Thus far, we have described how ceramide is generated from SM by SMases, and how an excess or defect in ceramide or sphingomyelinase activity may result in pathobiologic effects. However, ceramide and S1P have been shown to often exert opposite effects (Cuvillier O et al., 1998; Huwiler and Pfeilschifter, 2006; Taha et al., 2006b; Canals et al., 2010). There are only two enzymatic steps separating ceramide and S1P: ceramidases provide the first node of this regulatory connection, and SphK the second one. It should not be surprising that both of these enzymes have also been involved in cancer progression and inflammation (Horton, 1999; Nemoto et al., 2009; Pyne and Pyne, 2010; Snider et al., 2010). This section will discuss the ceramidases; the reader is referred to other reviews for discussion of SphKs (Taha et al., 2006a; Pitman and Pitson, 2010).
Five different human genes have been identified that encode proteins that hydrolyse ceramide to Sph and free fatty acid (N-acylsphingosine amidohydrolase). They have been grouped as acid, neutral or alkaline ceramidases (EC 184.108.40.206), depending on the optimum pH of their ceramidase activity being pH 4.5 for aCDase (gene name: ASAH1, NAAA), pH 7–9 for neutral ceramidase (nCDase, ASAH2) and pH 8.5–9.5 for alkaline ceramidases (alkCDase, three genes: ASAH3/ACER1, ACER2 and ACER3). A reverse activity has also been described for most of these ceramidases with that for aCDase showing a pH optimum slightly less acidic (5.5) than that of the forward reaction (Okino et al., 2003). Interestingly, and as predicted from their mechanism, the reverse activity of ceramidases uses a free fatty acid as a substrate, unlike ceramide synthases (EC 220.127.116.11) that use acyl-CoAs as substrates. These different ceramidases are found in diverse cellular compartments. Thus, aCDase resides in the lysosomes (Koch et al., 1996), nCDase at the PM, intracellular compartments and secreted in the intestinal lumen (El Bawab et al., 2000) and ACER family in the ER-Golgi network (Mao et al., 2001; Xu et al., 2006).
Acid ceramidase activity, as well as the reverse reaction, was first described in 1963 by Shimon Gatt using rat tissues (Gatt, 1963). In the early 1990s, human aCDase was purified to homogeneity from human urine (Bernardo et al., 1995), and a few years later, the cDNA for the gene was cloned (Li et al., 1998). aCDase has subsequently been purified and characterized from other tissues (Linke et al., 2001b). aCDase consists of a single precursor polypeptide of 53–55 kDa (Ferlinz et al., 2001) that is proteolytically processed into α- and β-subunits that migrate at 13 kDa and 40 kDa respectively. The α-subunit can be reduced to 29 KDa by N-glycanase F treatment (Koch et al., 1996). Of six individual potential N-glycosylation sites, five of them are used (Schulze et al., 2007), and some of them are required for correct lysosomal processing or enzymatic activity, and for the formation of the heterodimeric enzyme form (Ferlinz et al., 2001). Purification of human aCDase revealed that at least two β-subunits could be generated for aCDase differing by 2–4 kDa at the C-terminus (He et al., 2003). Although aCDase has been localized mainly in lysosomes, a portion of aCDase is secreted to the medium as a 47 kDa monomer (Bernardo et al., 1995).
Little is known about how the secretory form is regulated and processed. The generation of the lysosomal mature form involves cleavage of the precursor in endosonal/lysosomal compartments, as well as trafficking through the mannose-6-phosphate receptor. Interestingly, mutation of putative N-glycosylation sites did not alter the ratio of secreted : lysosomal aCDase (Ferlinz et al., 2001). The use of insect Sf21 cells overexpressing human aCDase showed that the secreted precursor can be processed to a mature form upon acidification of the cell culture supernatant to pH 4.2–4.3m causing the processing of the precursor and resulting in a homogeneous sample of mature aCDase.
aCDase and aSMase are metabolically consecutive enzymes in SM hydrolysis, and their expression and activity are regulated by each other. For example, aCDase activity enhances aSMase secretion (He et al., 2003). More recent work has shown that secreted aCDase can be found forming a multi-enzymatic complex with aSMase and β-galactosidase (He et al., 2003). The results showed strong enough interaction among these enzymes to allow co-precipitation, and there was significant specificity that other lysosomal enzymes were not detected in those complexes.
