New fluorogenic probes for neutral and alkaline ceramidases

Article abstract of DOI:10.1194/jlr.D092759

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

Full text of DOI:10.1194/jlr.D092759

New fluorogenic probes for neutral and alkaline ceramidases
Mireia Casasampere,a,b Núria Bielsa,a,b Daniel Riba,a Laura Bassas,a , Ruijuan Xuc, Cungui
Maoc, Gemma Fabriàs,a,d José Luis Abad,a Antonio Delgado,a,b* and Josefina Casas.a,d*
a Spanish National Research Council (CSIC), Institute for Advanced Chemistry of Catalonia
(IQAC-CSIC), Department of Biomedicinal Chemistry, Research Unit on Bioactive Molecules
(RUBAM), Jordi Girona 18-26, 08034 Barcelona, Spain.
b
University of Barcelona (UB), Faculty of Pharmacy and Food Sciences, Department of
Pharmacology, Toxicology and Medicinal Chemistry, Unit of Pharmaceutical Chemistry
(Associated Unit to CSIC), Avda. Joan XXIII 27-31, 08028 Barcelona, Spain.
c Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY
11794-8155, USA.
d Centro de Investigación Biomédica en Red (CIBEREHD).
1

Abstract
New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously
reported probes (RBM14) are reported. While none of the new probes were superior to the
known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide
(RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral
ceramidase, both in cell lysates and in intact cells. A second generation of substrates,
incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is
also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl
chain can be used as substrates to determine ACER1 and ACER2 activities. In particular,
compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure
ACER1 and ACER2 activities in a 96-well plate format.
Key words
Ceramidase, substrate, fluorogenic
2

Introduction
Besides their classically accepted role as structural cell components, sphingolipids are also a
well-recognized group of bioactive lipids with significant implications in cell signaling and
disease (1). This is the basis of the so-called sphingolipid rheostat, in which the relative levels
of sphingosine (So) and sphingosine-1-phosphate (S1P) dictate the cell fate (2). Thus, while
S1P results from phosphorylation of So by specific kinases (3), the intracellular levels of So are
regulated by ceramidases, a group of amidohydrolases that catalyze the hydrolysis of ceramides
(Cers) into So and a fatty acids (4). According to their optimum pH, ceramidases are classified
as acid, neutral or alkaline. They differ in their primary structures, since they are coded by
different genes. The five human ceramidases described so far, one acidic (AC), one neutral
(NC,) and three alkaline (ACERs) differ also on their tissue distribution.(5) Thus, while AC is
ubiquitously expressed, NC is highly expressed the small intestine, while the three ACERs are
exclusively expressed in the skin cells. Very recently, the structure of ACER3 has been
disclosed (6). Apart from their optimum pH, ceramidases show different selectivity for Cers
depending on the length and degree of saturation of the N-acyl side chain (4). Thus, AC prefers
C12 and C14 Cers, NC prefers C16 and C18, while ACER1 prefers C20 to C24 chains (7). The
remaining ACERs are less selective, since ACER2 deamidates Cers with saturated acyl chains
(8), as well as dihydroceramides (dhCers) with C18 and C20 monounsaturated chains (9), while
ACER3 also hydrolyses phytoceramides with unsaturated acyl chains (10).
The role of some CDases as therapeutic targets (4) raises the interest for selective CDase
inhibitors and, hence, the development of efficient protocols amenable to HTS formats. In this
context, in previous works (11, 12), a series of first generation fluorogenic substrates (RBM14)
with different N-acyl chains (from C8 to C16) were described as efficient CDase substrates with
different selectivities. However, while RBM14C12 has been characterized as an efficient AC
substrate (11, 13), the remaining substrates proved less selective towards NC, another
3

