A Potent and Specific Ceramide Kinase Inhibitor
931
70% and 80% respectively, but also increased NBD-Cer levels particular the profiling of endogenous Cer and C1P species,
by 90% (cЈ). In the presence of the two inhibitors, NBD-C1P will be required to thoroughly test this hypothesis. In the
was not detectable, NBD-GlcCer was marginally reduced; studies performed by others previously, the critical stress
remarkably, NBD-SM and NBD-Cer levels reached 300 and may have been the removal of M-CSF in macrophages de-
250% of control levels, respectively (dЈ). It is noteworthy that rived from CD-1 mice—these animals cannot synthesize this
the absolute level of every metabolite was comparable in both growth factor (Go´mez-Mun˜oz et al., 2004)—or the use of a
cell types after treatment with the two inhibitors (compare d low serum-containing culture medium (Pettus et al., 2003;
and dЈ).
Mitra et al., 2007; Tsuji et al., 2008); both conditions are
Because an increase in ceramide leads to growth inhibi- known to increase Cer levels. All together, this illustrates the
tion, we evaluated the influence of treatment with NVP-231 key role played by CerK in disposing of Cer, thereby in
and tamoxifen on cell growth in a second set of experiments keeping with the higher Cer levels measured in CerK-defi-
(Fig. 4B). Growth-inhibition induced by tamoxifen alone was cient mice (Graf et al., 2008a,b). The proposed role for CerK
more pronounced in COS cells (compare aЈ and bЈ) compared in controlling cell growth and survival via regulation of Cer
with COS-CerK cells (compare a and b). NVP-231 had a levels is summarized in Fig. 5. Our finding that a combi-
marginal effect alone on either cell population (c and cЈ). nation of ceramide metabolism inhibitors can synergisti-
When tamoxifen and NVP-231 were combined, no further cally increase ceramide levels and reduce cell growth may
growth inhibition was observed in COS cells compared with provide a valuable rationale for the treatment of prolifer-
tamoxifen alone (d). However, in COS-CerK cells, the two ative disorders.
compounds synergized to reduce growth (dЈ), yielding viabil-
ity scores similar to those observed in COS cells treated with
Acknowledgments
tamoxifen alone (compare dЈ and b).
We thank Peter Fu¨rst and Lorenz Mayr for continuous support;
Felix Freuler, Michael Forstner, Daniel Rotmann, and Micha Wicki
for cloning and protein production; Sigmar Dressler for hitlist anal-
ysis; Alistair Boath, Gregory Hollingworth, Emilie Lidome, and Lo¨ıc
Tauzin for contribution to the profiling of NVP-231; and Gabriele
Meder and Christine Niklaus for technical assistance. We are grate-
ful to Pr. F. Sakane (Sapporo Medical University) for providing an
anti-DAGK␣ antibody.
Discussion
In this work, we have discovered and characterized the
first potent inhibitor of CerK and one of the most potent lipid
kinase inhibitors to date. NVP-231 features a number of
remarkable properties: 1) it competes with ceramide binding
to CerK by a mechanism that does not rely on ceramide
mimicry (Fig. 1), 2) it is as potent and efficacious in cell based
assays as it is in vitro (Figs. 1 and 2), 3) it is highly specific
for CerK (Fig. 2 and Table 1), and 4) its action is fully
reversible (Supplemental Figs. 1 and 2). Based on these char-
acteristics, we anticipate that the discovery of NVP-231 has
provided a powerful tool to elucidate CerK biology and probe
whether its regulation may have therapeutic potential.
Our experiments based on a mechanistic cellular model
(Fig. 4) suggest that CerK inhibition can be detrimental to
cell growth provided a concomitant stress signal is applied.
We found that inhibition of glucosyl ceramide synthase by
tamoxifen allows for synergy with NVP-231 to increase NBD-
Cer levels and reduce cell growth. Further work, including in
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Stress signal
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Stress signal
NVP-231
+ NVP-231
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Cer
Cer
Cer
Cer
=
Low
=
=
=
Controlled
(CerK active)
Partly controlled
(CerK inactive)
Uncontrolled
(CerK inactive)
GROWTH/SURVIVAL
DEATH
Fig. 5. Role of CerK in the control of cell growth and survival. This
scheme recapitulates the findings of the present study together with the
conclusions of previous work from many laboratories (reviewed in Go´mez-
Mun˜oz, 2006; Mitra et al., 2007, Tsuji et al., 2008). Under resting condi-
tions, cellular Cer levels are low. Stress signals converge to increase Cer,
but Cer levels are kept under threshold levels in the presence of active
CerK. When CerK is down-regulated (e.g., inhibited by NVP-231), cells
are less efficient in disposing of Cer; consequently, they become more
sensitive to stress signals. If stress occurs, Cer will accumulate, leading to
cell death.