biol 2008, 9:162-176. Rains JW, Hall T, Chrencik J, Kraust M, Cronin CN et al.:

Modulation of cellular S1P levels with a novel, potent and

31. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ: Experimental speciﬁc inhibitor of sphingosine kinase-1. Biochem J 2012,

and computational approaches to estimate solubility and 444:79-88.

permeability in drug discovery and development settings. Adv This article reports the ﬁrst potent and selective inhibitor of SphK1 (100-

Drug Deliv Rev 1997, 23:3-25. fold selectivity over SphK2). This inhibitor highlighted that speciﬁc inhibi-

tion of SphK1 had no effect on the cellular proliferation and survival,

32. Dyckman AJ: Modulators of sphingosine-1-phosphate

despite a dramatic change in the cellular S1P/sphingosine ratio.

pathway biology: recent advances of sphingosine-1-

phosphate receptor 1 (S1P(1)) agonists and future 47. Wang J, Knapp S, Pyne NJ, Pyne S, Elkins JM: Crystal structure

perspectives. J Med Chem 2017, 60:5267-5289. of sphingosine kinase 1 with PF-543. ACS Med Chem Lett 2014,

5:1329-1333.

33. Kappos L, Bar-Or A, Cree BAC, Fox RJ, Giovannoni G, Gold R,

Vermersch P, Arnold DL, Arnould S, Scherz T et al.: Siponimod 48. Pecreaux J, Dobereiner HG, Prost J, Joanny JF, Bassereau P:

versus placebo in secondary progressive multiple sclerosis Reﬁned contour analysis of giant unilamellar vesicles. Eur Phys

(EXPAND): a double-blind, randomised, phase 3 study. Lancet J E Soft Matter 2004, 13:277-290.

2018, 391:1263-1273.

49. Morlot S, Galli V, Klein M, Chiaruttini N, Manzi J, Humbert F,

34. Hanson MA, Roth CB, Jo EJ, Grifﬁth MT, Scott FL, Reinhart G, Dinis L, Lenz M, Cappello G, Roux A: Membrane shape at the

Desale H, Clemons B, Cahalan SM, Schuerer SC et al.: Crystal edge of the dynamin helix sets location and duration of the

structure of a lipid G protein-coupled receptor. Science 2012, ﬁssion reaction. Cell 2012, 151:619-629.

335:851-855.

This structure provides a molecular rational for receptor subtype selec- 50. Dai J, Sheetz MP, Wan X, Morris CE: Membrane tension in

tivity by different agonist and is a landmark for a rational drug design. swelling and shrinking molluscan neurons. J Neurosci 1998,

18:6681-6692.

35. Scott FL, Clemons B, Brooks J, Brahmachary E, Powell R,

Dedman H, Desale HG, Timony GA, Martinborough E, Rosen H 51. Evans E, Heinrich V, Ludwig F, Rawicz W: Dynamic tension

et al.: Ozanimod (RPC1063) is a potent sphingosine-1- spectroscopy and strength of biomembranes. Biophys J 2003,

phosphate receptor-1 (S1P(1)) and receptor-5 (S1P(5)) agonist 85:2342-2350.

with autoimmune disease-modifying activity. Br J Pharmacol

2016, 173:1778-1792. 52. Raucher D, Sheetz MP: Characteristics of a membrane

reservoir buffering membrane tension. Biophys J 1999,

36. Kusumi K, Shinozaki K, Yamaura Y, Hashimoto A, Kurata H, 77:1992-2002.

Naganawa A, Ueda H, Otsuki K, Matsushita T, Sekiguchi T et al.:

Discovery of novel S1P(2) antagonists. Part 2: Improving the 53. Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D,

proﬁle of a series of 1,3-bis(aryloxy)benzene derivatives. Stan RV, Butler-Browne G, Vedie B, Johannes L et al.: Cells

Bioorg Med Chem Lett 2015, 25:4387-4392. respond to mechanical stress by rapid disassembly of

caveolae. Cell 2011, 144:402-413.

