Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT

Article abstract of DOI:10.1002/anie.201511301

We present a novel chemical scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We d

Full text of DOI:10.1002/anie.201511301

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International Edition: DOI: 10.1002/anie.201511301
German Edition: DOI: 10.1002/ange.201511301
Drug Discovery
Hot Paper
Chemoproteomics-Enabled Discovery of a Potent and Selective
Inhibitor of the DNA Repair Protein MGMT
Chao Wang+, Daniel Abegg+, Dominic G. Hoch, and Alexander Adibekian*
Abstract: We present a novel chemical scaffold for cysteine-
reactive covalent inhibitors. Chloromethyl triazoles (CMTs)
are readily accessed in only two chemical steps, thus enabling
the rapid optimization of the pharmacological properties of
these inhibitors. We demonstrate the tunability of the CMTs
towards a specific biological target by synthesizing AA-CW236
as the first potent non-pseudosubstrate inhibitor of the O6-
alkylguanine DNA methyltransferase (MGMT), a protein of
major clinical significance for the treatment of several severe
cancer forms. Using quantitative proteomics profiling tech-
niques, we show that AA-CW236 exhibits a high degree of
selectivity towards MGMT. Finally, we validate the effective-
ness of our MGMT inhibitor in combination with the DNA
alkylating drug temozolomide in breast and colon cancer cells
by fluorescence imaging and a cell-viability assay. Our results
may open a new avenue towards the development of a clinically
approved MGMT inhibitor.
higher formation of the desired O6-MeG adducts in human
patients with various cancer forms.[7] Unfortunately, DNA
alkylating agents such as temozolomide also cause severe
myelosuppression in patients, and this side effect is even
increased by the co-administration of MGMT pseudosub-
strates.[8] Interestingly, the level of myelosuppression varies
markedly between the different pseudosubstrates, making us
optimistic that this serious side effect can at least be improved
with new MGMT inhibitors.[9] Paradoxically, the reaction
between the highly dosed MGMT pseudosubstrates and the
protein itself creates free guanine base as a side product that
can be utilized by the cancer cells to synthesize new DNA,
which is exactly the opposite effect of what many anticancer
agents aim to achieve.[10] Thus, we believe that there is a need
for new, non-O6-alkylguanine-based MGMT inhibitors.
Herein, we present chloromethyl triazoles (CMTs) as a new,
tunable cysteine-reactive chemical scaffold that can be
accessed in only two chemical steps, enabling rapid prepara-
tion of a large series of compounds and also streamlining the
optimization of inhibitor potency and selectivity. By applying
methods from biochemistry, cell biology, and quantitative
proteomics, we discover and evaluate AA-CW236 as the first
potent and selective non-pseudosubstrate inhibitor of
MGMT.
D
NA is a chemically vulnerable molecule, and our cells
have developed sophisticated repair mechanisms to maintain
the integrity and stability of DNA.[1] The DNA repair protein
O6-methylguanine-DNA-methyltransferase (MGMT) trans-
forms the naturally occurring DNA lesion O6-methylguanine
back into guanine.[2] During this transformation, the O6-
methyl group is transferred to the active-site cysteine,
irreversibly inactivating MGMT. This attribute of MGMT
has been exploited for designing a self-labeling protein tag.[3]
Unfortunately, MGMT also repairs damage caused by DNA
alkylating agents in cancer cells.[4] For example, MGMT
activity levels in brain tumors can vary by a factor of up to
300, and there is a strong correlation between high MGMT
levels in patients and failed response to chemotherapy.[5]
Pseudosubstrate inhibitors, such as O6-benzylguanine and
O6-(4-bromothienyl)guanine (lomeguatrib), irreversibly alky-
late the active-site cysteine in MGMT and have thus been
tested in clinical trials in combination with DNA alkylating
agents.[6] Phase I trials based on DNA alkylating drug
temozolomide/lomeguatrib combinations were successful
and attested effective depletion of MGMT activity and
Covalent inhibitors currently enjoy a renaissance of
popularity in pharmaceutical research.[11] The proposal of
chloromethyl triazoles as a scaffold for cysteine-reactive
covalent inhibitors (Figure 1A) was inspired by the shape of
N-heterocyclic carbene ligands, which are widely used in
catalysis,[12] where both nitrogen substituents are sterically
protecting the reactive metal center by pointing towards it.
