Crystal Structures of Staphylococcus aureus Methionine Aminopeptidase Complexed with Keto Heterocycle and Aminoketone Inhibitors Reveal the Formation of a Tetrahedral Intermediate

Article abstract of DOI:10.1021/jm034188j

High-resolution crystal structures of Staphylococcus aureus methionine aminopeptidase I in complex with various keto heterocycles and aminoketones were determined, and the intermolecular ligand interactions with the enzyme are reported. The compounds are effective inhibitors of the S. aureus enzyme because of the formation of an uncleavable tetrahedral intermediate upon binding. The electron densities unequivocally show the enzyme-catalyzed transition-state analogue mimicking that for amide bond hydrolysis of substrates.

Full text of DOI:10.1021/jm034188j

J . Med. Chem. 2004, 47, 1325-1328
1325
Cr ysta l Str u ctu r es of Sta ph ylococcu s
a u r eu s Meth ion in e Am in op ep tid a se
Com p lexed w ith Keto Heter ocycle a n d
Am in ok eton e In h ibitor s Revea l th e
F or m a tion of a Tetr a h ed r a l In ter m ed ia te
Alice Douangamath, Glenn E. Dale, Allan D’Arcy,
Michael Almstetter,† Robert Eckl,†
Annabelle Frutos-Hoener, Bernd Henkel,†
Katrin Illgen,† Sven Nerdinger,† Henk Schulz,
Aengus MacSweeney, Michael Thormann,†
Andreas Treml,† Sabine Pierau, Sjoerd Wadman, and
Christian Oefner*
Morphochem AG, WRO-1055/ 388, Schwarzwaldallee 215,
CH-4058 Basel, Switzerland
F igu r e 1. Chemical structures of SaMetAP inhibitors. IC50
values are the average of three or more determinations
Received September 10, 2003
indicate a higher activity of the yeast MetAP I in the
presence of Zn2+, while the E.coli enzyme requires Mn2+
or Fe2+ 15-18
.
Abstr a ct: High-resolution crystal structures of Staphylococ-
cus aureus methionine aminopeptidase I in complex with
various keto heterocycles and aminoketones were determined,
and the intermolecular ligand interactions with the enzyme
are reported. The compounds are effective inhibitors of the S.
aureus enzyme because of the formation of an uncleavable
tetrahedral intermediate upon binding. The electron densities
unequivocally show the enzyme-catalyzed transition-state
analogue mimicking that for amide bond hydrolysis of sub-
strates.
Aldehydes, fluoroketones, keto esters, diketones, ke-
toamides as well as R-keto heterocycles have been
shown to be potent inhibitors for proteases. In all cases,
the scissile amide bond of the protease substrate is
replaced by an electron-deficient carbonyl group that
can readily hydrate. These molecules are expected to
be competitive protease inhibitors because the sp3-
hybridized hydrate resembles the transition state for
amide bond hydrolysis.19-23
Methionine aminopeptidases (MetAPs) are ubiquitous
enzymes found in both eukaryotic and prokaryotic cells
and play a critical role in the maturation of proteins
for proper function, targeting, and degradation.1 MetAP
removes the N-terminal methionine residue from newly
synthesized polypeptide chains that contain a small,
uncharged amino acid in the penultimate position.2,3 In
prokaryotes, mitochondria, and chloroplasts, translation
of proteins is initiated with N-formylmethionine, whereas
in the cytosol of eukaryotes protein biosynthesis begins
with methionine. However, the majority of mature
proteins do not retain this residue.4 The physiological
importance of MetAP is underscored by the lethality of
its absence in Escherichia coli, Salmonella typhimur-
ium, and Saccharomyces cerevisiae.5-7 Moreover, MetAPs
have been identified biochemically to be the molecular
target of the antiangiogenesis agent fumagillin and its
derivatives.8-10 Thus, as a result of their importance for
cell growth and proliferation, MetAPs are promising
antifungal, antibacterial, and antiangiogenesis targets.
