Subtype-selectivity of metal-dependent methionine aminopeptidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.093

Inhibitors of methionine aminopeptidases (MetAPs) are treatment options for various pathological conditions. Several inhibitor classes have been described previously, but only few data on the subtype selectivity, which is of crucial importance for these enzymes, is available. We present a systematic study on the subtype- and species-selectivity of MetAP inhibitors that require the binding of an auxiliary metal ion. This includes, in particular, compounds based on the benzimidazole pharmacophore, but also hydroxyquinoline and picolinic acid derivatives. Our data indicates that a significant degree of selectivity can be attained with metal-dependent MetAP inhibitors.

Full text of DOI:10.1016/j.bmcl.2010.05.093

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4038–4044
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Subtype-selectivity of metal-dependent methionine aminopeptidase inhibitors
*
Markus A. Altmeyer, Aline Marschner, Rolf Schiffmann, Christian D. Klein
Department of Medicinal Chemistry, Universität Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of methionine aminopeptidases (MetAPs) are treatment options for various pathological
conditions. Several inhibitor classes have been described previously, but only few data on the subtype
selectivity, which is of crucial importance for these enzymes, is available. We present a systematic study
on the subtype- and species-selectivity of MetAP inhibitors that require the binding of an auxiliary metal
ion. This includes, in particular, compounds based on the benzimidazole pharmacophore, but also
hydroxyquinoline and picolinic acid derivatives. Our data indicates that a significant degree of selectivity
can be attained with metal-dependent MetAP inhibitors.
Received 30 April 2010
Revised 21 May 2010
Accepted 22 May 2010
Available online 27 May 2010
Keywords:
Methionine aminopeptidase
Benzimidazole
Hydroxyquinoline
Picolinic acid
Ó 2010 Elsevier Ltd. All rights reserved.
Selectivity
Methionine aminopeptidases (MetAPs) are ubiquitous enzymes
that are pursued as drug targets in the context of various pharma-
cological interventions, including the treatment of parasitic and
bacterial infections and the therapy of cancer.1,2 The biological
function of MetAPs is to remove the N-terminal initiator methio-
nine from nascent polypeptide chains, in order to recycle methio-
nine and prepare further post-translational modifications.
MetAPs play a central role in the processing and quality control
of proteins and are therefore essential for cell survival.3
the benzimidazole derivatives first described in our group are potent
inhibitors of Escherichia coli MetAP that require an auxiliary metal
ion. The binding mode of this class of compounds has been
elucidated by X-ray crystallography.10 The application of a metal-
mediated binding mode for inhibitor development offers novel
pharmacological opportunities, since it couples the inhibition of an
enzyme to the presence of metal ions. Furthermore, recent work
from the group of Qi-Zhuang Ye indicates that metal-dependent
MetAP inhibitors show activity under in vivo-conditions.12
The active site of MetAPs contains two transition metal ions.
While the nature of the biologically relevant element is debated, co-
balt ions (CoII) are usually employed as the metal for in vitro enzy-
mological studies.4 There exist two major classes of MetAPs, one
associated with eubacteria (MetAP-1) and one associated with
archaebacteria (MetAP-2). Eukaryotic organisms possess both clas-
ses. The structural scaffold of the two classes of MetAPs is highly
conserved.5,6
The most prominent inhibitory compound in the context of
MetAPs is fumagillin, which selectively targets the human subtype
2 of the enzyme.2,7 Fumagillin and its derivative TNP-470 have been
studied as inhibitors of tumor angiogenesis.8,9 Various other classes
of compounds have been described as inhibitors of bacterial or hu-
man MetAPs, including compounds that bind via an auxiliary metal
ion.10,11 Under in vitro-conditions, the auxiliary metal ion may be
‘recruited’ from excess metal present in the buffer. For example,
Relatively few data—and, in particular, practically no systematic
SAR studies on groups of analogs—exist on the subtype- and spe-
cies-selectivity of MetAP inhibitors. We have established a panel
of different MetAPs which allows us to perform selectivity studies
under uniform conditions (concentrations of enzymes and buffer
constituents, assay-readout).13 We would like to report here on
the activity and selectivity of metal-dependent inhibitors, in par-
ticular from the benzimidazole class,14 in those MetAPs with the
largest therapeutical relevance.