[*] C. Wang,[+] D. Abegg,[+] D. G. Hoch, Prof. Dr. A. Adibekian
School of Chemistry and Biochemistry
NCCR Chemical Biology, University of Geneva
30 quai Ernest-Ansermet, Geneva (Switzerland)
E-mail: alexander.adibekian@unige.ch
Figure 1. Testing the reactivity of a clickable chloromethyl triazole
probe. A) A general reaction scheme showing the concept and the
synthetic accessibility of CMTs and the expected mechanism of MGMT
inhibition. B) Chemical structure of the clickable CMT probe 1 (AA-
CW159A). C) Gel-based fluorescence labeling of mock-transfected
MCF7 lysate and lysate with overexpressed GFP-hMGMT (the full-
length gel image is shown in Figure S2).
[+] These authors contributed equally to this work.
Supporting information for this article (synthesis of compounds, cell
culture, bioassays, labeling and sample preparation for proteomics,
and mass spectrometry) is available on the WWW under http://dx.
doi.org/10.1002/anie.201511301.
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We hypothesized that owing to the close proximity to the
chloromethyl group, groups R1 and R2 should strongly
influence both the chemical reactivity and selectivity of the
inhibitors.
To test whether CMTs covalently react with proteomic
cysteines, we synthesized the clickable probe AA-CW159A
(1; Figure 1B). We then treated breast-cancer-derived MCF7
cellular lysates with probe 1 at 10 mm concentration, which
were then reacted with TAMRA alkyne through copper(I)-
catalyzed alkyne–azide cycloaddition (CuAAC), separated by
SDS-PAGE, and visualized by in-gel fluorescence scanning.
Multiple bands were detectable, and the fluorescence labeling
was completely abolished by pretreatment with the broad-
profile cysteine-reactive probe iodoacetamide, indicating that
1 shows a strong preference for the labeling of cysteines over
other nucleophilic amino acids (Supporting Information,
Figure S1). We next performed a proteomics experiment to
explore whether MGMT can be labeled and enriched by
probe 1. Briefly, MCF7 cell lysates were treated with 10 mm of
probe 1, clicked to biotin alkyne, enriched over streptavidin
beads, and analyzed by LC-MS/MS. MGMT was identified as
a target of probe 1 along with many other proteins (Table S1).
We confirmed that the CMT model probe 1 is indeed capable
of covalently binding to MGMT by overexpressing the GFP-
tagged human MGMT in MCF7 cells and performing gel-
based fluorescence labeling with 1. A strong new fluorescent
band at 52 kDa indicates the formation of a covalent bond
between the CMT probe and MGMT (Figures 1C, S2).
Encouraged by these results, we prepared a small collec-
tion of 1,4-substituted CMTs 2–19 (Figure 2A). Starting from
commercially available alkyl azides and substituted propar-
gylic alcohols, 5-hydroxymethyl triazoles were accessed by
thermal azide–alkyne cycloaddition. Alternatively, triazoles
could also be efficiently prepared from more temperature-
sensitive substrates by ruthenium-catalyzed azide–alkyne
cycloaddition[13] (RuAAC). With both methods, the 1,4-
substituted isomer was generally obtained as the major
product and separated from the 1,5-substituted isomer by
flash chromatography. Finally, the hydroxymethyl triazoles
were converted into the corresponding chlorides by mesyla-
tion and nucleophilic substitution with tetrabutylammonium
chloride in one-pot fashion. The 1,4-substitution of the CMTs
was unambiguously confirmed by X-ray structural analysis
(for five CMTs) and by HMBC NMR analysis of the alcohol
precursors. In parallel, we established a gel-based competitive
proteomics profiling experiment to identify potent MGMT
binders. Briefly, cellular lysates with overexpressed hMGMT
were treated with compounds at 200 nm concentration,
followed by probe 1 (Figures 2A,B, and S3). Whereas most
of the CMTs did not compete the fluorescence labeling of
MGMT, > 70% competition was observed with triazole 14.
Some activity was also detectable with CMTs 17 and 18.