Several crystal structures of the catalytic domain of
type I E. coli and S. aureus MetAP11-13 indicate an
identical fold and topology of the enzyme. The active
site, located near the center of a central â-sheet,
constitutes a dinuclear metal center formed by highly
conserved acidic residues. Several metal ions Fe(II), Zn-
(II), Mn(II), Co(II), and Ni(II) can be substituted in vitro
and retain the enzymatic activity.12,14 In vivo studies
In an attempt to design novel Staphylococcus aureus
methionine aminopeptidase (SaMetAP) I inhibitors, we
have identified two series of molecules, the keto het-
erocycles and the aminoketones, that inhibit the target
enzyme (Figure 1). The R-keto heterocycles and ami-
noketones are potent inhibitors of the Co2+-loaded
staphylococcal enzyme with IC50 values in the low
micromolar range, employing an assay previously de-
scribed.12 The inhibition pattern of the compounds was
typical for a competitive inhibitor when analyzed using
Dixon and Cornish-Bowden plots26 (data not shown). To
determine the mode of enzyme inhibition, we have
solved the crystal structures of the binary complexes
in the presence of cobalt at atomic resolution. Crystal-
lographic data and refinement statistics are presented
in the Supporting Information. Atomic coordinates have
been deposited with the Protein Data Bank (1QXW,
1QXY, 1QXZ).
The three-dimensional structure of the S. aureus
enzyme has been described and resembles the conserved
pita bread fold, common to all known MetAPs.11-13,27-29
Briefly, the S. aureus ensyme is similar to the E. coli
enzyme; two cobalt ions are located in the active site
and are coordinated by the side chains of the conserved
residues D97, D108, H171, E204, and E235. The resi-
dues interact in a monodentate or bidentate manner
similar to that observed for the E. coli enzyme.11,13 The
amino acid side chains of P59, E63, F65, C70, H79,
L177, and W221 form the S1 subsite of the enzyme on
one site of the metal center as a gorge, which has largely
hydrophobic character. It recognizes the N-terminal
* To whom correspondence should be addressed. Phone: ++41 61
6952116.Fax: ++41616952122.E-mail: christian.oefner@morphochem.ch.
† Morphochem AG, Gmuenderstrasse, 37a, 81379, Munich, Ger-
many.
10.1021/jm034188j CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/07/2004

1326 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Letters
hydrophobic S1 subsite. Spatial rearrangements occur
in this subsite upon inhibitor binding. The side chain
of E63 becomes ordered and adopts a conformation that
enlarges the S1 pocket. This allows the methyl group
of each compound to adopt different orientations corre-
sponding to the possible rotamers (Figure 2). The
heterocyclic ring systems and the cyclopropyl moiety
form the P1′ residues. Compared to the thiazole and
pyridine groups of compounds 1 and 2, the cyclopropyl
substituent is rotated by approximately 80°, orienting
it toward the inner side of the S1′ pocket (Figure 2).
Surprisingly, in both binary R-ketoheterocycle com-
plexes, a third cobalt ion (CoIII) is pentacoordinated in
the active site (Figure 2A,B). The formation of the
tetrahedral intermediate results in a favorable metal
binding environment formed by the hydroxyl group of
the gem-diol, the NE2 of H178, the nitrogen in the keto
heterocycle ring moiety, and two solvent molecules and
thus allows the trapping of a cobalt ion. In turn, this
metal ion helps in the stabilization of the anion derived
from the geminal diol, in a similar way as does the
oxyanion hole in serine proteases. The position of the
nitrogen atom in the heterocyclic thiazole and pyridine
moiety plays a critical role in the activity of the keto-
heterocycle inhibitors 1 and 2. Displacement of the ring
nitrogen to a site different from the â-position leads to
a complete loss of activity (SAR data will be published
elsewhere), which is in agreement with our crystal-
lographic data. The importance of a nitrogen atom at
the â-position in R-ketoheterocycles has also been
observed for inhibitors of serine proteases where this
atom plays a critical role in hydrogen-bond formation
with the histidine residue of the catalytic triad.24,25,31
F igu r e 2. Inhibitor binding to the active site of S. aureus
MetAP I. The left column shows compounds 1 (A), 2 (B), and
3 (C) in ball-and-stick form. Electrostatic interactions are
indicated by dashed lines. The right column shows the mo-
lecular surface of the active site of the S. aureus enzyme
superimposed with inhibitors and their final, experimentally
determined 2Fo - Fc electron density contoured at 1.0σ. The
figures were generated using PyMOL.28
The aminoketone inhibitor 3 interacts with the en-
zyme in a way that the secondary amine is oriented
toward H79 to provide a strong hydrogen bond. More-
over, H178 is flipped away from the metal ion center
(Figure 2C). In this case, no third cobalt ion is observed.