The results are summarized in Table 1, sorted according to the
structural scaffold and the inhibitory potency against E. coli MetAP.
The structure–activity relationships can be summarized as follows:
A. Benzimidazoles15
ꢀ
Benzimidazoles with a chelating substructure are potent
inhibitors of E. coli MetAP. Compounds that lack chelating
properties are inactive against all tested MetAPs (com-
pounds 1, 2, 3, 4 and the clinical drugs except thiabenda-
zole). This clearly indicates that binding to an auxiliary
transition metal ion is important for the activity of benz-
imidazoles, irrespective of the enzyme subtype.
Abbreviations: MetAP, methionine aminopeptidase; AAO, amino acid oxidase;
HRP, horseradish peroxidase.
* Corresponding author. Tel.: +49 6221 544875; fax: +49 6221 546430.
E-mail address: c.klein@uni-heidelberg.de (C.D. Klein).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.093

M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
4039
Table 1
Structure of the compounds and their activities against various MetAPs, sorted with respect to structural class
Compound ID/
generic name
Structure
E. coli MetAP
S. aureus MetAP
H. sapiens MetAP-1
H. sapiens MetAP-2
IC50 M]
Ref.
% Inh. IC50
[l
M]
% Inh. IC50
[l
M] % Inh. IC50 M] % Inh.
[l
[l
(25
n.i.
lM)
(25
20
lM)
(25
n.i.
l
M)
(25
lM)
H
N
1
2
n.i.
14
14
a
N
N
H
N
n.i.
n.i.
n.i.
n.i.
N
S
H
N
H
N
Fenbendazole
n.i.
26
34
12.5
10
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
N
O
S
O
H
N
3
14
N
H
N
O
H
N
a
Carbendazim
n.i.
N
O
O
H
N
H
N
a
Albendazole
35
66
10.5
12
n.i.
n.i.
10
18
N
O
S
O
H
N
4
14
N
HO
H
N
H
N
a
Mebendazole
47
n.i.
17
31
n.i.
34
n.i.
N
O
O
O
H
N
S
5
6
10.0 3.5
9.0 1.3
38.8 0.7 n.i.
14
14
N
N
H
N
H
N
35.9 0.6 25
10
N
NH2
S
H
N
S
7
7.2 0.1
5.2 1.0
4.6 0.4
4.3 0.6
n.i.
15
n.i.
21
34
28
15
26
35.5 2.5 39
47.0 3.8 46
43
40.6 2.4 14
28.0 1.3 14
32.9 2.7 14
38.4 5.5 14
N
N
H
N
S
8
N
N
Cl
H
N
N
9
N
N
H
N
S
10
45.8 3.1 46
N
N
F
H
N
S
11
3.9 0.5
30.2
41.7 2.3 n.i.
14
14
N
N
(continued on next page)

4040
M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
Table 1 (continued)
Compound ID/
generic name
Structure
E. coli MetAP
S. aureus MetAP
% Inh. IC50
H. sapiens MetAP-1
H. sapiens MetAP-2
IC50 M]
Ref.
% Inh.
(25 M)
IC50
[l
M]
[l
M] % Inh. IC50 M] % Inh.
[l
[l
l
(25
l
M)
(25
14
l
M)
(25
lM)
H
N
S
12
3.4 0.2
92
2.3 0.7
78
10.1 1.3 14
N
N
N
H2N
H
N
13
14
2.6 0.4
2.4 0.3
40.9
87
38.9 3.7 n.i.