Interestingly, as seen for nSMase and aSMase, stress stimuli such as TNF-α also activate aCDase. Other stress stimuli such as UV and ionizing radiation have also been reported to induce aCDase activity. Moreover, aCDase requires anionic lysosomal lipids and SL activator proteins (saposin) as cofactors for efficient hydrolysis of ceramide in vivo (Azuma et al., 1994; Linke et al., 2001a).
Abnormal overexpression of aCDase has been related to tumour progression and protection from cell death; on the other hand, deficiency of aCDase results in Farber disease (first described in 1957 by Sidney Farber), an autosomal-recessively inherited LSD resulting in accumulation of ceramide. Farber disease is associated with distinct clinical phenotypes, involving painful swelling of the joints and tendons, pulmonary insufficiency, neurological and general deficient development and a shortened lifespan (Levade et al., 1995; Koch et al., 1996). Several point mutations and exon skipping in the aCDase gene have been identified in Farber disease. Moreover, although certain mutations in the aCDase gene mimic Farber disease, the generation of aCDase knockout mice (Li et al., 2002) resulted in embryonic lethality for the homozygous mice. Furthermore, aCDase has been implicated in other metabolic complications; for example, aCDase was shown to have a role in preventing type 2 diabetes and modulating insulin signalling (Chavez et al., 2005).
nCDase activity, and the reverse activity, were described as early as 1969 as a ceramide-cleaving activity found in humanduodenal contents, with an optimal pH of 7.6 (Nilsson, 1969), and in 1980, in microsomes from rat liver (Stoffel and Melzner, 1980). nCDases have been cloned from bacteria (Okino et al., 1999; 2010), human (El Bawab et al., 2000), mouse (Tani et al., 2000), rat (Mitsutake et al., 2001), Drosophila (Yoshimura et al., 2002), amoeba (Monjusho et al., 2003), Zebra fish (Yoshimura et al., 2004), plants (Pata et al., 2008) and fungus (Tada et al., 2009). Note that the Aspergillus and the amoeba nCDases showed an optimum acidic pH. nCDase does not share significant sequence identity with either acid or alkCDases, as well as other amidases, including proteases (Galadari et al., 2006). Northern blotting analysis also revealed that nCDase is expressed ubiquitously (El Bawab et al., 2000), but highly expressed in kidney, liver, heart (Tani et al., 2000) and intestine (Choi et al., 2003). The activity of purified intestinal rat (Olsson et al., 2004) and human (Ohlsson et al., 2008) nCDase, a glycosylated protein of molecular weight of 116 kDa, is not affected by Ca2+, Mg2+or Mn2+, but inhibited by Zn2+, Fe2+ and Cu2+ (Galadari et al., 2006). No cations are required to activate nCDase. nCDase was found to be secreted into the intestine lumen for SL digestion, being resistant to pancreatic proteases (Olsson et al., 2004; Ohlsson et al., 2008) and, as with aSMase, was found extracellularly attached to the PM, exposing the catalytic site to the intestinal lumen (Duan et al., 2007). In other tissues and in cell culture, nCDase can be secreted or localized at the PM (Hwang et al., 2005) as a type II integral membrane protein (Tani et al., 2003). In addition, it has also been localized in endosome-like structures (Mitsutake et al., 2001), and in mitochondria in MCF-7 and HEK293 cells (El Bawab et al., 2000). Thus, intestinal and intracellular nCDase are identical enzymes (Duan and Nilsson, 2009). Moreover, nCDase is also highly glycosylated. Even though deglycosylation did not affect its activity, mutants lacking the mucine box or O-glycosylation sites in the mucine box were secreted and not localized at the cell surface in HEK cells (Tani et al., 2003). This is consistent with bacterial and invertebrate nCDase, which lack the mucin box, being secreted proteins (Inoue et al., 2009). Overexpression of nCDase did not result in Sph accumulation in unstimulated human platelets, unless the PM SM was previously hydrolysed (Tani et al., 2005), suggesting that not much ceramide is found at PM in unstimulated conditions. The same conclusion was shown using combination of recombinant bacterial sphingomyelinase and recombinant bacterial ceramidase (Canals et al., 2010).