interesting therapeutic target (14) for which no selective inhibitors have been developed so far.
Moreover, selective substrates for the alkaline ceramidases ACER1-3 are also lacking.
Considering the above precedents and given our continued interest in the development of
selective probes for the different ceramidases, we want to report on a series of new fluorogenic
substrates derived from our previously reported RBM14 probes (11–13), as well as new probes
derived from an homologated version thereof (RBM15) that also incorporates the
2-(N-acylamino)-1,3-diol-4-ene framework present in the natural Cers (Figure 1).
In order to widen the scope of our previously reported RBM14 probes (11–13), longer and/or
unsaturated N-acyl chain amides have been used in this study, in particular those from oleic
acid (C18:1-ω9), erucic acid (C22:1-ω9), and nervonic acid (C24:1-ω9). In addition, results
from a recent report seem to indicate that ACER1-/- cells accumulate -hydroxy eicosanoic
ceramide (15). Since this observation can be related with a higher selectivity of ACER1 towards
this type of side chain, we have also synthesized the corresponding (R) and
(S)-N-(α-hydroxyeicosanoyl) amides. For the sake of comparison, the same N-acyl substitution
patterns have been applied to the new RBM15 series (Figure 1).
Experimental Procedures
Materials
The required carboxylic acids (oleic, erucic, nervonic and (±)-α-hydroxyeicosanoic were
obtained from commercial sources. Dulbecco’s modified Eagle’s medium (DMEM), FBS,
methyl--cyclodextrin (MBCD) and tetracycline were from Sigma. Zeocin was from Genaxxon
Bioscience and blasticidin from CalBiochem. Opti-MEM and lipofectamine 2000 were from
Invitrogen. Recombinant human NC was obtained from R&D Systems. pCMV6 plasmid
harboring the mouse Acer3 (MR218055) was from OriGene and the plasmid harboring ASAH2
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was kindly provided by Dr. Daniel Canals and Prof. Yussuf Hannun (Department of Medicine,
State University of New York at Stony Brook, Stony Brook, NY, USA).
Synthesis
The probes described in this work were obtained by acylation of RBM14 (11) or RBM15 with
the suitable carboxylic acid. Probe RBM15 was obtained from deprotection of the
corresponding N-Boc precursor (RBM15-N-Boc), obtained as described in (16).
Deprotection of RBM15-N-Boc: A solution of the starting material (382 mg, 942.2 µmol) in
MeOH (60 mL) was treated with CH3COCl (500 µL, 27.6 mmol) and the reaction was stirred
at room temperature overnight. The mixture was concentrated in vacuo and the resulting crude
was flash chromatographed (silica gel) with a step gradient of DCM/MeOH (from 0 to 5 %
MeOH at 1 % increments). Compound RBM15 (262 mg, 855 µmol, 91 % yield) was obtained
as a yellow oil; TLC (DCM:MeOH, 9:1; Rf: 0.13);
[α]D25: -3.86 (c 1.0, MeOH). IR: δ 3322 (νO-H and νN-H), 2934 (νCH3), 1698 (νC=O, ester);
1H NMR (CD3OD, 400 MHz): δ 7.89 (d, J = 9.5 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 6.97-6.89
(m, 2H), 6.25 (d, J = 9.5 Hz, 1H), 5.97 (dtd, J = 15.2, 6.8, 1.3 Hz, 1H), 5.67 (ddt, J = 15.5, 6.6,
1.5 Hz, 1H), 4.34 (ddd, J = 6.1, 4.7, 1.2 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.81 (dd, J = 11.6, 4.1
Hz, 1H), 3.68 (dd, J = 11.6, 8.2 Hz, 1H), 3.23 (dt, J = 8.6, 4.4 Hz, 1H), 2.65-2.57 (m, 2H); 13C
NMR (CDCl3, 400 MHz): δ 163.64, 163.31, 156.86, 145.79, 131.50, 131.31, 130.46, 114.04,
113.91, 113.22, 102.19, 70.77, 68.83, 59.48, 58.31, 32.92.
+
HRMS: Calculated for C16H20NO5 [M+H]+: 306.1341; Found: 306.1328.
General Method for acylation of RBM14 and RBM15: A solution of HOBt (40 mg, 285.3
µmol), the corresponding carboxylic acid (280 µmol) and EDC (70 mg, 380.4 µmol) in
5