17.9 1.9 37
36
44.1 1.6 14
28.4 2.9 14
N
H
N
32.6 2.4 39
41.0 2.8 14
N
N
H
N
S
15
2.4 0.5
n.i.
36
14
N
N
O
H
N
16
17
18
2.1 0.3
1.7 0.2
1.7 0.1
68
32
16
19.4 3.9 49
45.5 3.4 53
25
32.6 4.9 77
19.0 0.9 18
48.8 2.3 63
7.7 1.1
14
14
N
N
H
N
S
N
N
N
H
N
18.1 2.0 14
14.9 1.9 14
N
N
Cl
F
H
N
19
20
21
1.5 0.2
1.3 0.3
1.2 0.1
31
47
n.i.
40.1 1.5 43
29.2 1.3 15
26
34.7 3.3 64
N
N
H
N
11
14
14
N
N
H
O
H
N
S
48.5 0.5 15
N
N
O2N
22
0.99 0.13 n.i.
n.i.
n.i.
14
N
N
N
H
N
23
24
25
26
0.97 0.02 92
0.78 0.03 97
0.57 0.08 56
0.55 0.08 55
4.9 0.1
4.4 1.7
n.i.
15
92
22
9.3 0.4
14
14
N
N
H2N
H
HO
N
N
N
N
H
N
19.9 2.2 40
22.8 1.9 74
28.4 0.7 62
10.3 0.8 74
13.3 2.7 14
N
H
N
8.0 1.4
14
N
N
N

M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
4041
Table 1 (continued)
Compound ID/
generic name
Structure
E. coli MetAP
S. aureus MetAP
H. sapiens MetAP-1
H. sapiens MetAP-2
Ref.
% Inh.
(25 M)
IC50
[
l
M]
% Inh.
(25 M)
IC50
[
l
M] % Inh.
(25 M)
IC50
[l
M] % Inh. IC50 M]
[l
l
l
l
(25
12
lM)
H
N
H
N
27
0.54 0.03 52
22.4 2.4 25
14
N
S
N
N
N
S
28
0.50 0.01 n.i.
0.47 0.06 28
38
28
32.4 1.1 49
47.8 1.4 32
24.3 2.8 14
N
N
H
N
S
S
a
Thiabendazole
86.3
44.7 3.1
N
29
30
0.46 0.01 n.i.
0.43 0.02 29
n.i.
n.i.
14
N
N
N
H
N
33.0 4.0 33
37.3 3.6 42
32.9 1.7 14
N
N
NC
N
H
N
31
32
0.38 0.02 18
0.24 0.04 51
28
33.9 3.9 36
40.1 1.2 24
36.4 3.7 14
48.4 2.4 14
N
N
N
H
N
N
22.0 2.7 29
N
N
N
H
N
33
34
35
0.16 0.02 n.i.
0.11 0.00 76
0.08 0.01 21
45
18.8 0.7 39
26
28.8 1.6 n.i.
27.6 1.9 49
39.8 1.9 17
21.5 2.3 85
14
26.7 1.5 14
14
N
N
O2N
H
N
N
N
N
N
H
S
N
N
N
8-Hydroxy
quinoline
a
95
32
0.77
65
10
18.3 1.3 54
5.6 0.8
N
OH
N
N
N
N
36
37
38
39
29
42.8 1.9 39
34.8 2.8 19
O
O
H
N
n.i.
n.i.
n.i.
n.i.
62
n.i.
16.0 1.9 29
15
17
41.2 1.2 25
18
19
O
41.7 4.1 19
O
O
S
S
O
O
n.i.
19
O
O
(continued on next page)

4042
M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
Table 1 (continued)
Compound ID/
generic name
Structure
E. coli MetAP
S. aureus MetAP
% Inh. IC50
H. sapiens MetAP-1
H. sapiens MetAP-2
IC50 M]
Ref.
% Inh.
IC50
[l
M]
[l
M] % Inh. IC50 M] % Inh.