Galadari et al. identified a nCDase motif comprised of six amino acids core (GDVSPN) from a comparison of diverse nCDase sequences, and identified the serine residue (Ser 354) in that hexapeptide as the nucleophile attacking the amide bond of ceramide in the catalytic site. The mutation of that serine, as well as the aspartate or the cysteine of the hexapeptide led to complete loss of nCDase activity (Galadari et al., 2006). However, Inoue et al., based on Pseusomona aeruginosa nCDase crystal structure, suggested that Ser-354 is involved in Zn2+ binding (Inoue et al., 2009). The crystal structure of bacterial P. aeruginosa nCDase also allowed the identification of the Zn2+ and the Mg2+/Ca2+ binding sites. Modelling the rat sequence on the P. aeruginosa crystal structure, other amino acids were identified in rat nCDase to be indispensable for Zn2+ binding and formation of the active site (His 175 and Tyr 160), for catalysis (Arg 238) and for the ceramide binding site (Tyr 572). Single mutations of these residues led to the loss of rat nCDase forward and reverse activity (Inoue et al., 2009).
The substrate specificity of nCDase was studied by El Bawab et al. (El Bawab et al., 2000), finding that only the natural d-erythro-ceramide isomer was used as substrate of the four stereoisomers of ceramide. Dihydroceramide or phytoceramide forms, shortening the length of the alkyl backbone, methylation of the primary or secondary hydroxyl groups resulted in reduction or loss of the nCDase activity (el Bawab et al., 2002).
Physiologically, IL-1β increased the activity of acid, but also neutral ceramidase in rat hepatocytes (Nikolova-Karakashian et al., 1997) and in renal mesangial cells, describing not only an increase in nCDase activity but also in mRNA levels and protein synthesis. This protein induction by IL-1β was blocked by the p38 mitogen-activated protein kinase inhibitor SB 202190 (Franzen et al., 2001). Moreover, other cytokines such as TNF and interferon-gamma also increased activity, mRNA and nCDase protein synthesis, where this elevation was reported to protect cells from cytokine-induced cell death (Zhu et al., 2008).
In 1975, it was reported that the hydrolysis of ceramide was observed not only at pH 4.0 but also at pH 9.0 in normal cerebellum (Sugita et al., 1975). Acid and alkCDase activity were also found in fibroblasts and leukocytes (Dulaney et al., 1976), and in different rat tissues (Spence et al., 1986).
In a study in yeast, aimed at molecular identification of enzymes of ceramide metabolism, Mao et al. identified two novel alkaline ceramidases (YPC1 and YDC1). These then became the founding members of a novel family of alkCDases. Indeed, three different genes comprise the known human alkCDases, ACER1, 2 and 3, which contain 264 (Sun et al., 2008), 275 (Xu et al., 2006) and 267 (Mao et al., 2001) amino acid, respectively, with a similar molecular weight around 31 kDa. All of them present multiple transmembrane domains. ACER1 is localized in the ER and is mainly expressed in the skin (Mao et al., 2003). ACER2 is localized in the Golgi apparatus and is highly expressed in the placenta and modestly expressed in many other tissues. ACER3 is localized in both ER and Golgi apparatus, and is highly expressed in most tissues, especially in the placenta (Mao and Obeid, 2008). All three ACER activities are enhanced by Ca2+ (Mao et al., 2001).
The substrate specificity among aCDases, nCDases and alkCDases was first studied in 1982, finding short acyl-chain ceramides (i.e. C16 acyl chain) being poor substrates for acid, but not for neutral and alkCDases. Both, alkaline and nCDase have been reported to be inhibited by phosphatidylcholine and SM (Yada et al., 1995).
Recently, plasma S1P has been reported to derive from Sph generated from alkCDase activity in erythrocytes. Moreover, this activity is the only ceramidase activity found in erythrocytes. Furthermore, alkCDase has also been shown to be important for erythroid differentiation inK562 erythroleukaemic cells (Xu et al., 2010).
ACER1 is upregulated in epidermal keratinocytes in response to increasing the concentration of Ca2+ in tissue culture medium. Using RNAi technology, ACER1 has been implicated in mediating extracellular Ca2+-induced differentiation of human keratinocytes. The mechanism by which ACER1 mediates keratinocyte differentiation remains unclear although an increase in the generation of Sph, S1P or both may be involved. ACER2 has been shown to be upregulated in tumours, and its upregulation promotes tumour cell proliferation and survival likely through increasing the generation of S1P and activating the S1P receptor S1P1. Interestingly, over-expression of ACER2 may also result in cell proliferation inhibition or cell death due to an accumulation of Sph, which is highly cytotoxic. ACER2 has also been implicated in the regulation ofβ1 integrin maturation and cell adhesion (Sun et al., 2009).