Instead, a network of water molecules connects the
hydroxyl group of the gem-diol to the carbonyl group of
L177 as well as to the carbonyl groups of T166 and A179
at the surface of the molecule with distances from 2.6
to 2.9 Å (Figure 2C). Similar stabilization of R-keto-
amide transition-state analogues by a network of water
molecules has been reported for thrombin inhibitors.33
Histidines 178 and 79, which are involved in the key
interactions with compounds 1-3, are strictly conserved
residues in all MetAPs sequenced to date. An H178A
mutant of the EcMetAP I enzyme has been shown to
exhibit a 50-fold lower activity than the wild-type
enzyme, supporting that H178 is not a catalytically
required residue.11 Recent kinetic and spectroscopic
analyses of H178A EcMetAP I suggests the implication
of H178 in regulating the pKa of the metal-bound
nucleophile.34 In contrast, the mutation of H79 to
alanine leads to a nearly complete loss of activity.11 This
result, combined with the structural information pro-
vided by the X-ray structures of EcMetAP I with
phorphorous-based transition-state analogues,13 sup-
ports the idea that H79 interacts with the nitrogen atom
of the scissile bond. This interaction seems to be crucial
for catalysis. Despite the loss of direct interactions with
H178, the aminoketone inhibitor 3 has an IC50 similar
to that of the ketoheterocycles 1 and 2. The loss of the
methionine residue of the substrate, whose binding
includes direct interactions with both metals.11 A sec-
ond, shallower binding pocket, located toward the
protein surface on the other site of the central metal
binding site, is composed of the residues A78, H79, and
F206 and the â-sheet residues L168, T169, and G170
and forms the S1′ subsite of the enzyme. It is believed
to accommodate the small, uncharged residue of the
substrate at the penultimate position.11
All three inhibitors show a common mode of inhibi-
tion. The high-resolution structures of the binary com-
plexes clearly indicate that the R-keto heterocycle and
aminoketone inhibitors of S. aureus MetAP I bind as
transition-state analogues. The sp2 carbonyl carbon of
all inhibitors undergoes a conversion to an sp3 carbon
in the tetrahedral intermediate upon binding (Figure
2). The transition-state analogues coordinate two cobalt
metal ions with one of the geminal hydroxyl groups
deriving from the activated solvent molecule, which
bridges the two cobalt ions in the apo structure.12 The
second hydroxyl function of the gem-diol provides the
sixth coordination to CoI. The common N-terminal
amino group of the inhibitors coordinates CoII, whereas
the methionyl side chain of the keto heterocycles and
the alkyl moiety of the aminoketone are located in the

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1327
(7) Miller, C. G.; Kukral, A. M.; Miller, J . L.; Movva, N. R. pepM is
an essential gene in Salmonella typhimurium. J . Bacteriol. 1989,
171, 5215-5217.
(8) Bradshaw, R. A.; Yi, E. Methionine aminopeptidases and an-
giogenesis. Essays Biochem. 2002, 38, 65-78.
interaction with H178 is sufficiently compensated by a
strong hydrogen bond with the side chain of H79.
In our search for inhibitors of SaMetAP I, we have
identified two series of compounds containing a keto
group, which are the keto heterocycle and the aminoke-
tone inhibitors. Trifluoroketones, R-keto esters, and
R-diketones, which are potent inhibitors of several
proteases, were reported to hydrate in aqueous DMSO
solution because of the electron-withdrawing effect of
the group neighboring the ketone moiety.19-23 Here, the
ketone group of the inhibitors 1 and 2 is not hydrated
in an aqueous DMSO solution31 (data not shown);
however, the compounds form an enzyme-catalyzed
transition-state analogue, mimicking that for amide
bond hydrolysis of substrates.
The activated water (or hydroxide ion) located be-
tween both metal ions attacks the keto group of the
inhibitors, which is further transformed into a tetrahe-
dral intermediate. Although isothermal titration calo-
rimetry studies on EcMetAP has indicated that the
active site can accommodate three cobalt ions, one with
high and two with low affinity,34 the stabilization of the
keto heterocycle transition-state analogues by a third
metal ion is questionnable because our experimental
conditions include 10 mM Co2+, a concentration that
certainly differs from the biological conditions.16,32 This
stabilization might as well occur through a network of
solvent molecules as observed for the aminoketone
inhibitor 3.
(9) Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.;
Brem, H.; Folkman, J . Synthetic analogues of fumagillin that
inhibit angiogenesis and suppress tumour growth. Nature 1990,
348, 555-557.
(10) Sin, N.; Meng, L.; Wang, M. Q.; Wen, J . J .; Bornmann, W. G.;
Crews, C. M. The anti-angiogenic agent fumagillin covalently
binds and inhibits the methionine aminopeptidase, MetAP-2.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6099-6103.