[l
[l
(25
lM)
(25
l
M)
(25
15
l
M)
(25
lM)
N
40
38
n.i.
24
19
N
O
O
S
S
O
O
N
H
N
41
42
93
11.0
47
22.9 1.9 28
44.9 1.9 32
39.8 2.4 19
O
O2N
H
N
S
n.i.
n.i.
n.i.
n.i.
n.i.
16
N
O
H
N
O
43
44
45
46
21
23
15
12
14
20
S
N
N
O
O
O
H
N
N
23
24
n.i.
21
16
N
N
S
S
N
O
H
N
80
2.8
20
O
O
H
N
N
100
0.06 0.01 58
21.2 1.3 89
8.1 3.0
48
25.8 1.3 16
N
O
S
Most compounds were screened at a single concentration (25
lM) and IC50 values were determined only if the initial screen showed relevant activity. To ease the
interpretation of the data, IC50 values below 10
l
M are highlighted with borders and IC50 values between 10 and 20 M are highlighted with dashed borders. The enzyme
l
concentration was 22 nM. n.i., no inhibition.
a
These compounds are part of the in-house compound collection and were originally purchased or donated by commercial suppliers.
ꢀ
ꢀ
Only a few compounds show activity against the MetAPs
from organisms other than E. coli. These are, in particular,
24, 23, 16, and 12. These compounds tend to have hydro-
gen-bonding functional groups in a position that allows
specific interactions at the entrance of the MetAP active
site, as is illustrated in Figure 1 for compound 24. A
detailed discussion of the binding mode and its implica-
tions is given in the figure legend.
Most notably, it is possible to achieve subtype selectivity
with metal-dependent inhibitors. This is illustrated by
compound 24, which has a submicromolar IC50 at the
bacterial MetAPs and practically no affinity towards
eukaryotic MetAPs. This is particularly relevant for the
suggested application of MetAP inhibitors as antibacterial
drugs. In this context, compound 24 is one of the very few
compounds with documented selectivity for bacterial
MetAPs and may serve as a starting point for further
optimization.
It is very remarkable that the N-benzyl-substituted ana-
logs (22 and 29) are highly selective at the E. coli enzyme
and possess no activity against the other MetAPs. The
selectivity is not attributable to differences in the hydro-
gen-bonding or metal-binding properties but rather to
the steric bulk of the benzyl group, as the methyl analog
28 retains some activity against the human MetAPs.
Because of their significant selectivity, the N-benzyl-
congeners may serve as leads for the development of
MetAP inhibitors with selective, antibiotic activity
against E. coli or other Gram-negative pathogens.
ꢀ
Compound 20 is tautomeric and may, in theory, be
present as the pyridone or hydroxypyridine species.
The pyridone tautomer is not expected to bind to
MetAPs via the chelating mechanism and the auxiliary
metal ion. We therefore studied the interaction of com-
pound 20 with cobalt ions in solution by UV–vis spec-
trometry and compared this to the non-hydroxylated
analog 25. The latter forms a cobalt complex, which
is evident from a bathochromic shift of the main UV–
vis absorption band from 320 to 350 nm. No changes
in the UV–vis spectrum upon addition of cobalt ions
could be observed for compound 20. We can therefore
conclude that this compound exists exclusively or pre-
dominantly as the pyridone tautomer and most likely
has an alternative binding mode. This would also
explain the SAR of this compound, which is, in compar-
ison to compound 25, practically inactive against the
human MetAP-1 and MetAP-2. It may therefore be con-
sidered as a novel starting point for the development of
MetAP inhibitors with selectivity for the bacterial iso-
forms. The logical next step in that direction would
be to combine this structural feature with the
N-benzyl substitution discussed above.
