A photocaged inhibitor of acid sphingomyelinase

Article abstract of DOI:10.1039/d0cc06661c

Acid sphingomyelinase (ASM) is a potential drug target and involved in rapid lipid signalling events. However, there are no tools available to adequately study such processes. Based on a non cell-permeable PtdIns(3,5)P2 inhibitor of ASM, we developed a co

Full text of DOI:10.1039/d0cc06661c

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A photocaged inhibitor of acid sphingomyelinase†
a
bc
Kevin Prause,a Gita Naseri,
Fabian Schumacher,
Christian Kappe,a
Cite this: Chem. Commun., 2020,
56, 14885
Burkhard Kleuserb and Christoph Arenz
*
a
Received 5th October 2020,
Accepted 5th November 2020
DOI: 10.1039/d0cc06661c
rsc.li/chemcomm
Acid sphingomyelinase (ASM) is a potential drug target and involved ASM8,9 Rapid cleavage of sphingomyelin results in transient and
in rapid lipid signalling events. However, there are no tools available local elevation of downstream metabolites, such as ceramide
to adequately study such processes. Based on a non cell-permeable and sphingosine, which are likely responsible for ASM-mediated
PtdIns(3,5)P2 inhibitor of ASM, we developed a compound with o-nitro- effects. ASM is being discussed as a drug target for treating
benzyl photocages and butyryl esters to transiently mask hydroxyl acute-lung injury,10 sepsis,11 metastasis of melanoma12 and
groups. This resulted in a potent light-inducible photocaged ASM major depression.13 However, no potent drug-like inhibitors of
inhibitor (PCAI). The first example of a time-resolved inhibition of ASM are known.14 Currently, functional inhibitors of acid sphin-
ASM was shown in intact living cells.
gomyelinase (FIASMA) are the first choice to inhibit ASM,
although they are known to be indirect and non-specific.15 The
Sphingolipids are ubiquitous and important in eukaryotic cells bisphosphonate Arc39 is the most potent inhibitor known.16 The
as structural and signaling lipids.1,2 Due to the lipid nature of compound acts on ASM with high potency in cells, but not in
the intermediates, investigation into this class of metabolites is mice.16 Recently, a novel hydroxamic acid inhibitor showed
not straight forward, and usually requires tedious extraction antidepressant activity in a mouse model,17 but not much is
procedures. As a consequence, topological as well as time- known about its specificity.
resolution is often lost during their analysis, although the exact
One of the first ASM inhibitors discovered, is the naturally
localization of sphingolipids is key to their function. This occurring phosphatidyl inositol-3,5-bisphosphate (PtdIns(3,5)P2),
fundamental problem has made research on sphingolipids but its structure prevents its use in cell culture or in vivo.18 Our
largely dependent on functionalized lipid analogues, including goal was to synthesize a cell-compatible analogue of PtdIns(3,5)P2,
radioactively or fluorescently labeled compounds,3 cross-link based on the previously published analogue rac-12.19 In the latter
compounds,4,5 or photo-switchable versions of metabolites.6 compound, the phosphatidic acid is replaced by a simple sulfonic
Acid sphingomyelinase (ASM) is an important enzyme involved ester (Fig. 1). We now wanted to mask the phosphate groups at the
in sphingolipid metabolism, as it cleaves the major plasma
membrane lipid sphingomyelin during lysosomal membrane
digestion.7 In addition to this constitutive degradation process,
the activity of ASM can be stimulated by various exogenic
triggers such as cytokines, ionizing radiation and reactive
oxygen species (ROS). As a result, recent evidence suggests a
fusion of lysosomes with the plasma membrane within seconds
after stimulation being the major source of a secreted form of
a
¨
Institute for Chemistry, Humboldt Universitat zu Berlin, 12437 Berlin, Germany.
E-mail: arenzchr@hu-berlin.de
b
¨
¨
Institute of Pharmacy, Freie Universitat Berlin, Konigin-Luise-Str. 2+4,
14195 Berlin, Germany
c Department of Molecular Biology, University of Duisburg-Essen, Hufelandstr. 55,
Fig. 1 Stepwise modification of the initial natural compound inhibitor
PtdIns3,5-bP, into 1-O-sulfonyl-myo-inositol rac-12,19 the photocaged
phosphoramide 1 and the completely masked analogue 2 (PCAI).
45147 Essen, Germany
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc06661c
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30 and 50 position with non-polar residues to allow efficient pene-
tration of cells.
We were well aware of the fact that such a masking strategy
would also offer the possibility of installing photo-labile
groups, enabling a light-induced inhibition.
Such photocaging strategies are ideal to study lipid-signaling
events, as they provide the spatiotemporal resolution needed.