Finally, the best competition of MGMT fluorescence labeling
(88%) was achieved with compound 19 (AA-CW236), which
includes both active structural elements, the 3,5-dimethyl-
isoxazole group and the 4-(trifluoromethoxy)phenyl substitu-
ent (Figures 2B and S4).
Figure 2. Synthesis and gel-based screening of 1,4-substituted CMTs.
A) General synthetic route to 1,4-substituted CMTs and chemical
structures of CMTs 2–19 (method A: PhMe, 1258C; method B: Cp*Ru-
(PPh3)2Cl (2 mol%), PhMe, 808C). Values in parentheses indicate the
competition (in %) of GFP-hMGMT labeling with probe 1 (AA-
CW159A). All compounds were screened at a concentration of 200 nm.
B) Images from the gel-based competitive screening of CMTs 2–19.
active-site Cys145. In contrast to the overexpressed wild type
(WT) protein, the C145A MGMT mutant was not labeled by
probe 1, meaning that CMTs bind exclusively to Cys145
(Figure 3A). As AA-CW236 binds to MGMT covalently
and irreversibly, we applied the method of Kitz and Wilson to
[14]
determine the binding constants KI and kinact
.
Our kinetics
assay relies on measuring the competition of probe 1 and not
on substrate turnover and is therefore applicable to non-
enzyme proteins such as MGMT. The calculated KI value of
24 nm demonstrates that AA-CW236 is a highly potent
inactivator of human MGMT. For comparison, AA-CW236
is as potent as lomeguatrib and approximately ten times more
potent than O6-benzylguanine (Figure S5). However, we
believe that selectivity is more relevant for predicting the
clinical potential of covalent inhibitors, which are tradition-
ally considered to be more promiscuous.[11] First, we evaluated
the selectivity of AA-CW236 in a gel-based assay using MCF7
As hMGMT contains five different cysteines, it was
important to ascertain that the CMTs indeed react with the
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peted after treatment with 200 nm AA-CW236. Finally, probe
20 also proved useful for measuring the activity of AA-
CW236 directly in living MCF7 cells (Figure S7). The gel-
based profile indicates that MGMT was completely inhibited
after treatment of cells with 100 nm of AA-CW236 for ten
hours.
MS-based profiling methods offer a clear advantage over
gel-based techniques, because a much higher number of
proteins can be detected and quantified in one single experi-
ment.[17] Therefore, we performed a competitive activity-
based protein profiling experiment in MCF7 proteomes in
combination with quantification by stable isotope labeling of
amino acids in culture (SILAC). We used the MGMT
inhibitor AA-CW236 at two different concentrations,
200 nm (Table S2) and 1 mm (Table S3), and the broad-profile
CMT probe 1 for subsequent proteomic enrichment. Intrigu-
ingly, among the combined 1039 quantified proteins and even
at 1 mm concentration, only MGMT was found to be greater
than 66% competed (SILAC ratio of 0.12 = 88% competi-
tion; Figure 3D). Moreover, we conducted an additional
competitive proteomics experiment based on the catch-and-
release strategy recently reported by us, using iodoacetamide
(IAA) alkyne as a general cysteine-reactive enrichment
probe,[18] but applying dimethyl labeling (DML) as an
alternative method for stable-isotope-based quantification.
This method allows selective enrichment and quantification
of cysteine-containing and IAA-modified peptides rather
than of whole proteins. Again, after treatment with 200 nm
AA-CW236, among all 1245 enriched and quantified pep-
tides, only one peptide, which contained the MGMT active-
site cysteine, was found to be greater than 66% competed
(Figure 3D, Table S4). This is remarkable considering that the
human proteome contains thousands of cysteines that are
capable of reacting with electrophilic probes.[18,19] In fact, even
highly optimized and clinically approved kinase inhibitors
bind to numerous off-target cysteines.[20] For comparison,
when lomeguatrib (200 nm) was used as a competitor, we
observed several other > 66% competed peptides originating
from proteins such as the arginine tRNA ligase RARS,
guanine nucleotide binding protein GNB2, and dynamin-2
(Figure S8, Table S5).