37. Santos WL, Lynch KR: Drugging sphingosine kinases. ACS

Chem Biol 2015, 10:225-233. 54. Dai J, Sheetz MP: Mechanical properties of neuronal growth

cone membranes studied by tether formation with laser

38. Laviad EL, Albee L, Pankova-Kholmyansky I, Epstein S, Park H, optical tweezers. Biophys J 1995, 68:988-996.

Merrill AH, Futerman AH: Characterization of ceramide

synthase 2: tissue distribution, substrate speciﬁcity, and 55. Lieber AD, Yehudai-Resheff S, Barnhart EL, Theriot JA, Keren K:

inhibition by sphingosine 1-phosphate. J Biol Chem 2008, Membrane tension in rapidly moving cells is determined by

283:5677-5684. cytoskeletal forces. Curr Biol 2013, 23:1409-1417.

www.sciencedirect.com Current Opinion in Chemical Biology 2019, 50:19–28

28 Next generation therapeutics

56. Morris CE, Homann U: Cell surface area regulation and First demonstration that TORC2 activity is regulated speciﬁcally by

membrane tension. J Membr Biol 2001, 179:79-102. changes in plasma membrane tension.

57. Diz-Munoz A, Thurley K, Chintamen S, Altschuler SJ, Wu LF, 66. Riggi M, Niewola-Staszkowska K, Chiaruttini N, Colom A,

Fletcher DA, Weiner OD: Membrane tension acts through PLD2 Kusmider B, Mercier V, Soleimanpour S, Stahl M, Matile S, Roux A

and mTORC2 to limit actin network assembly during et al.: Decrease in plasma membrane tension triggers PtdIns

neutrophil migration. PLoS Biol 2016, 14:e1002474. (4,5)P2 phase separation to inactivate TORC2. Nat Cell Biol

2018, 20:1043-1051.

58. Paluch E, Heisenberg CP: Biology and physics of cell shape

This work demonstrates that a membrane can be the relevant target of a

changes in development. Curr Biol 2009, 19:R790-R799.

drug-like small molecule.

59. Yu H, Mouw JK, Weaver VM: Forcing form and function:

67. Colom A, Derivery E, Soleimanpour S, Tomba C, Molin MD,

biomechanical regulation of tumor evolution. Trends Cell Biol

Sakai N, Gonzalez-Gaitan M, Matile S, Roux A: A ﬂuorescent

2011, 21:47-56.

membrane tension probe. Nat Chem 2018, 10:1118-1125.

The ﬁrst in class ﬂuorescent probe to detect membrane tension is

60. Lieber AD, Schweitzer Y, Kozlov MM, Keren K: Front-to-rear

calibrated in vitro and applied in vivo to measure membrane tension.

membrane tension gradient in rapidly moving cells. Biophys J

2015, 108:1599-1603.

68. Eger EI 2nd, Raines DE, Shafer SL, Hemmings HC Jr,

Sonner JM: Is a new paradigm needed to explain how

61. Shi Z, Graber ZT, Baumgart T, Stone HA, Cohen AE: Cell

inhaled anesthetics produce immobility? Anesth Analg 2008,

membranes resist ﬂow. Cell 2018, 175:1769-1779 e1713.

107:832-848.

62. Parton RG: Caveolae: structure, function, and relationship to

69. Pickholz M, Oliveira ON Jr, Skaf MS: Interactions of

disease. Annu Rev Cell Dev Biol 2018, 34:111-136.

chlorpromazine with phospholipid monolayers: effects of

An excellent, recent review illustrating through clinical examples how,

the ionization state of the drug. Biophys Chem 2007, by caching plasma membrane, caveolae protect cells/tissues from 125:425-434.

mechanical stress.

63. Petty HR: Frontiers of complex disease mechanisms: 70. Franks NP, Lieb WR: Molecular mechanisms of general

membrane surface tension may link genotype to phenotype in anaesthesia. Nature 1982, 300:487-493.

glaucoma. Front Cell Dev Biol 2018, 6:32.

71. Herold KF, Sanford RL, Lee W, Andersen OS, Hemmings HC Jr:

64. Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and Clinical concentrations of chemically diverse general

metabolism. Cell 2006, 124:471-484. anesthetics minimally affect lipid bilayer properties. Proc Natl

Acad Sci U S A 2017, 114:3109-3114.

65. Berchtold D, Piccolis M, Chiaruttini N, Riezman I, Riezman H,

Roux A, Walther TC, Loewith R: Plasma membrane stress 72. He C, Hu X, Weston TA, Jung RS, Sandhu J, Huang S, Heizer P,

induces relocalization of Slm proteins and activation of Kim J, Ellison R, Xu J et al.: Macrophages release plasma

TORC2 to promote sphingolipid synthesis. Nat Cell Biol 2012, membrane-derived particles rich in accessible cholesterol.

14:542-547. Proc Natl Acad Sci U S A 2018, 115:E8499-E8508.

Current Opinion in Chemical Biology 2019, 50:19–28 www.sciencedirect.com