anhydrous DCM (5 mL) was added dropwise to a solution of RBM14 or RBM15 (190 µmol)
in DCM (5 mL) and Et3N (100 µL, equivalent to 717 µmol) under argon atmosphere. For the
amides of (±)-α-hydroxyeicosanoic acids, the solution of EDC and HOBt was added dropwise
over a mixture of the starting amine and the carboxylic acid. In all cases, the reaction mixture
was stirred at room temperature overnight and the mixture was concentrated in vacuo. The
resulted crude was flash chromatographed (silica gel) using a step gradient of DCM/MeOH
(from 0 to 3 % MeOH at 1 % increments). The required amides were obtained as oils or waxy
solids. Full characterization and NMR spectra can be found in the Supplemental Data.
Cells
FD-AC cell line stably overexpressing AC (provided by Prof. Thierry Levade, INSERM
UMR1037 CRCT, Toulouse, France), HeLa T-Rex ACER1-TET-ON, HeLa T-Rex
ACER2-TET-ON, HEK293T and HT29 cells were cultured at 37°C in 5% CO2 in DMEM
medium supplemented with 10% FBS. The antibiotic selection of HeLa T-Rex TET-ON cells
was performed with Zeocin (25 µg/ml) and Blasticidin (5 µg/ml). These antibiotics were
removed during treatments.
Enzyme source
Experiments with AC were performed in lysates of FD-AC cell line stably overexpressing
ASAH1. NC experiments were carried out in recombinant human NC and in HT29
overexpressing NC cells. To overexpress human NC, HT29 cells were transfected with 2.5
µg/well of a plasmid harboring the ASAH2 gene using 5 µl/well lipofectamine and opti-MEM,
following the manufacturer’s instructions. Then tetracycline 0.05 µM were added for 24 h
before use. In the case of ACERs, microsomes from three different cell lines were used. To
overexpress mouse ACER3, HEK293T cells were transfected with 2.5 µg/well of a plasmid
6

harboring the mouse ACER3 gene using 5 µl/well lipofectamine and opti-MEM, following the
manufacturer’s instructions. To overexpress ACER1 and ACER2, tetracycline 0.05 µM were
added to HeLa T-Rex TET-ON cells for 24 h before use.
Cell lysates
Cell pellets were resuspended in the appropriate volume of a 0.25 M saccharose solution with
the protease inhibitors aprotinin (1 mg/ml), leupeptin (1 mg/mml) and PMSF (100 mM). The
suspension was submitted to three cycles of a 5 s sonication (probe) at 10 watts/5s resting on
ice. The cell lysate was centrifuged at 600 g for 5 min. The supernatant was collected, and
protein concentration was determined as specified below.
Microsomal preparations
Cell lysates obtained from HeLa T-Rex ACER1-TET-ON, HeLa T-Rex ACER2-TET-ON and
HEK293T overexpressing ACER3 cells were transferred to ultracentrifuge tubes and were spun
at 100000 g for 1 h at 4°C. Pellets were resuspended in a 0.25 M saccharose solution and protein
concentration was determined as specified below.
Determination of protein concentration
Protein concentrations were determined with BSA as a standard using a BCA protein
determination kit (Thermo Scientific) according to the manufacturer’s instructions.
Ceramidase activities
Activity assays were carried out in 96-well plates at a final volume of 100 µl/well. Reaction
buffers were: 25 mM sodium acetate buffer pH 4.5 (AC), 50 mM HEPES 150 mM NaCl 1%
7