Indeed, photocaged biologically active compounds like photo-
caged enzyme inhibitors have shown to be invaluable tools to
study biological phenomena.20 For sphingolipid biology, a num-
ber of light-activated probes have been developed, including
caged21 and photo-switchable6 sphingosine-1-phosphate, photo-
caged sphingosine,22 photocaged ceramide23 and ceramide-1-
phosphate.24 However, to the best of our knowledge no tools for
light-activated control over ASM activity exist.
Scheme 1 (a) N3SO2Im*HCl, Cu(II)SO4, K2CO3, rt, 12 h; (b) ClSO2C16H33
DMAP, THF, 0 1C to RT, 2 h (c) pyridine, Bt2O, 0 1C to rt, 2 d. (d) (1) P(ONB)3,
CH2Cl2, 45–50 1C, 24–50 h (2) MeCN/H2O (1 : 1)
,
For the synthesis of the envisioned photocaged inhibitor, we
planned to replace the phosphoester bonds found in PtdIns(3,5)P2
and our previously synthesized analogues rac-12 (Fig. 1) by chemi-
cally less stable phosphoramidate bonds. Half-life times of phos-
phoramidates under mild acidic pH values are typically in the
range of 30 minutes up to a few hours.25 Due to the acidic pH in the
lysosomes, this could potentially cause a transient inhibition rather
than a long-term depletion of ASM, like it is observed for FIASMAs.
This would potentially be more in line with the idea of a time-
resolved inhibition.
We therefore planned the synthesis of compound 1 carrying
photolabile masking groups of the phosphoramidate moieties to
facilitate cell penetration (Fig. 1). As compound 1 – like other
phosphoinositides – would be still a rather polar molecule, we
planned in addition, to synthesize compound 2, which is also
masked at the hydroxyl groups by biologically labile butyl
ester groups, similar to earlier described cell-permeable
phosphoinositides.26
the purpose of uncaging would also be associated with bleaching
of the FRET probes. When compound 1 (10 mM) was incubated
with ASM in presence of the visual range FRET probe,30 similar
slopes for caged inhibitor-containing reaction and the control
reaction were observed (Fig. 2). Then, both reactions were illumi-
nated with UV light for one minute, respectively. As expected, bot
reactions showed a drop in fluorescence as expected due to FRET
probe bleaching, but now a clear difference in slopes, indicating
ASM activity, was observed.
While the fluorescence intensity for the reaction without
inhibitor increased further, the fluorescence for reaction
containing compound 1 remained almost constant. The slope
for the latter reaction was now similar to the slope for the
reaction containing 1, which was illuminated beforehand.
However, it was also clearly visible, that even the caged
compound 1 showed some inhibition at this concentration.
These observations suggested that a minor portion of fully or
partially uncaged inhibitor was formed either during handling
or during excitation of the FRET probe.
For synthesis of the desired compounds, the literature-known
3,5-diamino-D-myo-inositol 10 was synthesized from N-acetyl-D-
glucosamine (GlcNAc) in ten steps, following a route described by
Ogawa et al. (Scheme S1, ESI†).27 Compound 10 was converted into
the novel bisazide 11 by copper(II) – mediated diazo transfer. Careful
sulfonylation yielded the desired regioisomer 12a as a main product
(Scheme 1).
The latter was either directly subjected to a Staudinger
phosphite reaction28 to afford 1 or previously treated with
butyric acid anhydride to obtain the more lipophilic compound
2. The overall synthetic yields starting from 10 (Scheme S1,
ESI†) were 6.7% (1) and 18.3% (2), respectively.
After having accomplished the synthesis of the desired
compounds, inhibition of ASM by the UV-treated compounds
was tested. As expected, ASM inhibition was confirmed for
uncaged phosphoramidite derived from 1 at 5 mM. Noteworthy,
the IC50 value for ASM inhibition for the previously reported
phosphoinositide mimic rac-12 was found to be 0.5 mM
(C12 sulfonic ester)19 and B200 nM for the C16 sulfonic ester
(unpublished results). In the next step, we wanted to monitor
ASM inhibition in a continuous, time-resolved assay in vitro,
making use of ASM FRET substrates, which have been
published recently.29,30 It was clear to us that irradiation for
Fig. 2 In vitro de-caging experiment in real time using 10 mM of
compound 1. After 60 minutes, the reactions were illuminated for
60 seconds. Orange: 1; blue: w/o inhibitor. Red: reaction with 1, but
illuminated before. Black = red, but with an additional illumination.