The cytotoxic compound retinoid N- (4-hydroxyphenyl)retinamide (4-HPR), an inhibitor of dihydroceramide desaturaseactivity (Kraveka et al., 2007), has recently been shown to increase ACER2 activity and mRNA, but not ACER 1 or 3. Over-expression of ACER2 (but neither ACER3, nor aCDase nor nCDase) enhanced 4-HPR-induced (same with GT11 another dihydorceramide desaturase inhibitor) dihydroSph formation, and cell death (Mao et al., 2010), suggesting that ACER2 also regulates the levels of dihdyrosphingosine (DHS) as well as DHS-mediated cell death by controling the hydrolysis of certain dihydroceramides with unsaturated acyl chains. ACER3 prefers unsaturated long-chain ceramides, minor ceramide species in mammalian cells and tissues, so ACER3 knockdown results in an increase in the levels of unsaturated long-chain ceramides (d-e-C18:1-ceramide and d-e-C20:1-ceramide) in tumour cells. Interestingly, ACER3 down-regulation decreases the levels of other ceramide species while increasing the levels of both SPH and S1P by increasing the expression of ACER2, which hydrolyses most mammalian ceramide species. ACER3 knockdown inhibited cell proliferation by up-regulation of the cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), but also inhibited serum-deprivation induced apoptosis (Hu et al., 2010).
The physiologic regulation of ceramidase activity depends on the specific ceramidase (aCDase, nCDase or alkaline) and their localization in the cell. Thus, aCDase has been the most studied ceramidase, shown to be inhibited by anionic lipids such as phosphatidic acid (PA) and phosphatidylserine (PS) for the forward reaction, but these lipids appear to promote the reverse reaction (El Bawab et al., 1999). Conversely, SM activates aCDase in the forward reaction, inhibiting the reverse activity. Zinc cations strongly inhibit the reverse activity. In addition, the product of the forward activity, Sph and oleic acid, as well as other complex SLs such as HexCers have been found to inhibit forward ceramidase activity (Sugita et al., 1975). Furthermore, pH also plays an important regulatory role in ceramidase activity as aCDase can reach both endosome-lysosomal and extracellular matrix compartments. Not only the optimum pH of the enzyme is important for its activity, but also the processing requires low pH to produce the mature form, as seen before.
In vitro studies have shown that nCDase activity was inhibited by reducing agents such as dithiothreitol ans β-mercaptoethanol (Galadari et al., 2006). Natural Sph acted as a competitive inhibitor, and the reverse activity of nCDase was inhibited by L-erythro-Sph and myristaldehyde in a competitive mechanism (El Bawab et al., 2001). Phosphatidic acid and cardiolipin showed moderate inhibition as well. Interestingly, cardiolipin enhanced ceramidase activity, and, importantly, the reverse activity was not inhibited by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase enzymes (El Bawab et al., 2001). As seen for aSMase, low concentrations of cholesterol also inhibited intestinal nCDase (Ohlsson et al., 2007).
Synthetic ceramidases inhibitors
The design and screening of structural analogues of the sphingoid bases and ceramides has led to a few specific inhibitors for ceramidases.
The first aCDase inhibitor described was N-oleoylethanolamine (NOE) (Sugita et al., 1975), now better known as an endocannabinoid-related molecule, and has been one of the most used in vitro inhibitors for aCDase. In vitrostudies showed that NOE increased ceramide levels and enhanced apoptosis in L929 cells (Strelow et al., 2000), glioma cells (Hara et al., 2004), primary placenta trophoblast (Payne et al., 1999), and dendritic cells (Kanto et al., 2001). Although those effects were attributed to an increase in ceramide levels, they cannot be specificallyattributed to inhibition of aCDase, as NOE, afterwards, was found to inhibit ceramide glucosylation in neuroepithelioma cells as well (Spinedi et al., 1999). Furthermore, structural analogs of NOE were explored as aCDase inhibitors by Fabrias G (Grijalvo et al., 2006), defining NOE as a weak aCDase inhibitorin vitro and in vivo. Thus, NOE structure was improved for the development of aCDase inhibitors using shorter oxoacyl groups, adding the missing sphingoid alkyl tail and adding also a C3 hydroxyl group. Although the (E)-4,5 double bound was not required for inhibition, a (Z)-4,5 geometry was found to revert the inhibition. Due to its lack of selectivity (NOE can also inhibit neutral and alkaline ceramidase activities), and low potency, the canonical structure of NOE has been avoided for therapeutic use, although structural analogues of it are current candidates for aCDase inhibitors.