ꢀ

M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
4043
auxiliary metal ion. This can be realized with various structural scaf-
folds, including the highly drug-like benzimidazole pharmacophore
and the hydroxyquinoline moiety. Furthermore, the data shows that
even very moderate structural variations, like the N-benzyl group in
compounds 29 and 22, lead to significant changes in the selectivity
profiles of the compounds. When applied to the hydroxyquinoline
moiety, analogous modifications in the positions opposite to the
chelator function may lead to highly potent and subtype-selective
MetAP inhibitors.
Compound 20 has significant selectivity for the bacterial
MetAPs and is not a metal chelator. A combination of the pyridone
group with a N-benzyl substituted benzimidazole core with may
yield potent and selective inhibitors of bacterial MetAPs that are
no longer prone to form undesired metal complexes.
The binding of inhibitors to auxiliary metal ions in MetAPs may
be advantageous with respect to subtype- and species-selectivity:
Whereas the core of the active site with its two ‘native’ metal ions
and the methionine binding pocket is highly conserved among dif-
ferent MetAPs, this is not the case for the entrance region of the
active site. First, the binding affinity of the auxiliary metal ion is al-
ready highly variable among different MetAPs; Second, inhibitors
that rely on the auxiliary metal ion will experience a highly vari-
able binding environment, as has been discussed in this article
for several selective compounds. Therefore, it is the entrance
region of MetAPs and not the catalytic core that needs to be
addressed to generate subtype-selective compounds, and metal-
dependent inhibitors may be a viable approach to attain this goal.
Figure 1. Compound 24 docked at the auxiliary cobalt ion at the entrance of the
E. coli MetAP active site. The docking pose is identical to the X-ray structure of
thiabendazole complexed with E. coli MetAP (PDB code 1YVM).10,17 His79 is highly
conserved in MetAPs and it appears likely that all MetAPs can, in principle, bind a
third metal ion at this residue.18 The binding of inhibitors to this metal ion,
however, depends on additional, spatial requirements at the active site entry region
of MetAPs. This is clearly demonstrated by the selectivity profiles of the benzimid-
azole derivatives and the other compound classes presented in this article. In the
case of E. coli MetAP, there is an additional histidine residue (His63) which is not
conserved among the MetAPs studied here and may (upon rotation of the side-
chain) form a hydrogen bond to the phenolic hydroxy group of 24, thus explaining
the high affinity of the compound to this enzyme.
B. Quinolines
ꢀ
Our lead compound for this small set of compounds was
8-hydroxyquinoline. We initially tested this compound
because it is a well-known, bi-dentate complexing agent
used in analytical chemistry. The significant activity
against E. coli MetAP prompted us to perform a very lim-
ited exploration of the SAR (compounds 36–41), which—
not unexpectedly—indicated that the activity depends on
the chelator properties of the molecule. Because of the
ubiquitous binding of the unsubstituted 8-hydroxyquino-
line to various metal ions, we did not further pursue it as
a lead structure. Independent of our work, Huang et al.
described quinolinyl sulfonamides such as 41 as inhibi-
tors of Mn-loaded E. coli MetAP11 that bind to an auxil-
iary, third manganese ion.
Acknowledgments
This work was supported by the Deutsche Forschungsgeme-
inschaft (KL 1356). The authors thank Dr. S. Schütt of the Med-
ical Microbiology Department at Heidelberg University Clinic for
the S. aureus DNA and Dr. D. Kuck of the German Cancer Re-
search Center for advice on the expression of H. sapiens Me-
tAP-2 in Sf9 cells. E. coli MetAP was obtained using the
expression plasmid generously provided by Profs. B. Matthews
and T. Lowther.
Supplementary data
ꢀ
The selectivity data shown here indicates that 8-hydroxy-
quinoline has a remarkable selectivity towards HsMetAP-
2 (as compared to HsMetAP-1) and is the most potent
inhibitor of this enzyme in the present dataset. Thus,
chelating quinoline derivatives, where the chelator phar-
macophore is conserved, may be promising lead com-
pounds for the development of metal-dependent
HsMetAP-2 inhibitors. Since a wealth of structural data
is already available on this compound class, the struc-
ture-guided development of inhibitors with certain selec-
tivity profiles may be a promising approach.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.093.