sodium cholate (NaChol) pH 7.4 (NC) and 50 mM HEPES 1 mM CaCl2 pH 9 (ACERs). The
reaction mixtures contained 25 µl/well of protein (recombinant NC or cell lysate) and 75 µl/well
of substrate. Substrates were tested at 40 µM concentration, prepared from 4 mM stock
solutions in ethanol. The reaction mixture was incubated at 37°C for 3 h, except for the
determination of Km and Vmax parameters (between 20 and 60 min, depending on the substrate).
For the determination of ceramidases activity with RBM14 substrates, reactions were stopped
with 25 µl/well of MeOH and then 100 µl/well of NaIO4 (2.5 mg/ml in glycine-NaOH buffer,
pH 10.6) was added. After incubation at 37°C for 30 min in the dark, 100 µl/well of 0.1 M
glycine-NaOH buffer (pH 10.6) was added. To determinate ceramidase activity with RBM15
substrates, reactions were stopped with 50 µl/well of NaIO4 (2.5 mg/ml in MeOH) and then 100
µl/well of KOH-MeOH was added (0.1 M KOH-MeOH solution in the case of NC and ACERs,
or 0.2 M KOH-MeOH for AC). After incubation at 37°C for 30 min in the dark, 50 µl/well of
0.1 M glycine-NaOH buffer (pH 10.6) was added. In all cases, fluorescence was measured
spectrophotometrically at excitation and emission wavelengths of 355 and 460 nm,
respectively. The same reaction mixtures without enzymes were used as blanks.
Ceramidase activity in intact cells
To determine ceramidase activity in intact cells, 5x105 cells per well were seeded in a 12-well
plate. HT29 cells were transfected with 1 µg/well of a plasmid harboring the ASAH2 gene using
2 µl/well lipofectamine and opti-MEM, following the manufacter’s instructions. Then
tetracycline 0.05 µM were added for 24 h before use. Medium was replaced by MBCD at 1, 5,
and 10 mM in DMEM with NaChol 500 M for 30 min. Then RBM14-C24:1 was added at a
final concentration of 40 µM. The plate was incubated for 3 h at 37°C in 5% CO2. The reaction
was stopped with 125 µl/well of MeOH and then 500 µl/well of NaIO4 (2.5 mg/ml in glycine-
NaOH buffer, pH 10.6) was added. After incubation at 37°C for 1 h in the dark, 500 µl/well of
8

0.1 M glycine-NaOH buffer (pH 10.6) was added and fluorescence was measured
spectrophotometrically at excitation and emission wavelengths of 355 and 460 nm,
respectively. The same reaction mixtures without cells were used as blanks.
Statistics
Comparison between two means has been carried out with the unpaired two-tailed t-test and
statistical differences are marked with asterisks. For comparison of more than two means, data
have been analyzed by one-way ANOVA test followed by Bonferroni’s multiple comparison
test. Statistical differences between means are pointed out with different letters atop each bar
(same letter indicates no statistical difference).
Results
Design and synthesis of the probes
Probes RBM14 were obtained by N-acylation of the free base with the required carboxylic
acids, following previously reported protocols (12, 13). Probes RBM15 were inspired in our
“second generation” probes for S1P lyase, in which the installation of a vinyl unit between the
amino diol moiety and the umbelliferone reporter led to a significant increase of affinity,
probably due to the closer similarity of the vinylogated probe with the natural substrate (16).
The retro oxa-Michael reaction required for the release of the fluorescent reporter from probes
RBM15 can be promoted under basic conditions (see below) by abstraction of one of the
γ-protons of the intermediate aldehyde RBM7-083 (16) resulting from NaIO4 oxidation of the
amino diol RBM15 arising from hydrolysis of the probe by the target ceramidase (Figure 2).
9

Optimization conditions for umbelliferone release
Unlike our previously reported protocol with substrates RBM14 (11–13), the standard Gly-
NaOH buffer (pH 10.6) proved inefficient to promote the complete release of the umbelliferone
from the transient aldehyde RBM7-083 arising from oxidation of the aminodiol RBM15 (Figure
2). Harsher basic conditions were required in this case to ensure acceptable levels of
umbelliferone. The optimized conditions from RBM15 required the use of 0.2 M KOH-MeOH
(for AC) or 0.1 M KOH-MeOH (for NC and ACERs), followed by 30 min incubation at 37°C
in the dark, prior to the addition of 0.2 M glycine-NaOH buffer (pH 10.6) (50 µl/well).
However, even under these optimized conditions, a fluorescence of around 65% of the
theoretical maximum was observed (Supplemental Figure S2).
Probes RBM14 and RBM15 as AC substrates
Probes RBM14 and RBM15 (Figure 1) were tested as AC substrates in FD-AC cell lysates
overexpressing AC. These probes were compared with RBM14C12, reported as AC substrate
in a previous paper (12, 13). None of the new RBM14 probes were superior to RBM14C12 as
AC substrates in lysates from FD-AC cells, as evidenced by the fluorescence values observed
for the released umbelliferone (Figure 3A). This was also true for probes RBM15, since
RBM15C16, the best AC substrate of this series, gave values around three times lower than
those from RBM14C12 (Figure 3B). These results are in agreement with the known preference
of AC for C12-C14 Cers (7) and also with the fact that long chain Cers are not AC substrates
(17).
Probes RBM14 and RBM15 as NC substrates
Probes RBM14 and RBM15 were tested as substrates in human recombinant NC and HT29 cell
lysates overexpressing NC and compared with RBM14C16, which was reported in a previous
10