Not long after, another ceramidase inhibitor was developed; d-e-MAPP [(1S, 2R)-D-erythro-2- (N-Myristoylamino)-1-phenyl-1-propanol], was synthesized by Bielawska et al. as a ceramide analogue, causing cell cycle arrest in HL-60 cells, elevating intracellular ceramide levels up to 3-fold. In vitro studies showed that d-e-MAPP inhibited, at low micromolar concentrations, alkaline and neutral ceramidase, but had no effect on aCDase activity. Moreover, its enantiomer L-e-MAPP, although undergoing similar cellular uptake, was metabolized and had no effect on ceramidase activity (Bielawska et al., 1996). A recent study demonstrated that d-e-MAPP inhibits cellular activities of ACER1-3. Other authors have also reported increases in cellular ceramide levels after d-e-MAPP treatment (Raisova et al., 2002; Rodriguez-Lafrasse et al., 2002; Alphonse et al., 2004), and this increase in ceramide levels by d-e-MAPP has been involved in apoptosis induction (Rodriguez-Lafrasse et al., 2002; Choi et al., 2003; Lepine et al., 2004) and in radiation treatment, overcoming the resistance to radiation of cancer cells over-expressing ceramidases. Furthermore, d-e-MAPP was seen to block the mitogenic effect of oxidized low density lipoprotein (oxLDL), suggesting S1P as the responsible molecule of the mitogenic effect, coming from LDL-ceramide (Auge et al., 1999). Other pathways requiring S1P were also blocked by d-e-MAPP, such as DNA synthesis in smooth muscle cells (Maupas-Schwalm et al., 2004). In other cases, an acid ceramidase inhibitor was required to induce apoptosis, where d-e-MAPP had no effect on induction of apoptosis, although ceramide levels increased in both treatments (Payne et al., 1999). In fact, d-e-MAPP has been shown to protect cells from apoptosis in response to serum deprivation, the synthetic glucocorticoid dexamethasone, and the herbicide paraquat by preventing the production of Sph. d-e-MAPP resulted in blockage of progestin and adiponectinreceptor (PAQR), involving SLs, and ceramidases downstream of PAQR signalling, an important hormone receptor related to pathological conditions, including obesity, diabetes and coronary artery disease (Kupchak et al., 2009). Other studies have shown d-e-MAPP, as well as exogenous short length acyl-ceramides, to enhance the A23187 (Ca2+ionophore)-induced release of arachidonic acid, associated with an increase of endogenous ceramide accumulation (Shimizu et al., 2009). Modifications of the structure of d-e-MAPP led to B13 ((1R,2R)-2- (N-tetradecanoylamino)-1- (4-nitrophenyl)-1,3- propanediol), a more water soluble form. Interestingly, B13,as shown for d-e-MAPP, also increased intracellular levels of ceramide, but unlike d-e-MAPP, B13 was shown to be an inhibitor of acid ceramidase (Bielawska et al., 2008).
Although B13 showed a high potency in aCDase inhibition in vitro, the effect on the enzyme in vivo may not be direct; its neutral nature makes it difficult to accumulate in the lysosome. Thus, B13 was taken as a scaffold to design new ceramidase inhibitor structures, and the uptake by the lysosome was enhanced with a series of lysomotrophic molecules such as LCL204 (also known as Ad 2646). Thus, LCL204 significantly decreased cell migration caused by over-expressing aCDase, and it also sensitized head and neck cancer cells to FAS induced apoptosis.LCL204 overcame the resistance to apoptin mediated by the over-expression of aCDase in DU145, PC-3 and LNCaP cells. However, LCL204, although targeted to the lysosomes, was found to eventually destroy the lysosome and to cause degradation of aCDase by cathepsin B/L, and it also inhibited aSMase (Holman et al., 2008), as previously was observed with desipramine. A new family of LCL204 analogues, which also target the lysosome without destabilizing them and without inducing degradation of aCDase were developed, and these include LCL 433, 449, 463, 464, 488 and LCL506 (Bai et al., 2009). Another B13 analogue, LCL385 (Mahdy et al., 2009) showed aCDase inhibition in vivo, with similar effects as seen with knocking down aCDase, sensitizing prostatic cancer cells to radiation, and reducing xenografts tumour growth.