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3620.
C. Picolinic acid derivatives
ꢀ
This class of compounds, first described by Luo et al.,16
also has metal-chelating properties. 46 is the most potent
inhibitor of E. coli MetAP in the present dataset. It is also a
moderately active inhibitor of the Homo sapiens MetAP-1,
but has a 100-fold selectivity for the bacterial enzyme.
We prepared and tested a few compounds from this class,
including sulfonamide analogs, but were unable to
increase their activity or modify the selectivity profile.
11. Huang, M.; Xie, S. X.; Ma, Z. Q.; Hanzlik, R. P.; Ye, Q. Z. Biochem. Biophys. Res.
Commun. 2006, 339, 506.
12. Chai, S. C.; Ye, Q. Z. Bioorg. Med. Chem. Lett. 2009, 19, 6862.
13. The enzymes were obtained by heterologous expression in E. coli
(Staphylococcus aureus MetAP, E. coli MetAP, H. sapiens MetAP-1) or in Sf9
cells (H. sapiens MetAP-2) and subsequent chromatographic purification. For
the cloning of the S. aureus MetAP gene, genomic DNA from a multi-resistant
COL strain was used. E. coli MetAP was obtained using the expression plasmid
The data demonstrates that a subtype- and species-selective
inhibition of MetAPs is possible with inhibitors that bind to an

4044
M. A. Altmeyer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4038–4044
described by Profs. B. Matthews and T. Lowther (Lowther, W. T.; McMillen, D.
16. Luo, Q. L.; Li, J. Y.; Liu, Z. Y.; Chen, L. L.; Li, J.; Qian, Z.; Shen, Q.; Li, Y.;
Lushington, G. H.; Ye, Q. Z.; Nan, F. J. J. Med. Chem. 2003, 46, 2631.
A.; Orville, A. M.; Matthews, B. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12153)
The genes of the H. sapiens MetAPs were synthesized by a commercial supplier.
A detailed description of the cloning and expression of H. sapiens MetAP-2 in
Sf9 cells is given in the supporting information. Enzymatic assays were
performed as described previously (Schiffmann, R.; Neugebauer, A.; Klein, C. D.
J. Med. Chem. 2006, 49, 511). The concentration of all MetAPs in the enzymatic
assays was 22 nM. Interactions with the detection enzymes AAO and HRP were
excluded for all tested compounds.
17. Docking was based on the 1YVM structure of E. coli MetAP (without the
thiabendazole ligand) as receptor and the GOLD 4.1 software (Jones, G.; Willett,
P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43) with default settings. Visualization was
performed using the Chimera software from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San Francisco
(Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.;
Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605).
14. Schiffmann, R.; Neugebauer, A.; Klein, C. D. J. Med. Chem. 2006, 49, 511.
15. The syntheses of some of the compounds presented here (cf. Table 1) were
described previously. Synthetic and analytical data for the other compounds
are given in the supporting information. Commercial drug substances
(thiabendazole, fenbendazole, etc.) were taken from the in-house compound
collection.
18. Multiple sequence alignments were generated by ClustalW (Thompson, J. D.;
Higgins, D. G.; Gibson, T. J., Nucleic Acids Res. 1994, 22, 4673) and analyzed with
BioEdit (Hall, T. A. Nucleic Acids Symp. Ser. 1999, 41, 95).
19. Clark-Lewis, J. W.; Thompson, M. J. J. Chem. Soc. 1957, 442.
20. Caldwell, W. T.; Kornfeld, E. C. J. Am. Chem. Soc. 1942, 64, 1695.
21. Goulaouic-Dubois, C. A.; Guggisberg, A.; Hesse, M. J. Org. Chem. 1995, 60, 5969.