paper as the most efficient NC substrate, among the RBM14 probes (12). In all the experiments,
similar results were obtained with recombinant NC (Figure 3C,D) and with cell lysates (Figure
3E,F), although the corresponding C16 and C18:1 probes of both series (RBM14 and RBM15)
were somewhat better substrates in cell lysates, in comparative terms with the remaining probes.
The kinetic parameters in human recombinant NC are shown in Table 1. Among RBM14
substrates, RBM14C24:1 proved slightly superior in terms of affinity (KM) and efficiency (Kcat).
The preference of human recombinant NC for this N-acyl side chain was surprising, given the
preference of the enzyme for C16 and C18 Cers (7, 18). On the other hand, the vinylogated
probes RBM15 showed a slightly lower affinity in comparison with the corresponding RBM14
probes, albeit with similar or even superior Kcat/KM parameters. Interestingly, despite the
vinylogated probes RBM15 were generally well tolerated by human recombinant NC, a striking
drop in affinity was observed for RBM15C16, with KM>250. Finally, both (R) and
(S)-α-hydroxyeicosanoyl amides were well tolerated by NC, which showed a slight selectivity,
in terms of Kcat, for the (R) isomer in the RBM15 series.
Probes RBM14 and RBM15 as ACERs substrates
Three different ACERs have been reported with an optimum activity at pH around 9. Because
NC exhibits some residual CDase activity at basic pH, ACER overexpressing systems were
used to test the ability of our probes to act as ACER substrates. In a previous work (12), we
showed that RBM14 probes of short to medium N-acyl chain lengths (C8 to C16) were inactive
as ACER1 and ACER2 substrates, showing only a moderate activity as ACER3 substrates. In
this work, RBM14 analogues with longer saturated or unsaturated N-acyl chains were devoid
of activity as substrates towards all three ACER overexpressing systems (Figure 4 and
Supplemental Figure S3A,C,E). However, the vinylogated probes RBM15 have revealed as
new ACER1 and ACER2 substrates, being practically inactive towards ACER3. Only
11

RBM15C16 behaved as an ACER3 substrate, although weaker than its RBM14 counterpart
(Figure 4 and Supplemental Figure S3B,D,F). Overall, probes RBM15 having a long
unsaturated N-acyl chain (18:1 and 22:1) can be used as substrates to determine ACER1 and
ACER2 activities.
Selectivity of probe RBM14C24:1 for NC
The probe RBM14C24:1 turned out to be one of the best substrates in kinetic terms on the
recombinant human NC (Figure 5) with the lowest KM and a high Kcat/KM ratio compared to the
rest of substrates tested (Table 1) and a high selectivity for NC (Figure 5A). The above
promising results prompted us to test this substrate in intact HT29 cells. However, no activity
was observed after the initial assays (results not shown), which was attributed to the inability
of the probe to cross the cell membrane. Encapsulation into liposomes, similarly as reported by
us for a related probe (16), was not feasible due to the low solubility of RBM14C24:1 in the
required solvent system. Gratifyingly, the use of methyl-β-cyclodextrin (MBCD), a well-know
system used to increase the solubility of non-polar substances, such as fatty acids, lipids,
vitamins and cholesterol in several cell culture applications (19) proved efficient to allow the
use of RBM14C24:1 as NC substrate in intact cells (Figure 5B). A concentration-dependent
effect for MBCD was observed, and an optimal concentration of 5 mM could be established.
Discussion
Cer is considered the hub of the intricate set of pathways that are implicated in the biosynthesis
and degradation of sphingolipids (20). Apart from the biosynthesis from simple structural
moieties in the so-called de novo pathway, Cer can also be generated by the degradation of
complex sphingolipids. Moreover, Cer, as well as dihydroCer (21), can also be degraded by
12