To understand the molecular basis for the observed
potency of AA-CW236 towards hMGMT, we performed
covalent docking using a high-resolution X-ray structure of
hMGMT (PDB: 1EH6) and the protein-ligand docking
program GOLD[21] (Figure 4A). The predicted optimal bind-
ing mode suggests a key interaction between the trifluor-
omethoxy group of AA-CW236 and Tyr114 as well as an
additional potential interaction between the triazole group
and Ser159. Whereas the predicted binding mode for AA-
CW236 yielded a GoldScore of 46.0, significantly lower values
(35–40) were calculated for the less active derivatives 8, 9, and
11, thus supporting the proposed model. Moreover, to further
investigate the role of the CF3 group in hMGMT binding, we
prepared two structurally close derivatives of AA-CW236
that lack this functionality. Subsequent gel-based IC50 meas-
urements confirmed that these compounds are indeed sig-
nificantly less active than AA-CW236 (Figure S9).
Figure 3. Evaluation of the proteome-wide selectivity of AA-CW236.
A) The clickable CMT probe AA-CW159A labels WT GFP-hMGMT, but
not the C145A mutant. B) Chemical structure of the clickable CMT
probe 20 (AA-CW538). C) Gel-based profiling of the MGMT activity
and AA-CW236 selectivity in proteomes of different human cancer cell
lines. Proteomes were treated with DMSO or 200 nm AA-CW236 for
1 h followed by 30 mm AA-CW538. D) Evaluation of the AA-CW236
selectivity by quantitative proteomics. Shown are scatter plots with
SILAC and DML ratios. Left: SILAC ratios from the competitive
experiment with 200 nm (n=3) or 1 mm (n=4) AA-CW236 treatment
followed by 10 mm AA-CW159A. Right: DML ratios from the compet-
itive experiment with 200 nm AA-CW236 (n=6) followed by 10 mm IAA
alkyne. RatiosÆSEM for MGMT are shown.
lysates with overexpressed hMGMT and the CMT probe
1 (Figure S4). We observed complete competition of MGMT
after treatment with 300 nm AA-CW236, whereas no other
targets of probe 1 were visibly affected. Whereas the broad-
profile probe 1 proved useful for experiments with overex-
pressed MGMT, it did not allow gel-based detection of native
MGMTactivity owing to comparably low expression levels of
this protein. Therefore, we prepared the expectedly more
MGMT-directed probe 20 (AA-CW538), a clickable deriva-
tive of the inhibitor AA-CW236 (Scheme S1).
With the new probe in hand, we were finally able to detect
the native MGMT activity in various cancer cell lines
(Figures 3C, S6). Consistent with the literature,[15] high
MGMTactivity levels were observed in breast-cancer-derived
MCF7 cells. Furthermore, we found very high activity in colon
cancer Caco-2 cells and acute lymphoblastic leukemia
RPMI 8402 cells. The gel-based assay for profiling the
MGMT activity with probe AA-CW538 should also be
applicable to clinical samples and represent a significant
improvement in terms of convenience and throughput over
currently existing radioactive and northern blot based
MGMT activity assays.[16] Remarkably, although many other
proteins were also labeled by the probe AA-CW538 at 30 mm
concentration, only the MGMT band was selectively com-
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synthesis of structurally diverse chloromethyl triazoles led to
the discovery of AA-CW236 as a potent low-nanomolar non-
pseudosubstrate inhibitor of MGMT. By using various gel-
and MS-based proteomic profiling techniques, we have
demonstrated that AA-CW236 is active in cells and exhibits
a high degree of selectivity towards MGMT. Furthermore,
a strong sensitizing effect was observed in MCF7 and Caco-2
cells when AA-CW236 was co-administrated with the DN
alkylating agent TMZ. Finally, while there is no guarantee
that this particular compound will be successful in clinical
studies, we firmly believe that the results presented herein
justify additional efforts towards the identification of
improved MGMT inhibitors.
Acknowledgements
The University of Geneva, the Swiss National Science
Foundation, and the NCCR Chemical Biology are acknowl-
edged for financial support.
Keywords: activity-based protein profiling · drug discovery ·
mass spectrometry · MGMT · proteomics
Figure 4. Proposed binding mode and the cancer cell sensitization
effect of AA-CW236. A) Docking prediction of AA-CW236 covalently
bound to Cys145 of human MGMT. B) Detection of guanine O6-
alkylation with anti-O6-MeG antibody after 12 h treatment with DMSO
or AA-CW236 followed or not by the addition of temozolomide (shown
are confocal microscopy images; white bars indicate 10 mm). C) Quan-
tification of the guanine O6-alkylation staining (shown are relative
valuesÆSEM, n=3). The values were normalized against DAPI stain-
ing. D) Caco-2 cell viability curve after treatment with various concen-
trations of TMZ with or without 3 mm AA-CW236 (n=3ÆSD).