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In order to investigate this more thoroughly, we performed but significant reduction in activity at 50 mM. This could reflect
experiments using 10 mM (Fig. S7, ESI†) or 25 mM (Fig. S8, ESI†) the behavior of 1 in vitro, which also showed some inhibition at
of compound 1, which was illuminated prior to ASM assay for 25 mM, even in the presence of the photocage. In comparison to
different durations. Indeed, even illumination times as short as 1, compound 2 appeared to be more potent and therefore lower
5 seconds led to significantly increased inhibitions. However, concentrations were investigated (Fig. 3A). Indeed, between
25 mM of 1 not illuminated prior to reaction led to about 50% 2.5 mM and 50 mM, almost full inhibition of ASM was observed
inhibition, suggestion that (based on IC50 of rac-12) roughly 5% after 30 seconds of illumination, but with normal ASM
of 1 were uncaged during handling or excitation of the FRET activities when this step was omitted. Therefore we termed
probe (Fig. S8, ESI†). We also studied the uncaging kinetics and compound 2 as ‘‘photocaged ASM inhibitor’’ (PCAI). At 1 mM,
the stability of 2 under the assay conditions in more detail and only a moderate decrease in ASM activity was observed after
found that half-life times for uncaging in vitro were B10 s irradiation, which was not significant. However, when PCAI was
(5 mM) and B100 s (5 mM). The isolated yield for 2 was 93% illuminated three times for 60 seconds (each), a highly signifi-
(Fig. S1, ESI†). No decomposition of the phosphoamidate cant inhibition was observed (Fig. 3B). In another experiment,
bonds at pH 4.5 or pH 7.4 was observed even after 48 h we illuminated cells incubated with 5 mM of PCAI for 15, 30, 45,
(Fig. S2–S5, ESI†). When incubated for 30 minutes in cell 60 and 90 seconds, respectively. This experiment not only
lysates, a mixture of 2 and 1 but no other degradation products showed a highly significant inhibition after only 15 seconds
were recovered by HPLC analysis (Fig. S6, ESI†).
of illumination, but also that 60 seconds illumination was not
Next, we wanted to test the photocaged compounds in living enough to exploit the full inhibitory potential of PCAI. PCAI
cells. For analysis of ASM activity, we made use of the visible differs from 1 in butyryl groups masking the hydroxyl groups of
range FRET probe in combination with flow cytometry, as the inositol ring. We assume, that superior potency of PCAI
published recently.30 To this end, we incubated HEK293 cells over 1 is due to a higher level of cell penetration, which in turn
with different concentrations of both photocaged inhibitors. In is due to higher polarity of intact 1, but may be also a result of
order to avoid photo-bleaching of the ASM probe, we decided to partial decaging of 1.
incubate the cells with the caged inhibitors for a 16 h, then
In order to substantiate our finding that PCAI is a cell-
induce uncaging of the inhibitors by a light pulse and finally permeable photo-activatable inhibitor of ASM, the effect of
add the FRET probe for another 16 h to the cells for an PCAI on cellular lipid levels was investigated, although a recent
assessment of ASM activity. First, inhibition of both probes study showed that the overall effects are rather small, even in
was tested at concentrations of 25 mM and 50 mM, respectively presence of 50 mM FIASMA (imipramine).31 Towards this end,
(Fig. 3).
we incubated HEK293 cells with 10 mM of the compound,
For probe 1, only a slight but significant inhibition was followed by illumination for 60 seconds. Compared to cells
observed at 25 mM after irradiation, while inhibition at 50 mM devoid of the inhibitor, total sphingomyelin (SM) was increased
was close to the inhibition shown by Arc39 (10 mM), which (p = 0.06), as well as the ratios of total SM/Cer (p = 0.35) and
again was assumed to be close to 100%, based on previous total SM/Sph (p = 0.06), but none of these values increased
experiments.16 Notably, without illumination, ASM activity in significantly. In contrast, SM16/Cer16 ratio was increased
presence of 1 at 25 mM appeared to be normal, but with a slight significantly (p = 0.01), which represent the main substrate/
Fig. 3 (A) Photo-induced inhibition of ASM in living cells. Monitored by flow cytometry as the ratio of geometric mean fluorescence intensities (MFI) in
green and red channels. White columns: without photo-induction, yellow columns: with photo-induction (30 s). Control: white column: mock treated,
yellow column: 10 mM Arc39, leading almost complete ASM inhibition. If not indicated, all differences between respective white and yellow columns are
highly significant (p o 0.001). n.s. = not significant. (B) Inhibition of ASM in living cells, using PCAI (1 mM) without or with illumination for 1ꢀ 60 s or
3ꢀ 60 s. (C) Changes to the HEK293 lipidome in presence of PCAI (10 mM, 60 s illumination, followed by 24 h chase). All experiments were performed in
three distinct cell cultures as three technical replicates. SM = sphingomyelin, Cer16 = ceramide (N-palmitoylsphingosine), Sph = sphingosine.
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