CDases, a type of amido hydrolases that selectively convert Cers and dihydroCers into So and
sphinganine (Sa), respectively, by hydrolysis of the N-fatty acyl chain present in the above
sphingolipids. Interestingly, the removal of the N-acyl chain not only is important to control the
relative levels of sphingolipids inside the cell but also to dramatically alter their intracellular
mobility as a result of the changes in the physical properties after N-deacylation (22). Among
the different CDases, overexpression of NC has been implicated in colon carcinogenesis (14)
and, hence, it has emerged as a new therapeutic target. In this context, the discovery of selective
inhibitors of this enzyme should be boosted by the development of efficient, sensitive analytical
methods amenable for high-throughput screening (HTS) protocols. Based on our previous
works addressed at the development of fluorogenic probes for CDases (11–13), we were
interested in expanding the scope and selectivity of our first generation probes (RBM14) by
using alternative N-acyl chains, based on the reported preferences of CDases for the different
N-acyl Cers. Based on these premises, a collection of potential RBM14 substrates has been
synthesized and tested on our different CDase models. Interestingly, despite NC is selective for
C16 and C18 Cers (10), the N-acyl unsaturated probe RBM14C24:1 emerged as the first
selective NC substrate for recombinant hNC (Table 1 and Figure 5A). Moreover, by using
MBCD as adjuvant, this substrate is also suitable to measure NC activity in intact cells. The
fact that the C24:1 acyl chain of nervonic acid increases the selectivity of RBM14 probes
towards NC is in full agreement with the reported structural data for this enzyme, in which a
20Å deep, hydrophobic active site pocket determines the higher selectivity of NC towards long
chain acyl ceramide substrates (23). Based on these considerations, the C24:1 acyl chain would
be too long to fit into the 13Å cavity found in AC active site (24).
To further expand the applicability of our probes, we designed a second generation set of
potential substrates (compounds RBM15, see Figure 1). As a differential feature, these modified
substrates incorporate the characteristic C4-C5 vinyl unit present in the natural Cers. Since
13

probes RBM15 can be regarded as the vinylogated analogs of probes RBM14, their design also
fits into the essence of the well-established principle of vinylogy (25). In our effort to find
selective CDase substrates, probes RBM15 were tested in our five CDase models and compared
with the corresponding RBM14 counterparts. The most interesting results were obtained with
probes RBM15 having a long unsaturated N-acyl chain (18:1 and 22:1), which behaved as
useful ACER1 and ACER2 substrates (Figure 4A,B) and were totally devoid of activity on AC
(Figure 3A,B) and ACER3 (Figure 4C). Since RBM15C18:1 showed much weaker activity as
NC substrate (Figure 3C-F), this probe has emerged as the best fluorogenic probe reported so
far to measure ACER1 and ACER2 activities in a 96-well plate format. Although less studied
than other CDases, recent studies have shown the implication of ACER1 in mammalian skin
homeostasis and whole-body energy homeostasis (26) and that of ACER2 in DNA damage (27)
and cell differentiation processes (28).
In summary, the fluorogenic probes reported in this work complement our previously reported
probe for AC (RBM14C12) (11–13) by enlarging the scope of applicability with the discovery
of RBM14C24:1 as a highly selective probe for NC and probe RBM15C18:1 for ACER1 and
ACER2 with very low NC activity. These results represent a valuable addition to the available
toolkit of fluorogenic CDases substrates and may enable the development of efficient HTS
protocols for the screening of potential selective inhibitors of this relevant group of
amidohydrolases.
14

Acknowledgments
Partial financial support from Project CTQ2017-85378-R from the Spanish Ministry of Science,
Innovation and Universities is acknowledged. Experimental contributions from Mr. Alex
García and Mr. Pedro Rayo are also acknowledged.
15