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[1] T. Lindahl, R. D. Wood, Science 1999, 286, 1897 – 1905.
[2] A. E. Pegg, M. E. Dolan, R. C. Moschel, Prog. Nucleic Acid Res.
1995, 51, 167 – 223.
[3] A. Juillerat, T. Gronemeyer, A. Keppler, S. Gendreizig, H. Pick,
H. Vogel, K. Johnsson, Chem. Biol. 2003, 10, 313 – 317.
[4] M. Esteller, J. G. Herman, Oncogene 2004, 23, 1 – 8.
[5] J. R. Silber, A. Blank, M. S. Bobola, S. Ghatan, D. D. Kolstoe,
M. S. Berger, Clin. Cancer Res. 1999, 5, 807 – 814.
[6] a) M. R. Middleton, G. P. Margison, Lancet Oncol. 2003, 4, 37 –
44; b) A. Sabharwal, M. R. Middleton, Curr. Opin. Pharmacol.
2006, 6, 355 – 363.
[7] a) M. Ranson, M. R. Middleton, J. Bridgewater, S. M. Lee, M.
Dawson, D. Jowle, G. Halbert, S. Waller, H. McGrath, L.
Gumbrell, R. S. McElhinney, D. Donnelly, T. B. H. McMurry,
G. P. Margison, Clin. Cancer Res. 2006, 12, 1577 – 1584; b) A. J.
Watson, M. R. Middleton, G. McGown, M. Thorncroft, M.
Ranson, P. Hersey, G. McArthur, I. D. Davis, D. Thomson, J.
Beith, A. Haydon, R. Kefford, P. Lorigan, P. Mortimer, A.
Sabharwal, O. Hayward, G. P. Margison, Br. J. Cancer 2009, 100,
1250 – 1256.
[8] H. A. Tawbi, L. Villaruz, A. Tarhini, S. Moschos, M. Sulecki, F.
Viverette, J. Shipe-Spotloe, R. Radkowski, J. M. Kirkwood, Br. J.
Cancer 2011, 105, 773 – 777.
[9] O. Khan, M. R. Middleton, Expert Opin. Invest. Drugs 2007, 16,
1573 – 1584.
[10] W. B. Parker, Chem. Rev. 2009, 109, 2880 – 2893.
[11] a) L. Guterman, Chem. Eng. News 2011, 89, 19 – 26; b) J. Singh,
R. C. Petter, T. A. Baillie, A. Whitty, Nat. Rev. Drug Discovery
2011, 10, 307 – 317; c) D. S. Johnson, E. Weerapana, B. F. Cravatt,
Future Med. Chem. 2010, 2, 949 – 964.
Finally, we sought to investigate whether the potent
inhibition of MGMT by AA-CW236 also leads to increased
guanine O6-alkylation in cancer cells, especially in combina-
tion with the drug temozolomide (TMZ). TMZ is currently
clinically used as a first-line treatment for glioblastoma
multiforme and a second-line option for astrocytoma.[22]
Using an O6-alkylguanine-directed antibody, we established
a confocal fluorescence microscopy based method for the
direct visualization of nuclear O6-alkylated guanine in MCF7
cells (Figures 4B, S10). Treatment of the cells with 300 mm
TMZ for eight hours resulted only in an approximately 25%
increase in O6-alkylguanine staining. Administration of 1 mm
AA-CW236 to MCF7 cells led to a similarly low increase in
guanine alkylation. However, when both compounds were
added to cells together, we detected a robust threefold
increase in O6-alkylguanine staining (Figures 4C, S10). Intri-
guingly, the observed sensitization[15] of MCF7 cells was
significantly stronger than the one caused by combining TMZ
with the best reported[9] MGMT pseudosubstrate lomeguatrib
(Figure S10). Furthermore, a sensitization effect was also
detected in a cell-viability assay with Caco-2 cells, which
display comparably high MGMT activity (Figure 3C) and are
resistant to TMZ.[23] Co-treatment with 3 mm AA-CW236 and
TMZ significantly reduced the cell viability in comparison to
TMZ (Figure 4D) or AA-CW236 (Figure S11) treatments.