A series of modifications in ceramide and Sph were tested on nCDase activity, all stereoisomers of d-erythro-ceramide and Sph (L-threo, d-threo, and L-erythro isomers), N-methyl-D-erythro-Sph and d-erythro-urea-C16-ceramide showed significant inhibitory effects, whereas other ceramide and Sph analogues such as N-methyl ceramide, 1-O-methyl ceramide, cis-D-erythro ceramide or d N,N-dimethyl-D-erythro-sphingosine had no effect. Of note, C1P and S1P stimulated the enzyme (Usta et al., 2001). Diverse detergents such as taurocholate (TC), taurodeoxycholate (TDC), glycodeoxycholate (GDC), and (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) (CHAPS)inhibited in vitro nCDase activity (Ohlsson et al., 2007).
Functional effects of inhibitors of ceramidases
Together with aSMase, aCDase has also been related to the pathogenesis of cystic fibrosis, suggesting ceramide accumulation mediating inflammation and cell death in lungs, and enhancing bacterial infection which is the major cause of mortality in CF patients. Thus, CF patients treated with amitriptyline, another tricyclic antidepressant, showed improvement in their lung function (Riethmuller et al., 2009).
ACDase is involved in many cancers such as breast cancer (Ruckhaberle et al., 2008; 2009), prostate cancer (Saad et al., 2007; Holman et al., 2008; Mahdy et al., 2009), leukaemia (Furlong et al., 2008; Shah et al., 2008), colon cancer (Selzner et al., 2001), head and neck squamous cell tumours (Elojeimy et al., 2007), and individuals developing asbestos-induced malignant pleural mesothelioma (Archimandriti et al., 2009). Therefore, one of the important goals to control cancer progression has become the design of drugs targeting aCDase. Endogenous over-expression of aCDase has been shown to reduce ceramide levels and increase S1P levels, and both effects have been related to stimulate cancer progression (Huwiler and Pfeilschifter, 2006). Moreover, resistance to radiation and/or chemotherapy used in cancer treatment has been showed to be caused by induction of aCDase synthesis in some cancer cell culture models (Mahdy et al., 2009), thus pharmacological inhibition of aCDase sensitizes cancer cells to radiation and chemotherapy. The aCDase inhibitor B13 induced up to 90% apoptosis in colon cancer cells (Liu et al., 2008) and reduction of colon tumor growth in nude mice (Selzner et al., 2001).Furthermore, theB13 analogue, LCL 385, sensitized prostate cancer cells to radiation, and also decreased growth of xenograft tumour (Mahdy et al., 2009),and the analogue LCL204 sensitized head and neck squamous cell tumours to Fas-induced cell death in both in vitro an in vivo systems (Elojeimy et al., 2007).
On the other hand, the role of nCDase in cancer has not been studied. However, nCDase has been implicated in protective role in inflammation (Franzen et al., 2001) and pro-proliferation, as down-regulation of nCDase led to cell cycle arrest. ACER2 up-regulation has been implicated in tumour cell proliferation and survival whereas the role of ACER1 and ACER3 in cancer has not been studied.
Drug targeting of human ceramidases summary
The protein structure, the pH optimum, and the sub-cellular localization and topology of the ceramidases create, for one single reaction, different possible physiologies. Ceramidases have been implicated in many diseases, including various types of cancer, thus rendering ceramidases as attractive targets for drug developing. Thus, inhibitors of aCDase and nCDase have been shown to be promising drugs to overcome cell death resistance after prolonged anti-cancer treatments. Nevertheless, at the moment there are no inhibitors that combine specificity and the ability to reach the right cell compartment. For example, desipramine is used to inhibit aCDase, but it also inhibits aSMase, and other lysosomal enzymes. The inhibitor B13 is not efficient enough to reach the lysosome; encouragingly, other molecules such as LCL385 have been developed to solve this issue, and more studies are needed to show their in vivo specificity. There are no highly specific inhibitors for neutral or alkCDases, although d-e-MAPP seems to inhibit the latter enzymes, having no effect in the acidic form. More research is needed for selective inhibitors that would help to understand not only the function of different ceramidases, but also their development into more efficacious and promising therapeutics to control ceramide-related pathologies.