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18

Probe
RBM14C16
KM
16 ± 3.3 (*)
28 ± 3.0
14 ± 0.2
16 ± 1.7
28 ± 6.5
8 ± 1.7
Kcat
3281 ± 298
432 ± 64
3075 ± 61
2655 ± 317
1397 ± 219
1581 ± 100
-
Kcat/KM
205.1
15.4
219.6
165.9
49.9
RBM14C18:1
RBM14C20(S)OH
RBM14C20(R)OH
RBM14C22:1
RBM14C24:1
RBM15C16
197.6
-
> 250
RBM15C18:1
RBM15C20(S)OH
RBM15C20(R)OH
RBM15C22:1
RBM15C24:1
139 ± 7.2
21 ± 1.9
42 ± 0.02
33 ± 1.7
40 ± 0.9
3761 ± 465
7257 ± 1092
22954 ± 1004
4977 ± 515
8415 ± 1282
27.1
345.6
546.5
150.8
210.4
Table 1 Kinetic parameters for the NC catalyzed hydrolysis of substrates RBM14 and RBM15.
KM is given in M, Kcat as 10-3s-1 and Kcat/KM as M-1s-1. Recombinant NC (5 ng) was incubated
between 20 and 60 min (depending on the substrate) at graded concentrations. Experiments
were carried out as described in the methodology. Data are mean ± SD of three to five
independent experiments with triplicates. (*) see reference (12)
19

Figure 1. Probes described in this work
20

Figure 2. Metabolic degradation of probes RBM15 and release of umbelliferone after oxidation
of the free base and treatment of the transient aldehyde RBM7-083 with a base.
21

Figure 3. Determination of AC (A, B) and NC (C-F) activity using RBM14 and RBM15
substrates. AC activity was measured in cell lysates from FD-AC overexpressing cells.
Incubations were carried out with RBM14 (A) or RBM15 (B) substrates in 25 mM sodium
acetate buffer pH 4.5. NC activity was measured in recombinant NC (C, D) or cell lysates from
HT29 control cells (L2000) and HT29 overexpressing ASAH2 (NC) (E, F). Incubations were
carried out with RBM14 (C, E) or RBM15 (D, F) substrates in 50 mM HEPES 150 mM NaCl
1% sodium cholate pH 7.4. New RBM14 substrates were compared to RBM14C12 (for AC),
and RBM14C16 (for NC). Data correspond to the mean ± SD of at least three different
experiments with triplicates.
22

Figure 4. Determination of ACER1 (A), ACER2 (B) and ACER3 (C) activity using RBM14
and RBM15 substrates. ACER1 activity (A) was measured in microsomes isolated from mouse
HeLa T-Rex ACER1-TET-ON cells grown in the absence or presence of tetracycline (10 ng/ml)
for 24h. Incubations were carried out with RBM14 or RBM15 substrates in 50 mM HEPES
buffer with 1 mM CaCl2 at pH 9. ACER2 activity (B) was measured in microsomes isolated
from human HeLa T-Rex ACER2-TET-ON cells grown in the absence or presence of
tetracycline (10 ng/ml) for 24h. Incubations were carried out with RBM14 or RBM15 substrates
in 50 mM HEPES buffer with 1 mM CaCl2 at pH 9. ACER3 activity (C) was measured in
microsomes isolated from mouse HEK293T control cells or HEK293T ACER3 overexpressing
cells. Incubations were carried out with RBM14 or RBM15 substrates in 50 mM HEPES buffer
with 1 mM CaCl2 at pH 9. Hydrolysis of RBM14C16 (12) by the different ACERs is also shown
for comparison. Data correspond to the mean ± SD of at least three different experiments with
triplicates. Data correspond to difference between control cells and ACERs overexpressing
cells (See also Supplemental Figure S3).
23

Figure 5. Hydrolysis of RBM14C24:1 by CDase. A) Incubations with RBM14C24:1 were
carried out with cell lysates (AC and NC) or microsomes (ACERs) at acid, neutral of alkaline
pH, as described in the Materials and Methods. The amount (pmol) of umbelliferone released
from RBM14C24:1 was measured. Data (mean ± SD) were obtained from three different
experiments with triplicates. B) NC activity in intact cells using RBM14C24:1 as substrate.
HT29 cells were incubated for 30 min with MBCD at different concentrations in DMEM with
NaChol 500 M, followed by 3 h treatment with 40 M RBM14C24:1. Non-MBCD treated
cells were also tested. The amount (pmol) of umbelliferone released from RBM14C24:1 was
measured. Data correspond to the mean ± SD of two different experiments with duplicates. Data
were analyzed by one-way ANOVA test (P < 0.0001) followed by Bonferroni’s multiple
comparison test. Different letters denote a statistically significant difference between groups (P
< 0.05).
24