In summary, we have presented a novel chemical scaffold
for cysteine-reactive covalent inhibitors. Rapid and modular
[12] S. Díez-Gonzµlez, N. Marion, S. P. Nolan, Chem. Rev. 2009, 109,
3612 – 3676.
[13] L. K. Rasmussen, B. C. Boren, V. V. Fokin, Org. Lett. 2007, 9,
5337 – 5339.
[14] a) R. Kitz, I. B. Wilson, J. Biol. Chem. 1962, 237, 3245 – 3249;
b) P. Bey, F. N. Bolkenius, N. Seiler, P. Casara, J. Med. Chem.
1985, 28, 1 – 2; c) T. Wirth, K. Schmuck, L. F. Tietze, S. A. Sieber,
2914
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 2911 –2915

Angewandte
Communications
Chemie
Angew. Chem. Int. Ed. 2012, 51, 2874 – 2877; Angew. Chem. 2012,
apana, M. M. Blewett, B. F. Cravatt, Nat. Methods 2014, 11, 79 –
124, 2928 – 2931.
85; c) M. Abo, E. Weerapana, J. Am. Chem. Soc. 2015, 137,
7087 – 7090.
[15] M. Clemons, J. Kelly, A. J. Watson, A. Howell, R. S. McElhinney,
T. B. H. McMurry, G. P. Margison, Br. J. Cancer 2005, 93, 1152 –
1156.
[16] a) C. Robinson, J. Palomo, M. A. Vogelbaum, Anal. Biochem.
2010, 405, 263 – 265; b) K. Ishiguro, K. Shyam, P. G. Penketh,
A. C. Sartorelli, Anal. Biochem. 2008, 383, 44 – 51.
[20] B. R. Lanning, L. R. Whitby, M. M. Dix, J. Douhan, A. M.
Gilbert, E. C. Hett, T. Johnson, C. Joslynl, J. C. Kath, S. Niessen,
L. R. Roberts, M. E. Schnute, C. Wang, J. J. Hulce, B. X. Wei,
L. O. Whiteley, M. M. Hayward, B. F. Cravatt, Nat. Chem. Biol.
2014, 10, 760 – 767.
[17] a) M. Uttamchandani, C. H. S. Lu, S. Q. Yao, Acc. Chem. Res.
2009, 42, 1183 – 1192; b) L. I. Willems, W. A. Van der Linden, N.
Li, K. Y. Li, N. Liu, S. Hoogendoorn, G. A. Van der Marel, B. I.
Florea, H. S. Overkleeft, Acc. Chem. Res. 2011, 44, 718 – 729;
c) G. C. Rudolf, W. Heydenreuter, S. A. Sieber, Curr. Opin.
Chem. Biol. 2013, 17, 110 – 117.
[18] D. Abegg, R. Frei, L. Cerato, D. P. Hari, C. Wang, J. Waser, A.
Adibekian, Angew. Chem. Int. Ed. 2015, 54, 10852 – 10857;
Angew. Chem. 2015, 127, 11002 – 11007.
[21] M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray, R. D.
Taylor, Proteins Struct. Funct. Genet. 2003, 52, 609 – 623.
[22] M. E. Hegi, A. Diserens, T. Gorlia, M. Hamou, N. de Tribolet, M.
Weller, J. M. Kros, J. A. Hainfellner, W. Mason, L. Mariani,
J. E. C. Bromberg, P. Hau, R. O. Mirimanoff, J. G. Cairncross,
R. C. Janzer, R. Stupp, N. Engl. J. Med. 2005, 352, 997 – 1003.
[23] T. Strittmatter, A. Brockmann, M. Pott, A. Hantusch, T.
Brunner, A. Marx, ACS Chem. Biol. 2014, 9, 282 – 290.
[19] a) E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare,
M. B. D. Dillon, D. A. Bachovchin, K. Mowen, D. Baker, B. F.
Cravatt, Nature 2010, 468, 790 – U779; b) C. Wang, E. Weer-
Received: December 6, 2015
Published online: January 22, 2016
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