(11) Lowther, W. T.; Orville, A. M.; Madden, D. T.; Lim, S.; Rich, D.
H.; Matthews, B. W. Escherichia coli methionine aminopepti-
dase: implications of crystallographic analyses of the native,
mutant, and inhibited enzymes for the mechanism of catalysis.
Biochemistry 1999, 38, 7678-7688.
(12) Oefner, C.; Douangamath, A.; D’Arcy, A.; Haefeli, S.; Mareque,
D.; MacSweeney, A.; Padilla, J .; Pierau, S.; Schulz, H.; Thor-
mann, M.; Wadman, S.; Dale, G. E. The 1.15 Angstrom crystal
structure of the staphylococcus aureus methionyl-aminopepti-
dase and complexes with triazole based inhibitors. J . Mol. Biol.
2003, 332, 13-21.
(13) Lowther, W. T.; Zhang, Y.; Sampson, P. B.; Honek, J . F.;
Matthews, B. W. Insights into the mechanism of Escherichia
coli methionine aminopeptidase from the structural analysis of
reaction products and phosphorus-based transition-state ana-
logues. Biochemistry 1999, 38, 14810-14819.
(14) Li, J . Y.; Chen, L. L.; Cui, Y. M.; Luo, Q. L.; Li, J .; Nan, F. J .;
Ye, Q. Z. Specificity for inhibitors of metal-substituted methion-
ine aminopeptidase. Biochem. Biophys. Res. Commun. 2003, 307,
172-179.
(15) D’souza, V. M.; Holz, R. C. The methionyl aminopeptidase from
Escherichia coli can function as an iron(II) enzyme. Biochemistry
1999, 38, 11079-11085.
(16) D’souza, V. M.; Swierczek, S. I.; Cosper, N. J .; Meng, L.; Ruebush,
S.; Copik, A. J .; Scott, R. A.; Holz, R. C. Kinetic and structural
characterization of manganese(II)-loaded methionyl aminopep-
tidases. Biochemistry 2002, 41, 13096-13105.
(17) Walker, K. W.; Bradshaw, R. A. Yeast methionine aminopepti-
dase I can utilize either Zn2+ or Co2+ as a cofactor: a case of
mistaken identity? Protein Sci. 1998, 7, 2684-2687.
(18) Wang, J .; Sheppard, G. S.; Lou, P.; Kawai, M.; Park, C.; Egan,
D. A.; Schneider, A.; Bouska, J .; Lesniewski, R.; Henkin, J .
Physiologically relevant metal cofactor for methionine ami-
nopeptidase-2 is manganese. Biochemistry 2003, 42, 5035-
5042.
Finally, the structural data provide the foundation
for further optimization of these classes of inhibitors.
For example, the inhibitors could be optimized by
exploiting the S1 subsite, which can accommodate a
larger substituent, as observed with the triazole series
of inhibitors.12 Moreover, the cyclopropyl group allows
for extensions toward the S1′ subsite.
Ack n ow led gm en t. We gratefully acknowledge the
excellent technical assistance of S. Haefeli and Daniel
Mareque, and we thank Dr. C. Schulze-Briese at the
SLS protein crystallography beamline X06SA, Paul
Scherrer Institute, Villigen, Switzerland, for his experi-
mental help and advice.
(19) Andersson, L.; Isley, T. C.; Wolfenden, R. R-Aminoaldehydes:
transition state analogue inhibitors of leucine aminopeptidase.
Biochemistry 1982, 21, 4177-4180.
(20) Hu, L. Y.; Abeles, R. H. Inhibition of cathepsin B and papain by
peptidyl alpha-keto esters, alpha-keto amides, alpha-diketones,
and alpha-keto acids. Arch. Biochem. Biophys. 1990, 281, 271-
274.
(21) Mattis, J . A.; Henes, J . B.; Fruton, J . S. Interaction of papain
with derivatives of phenylalanylglycinal. J . Biol. Chem. 1977,
252, 6776-6782.
(22) Thaisrivongs, S.; Pals, D. T.; Kati, W. M.; Turner, S. R.;
Thomasco, L. M.; Watt, W. Design and synthesis of potent and
specific renin inhibitors containing difluorostatine, difluorosta-
tone, and related analogues. J . Med. Chem. 1986, 29, 2080-
2087.
Su p p or tin g In for m a tion Ava ila ble: Crystallographic
details and synthesis of the discussed compounds and their
spectral data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(23) Umezawa, H. Low-molecular-weight enzyme inhibitors of mi-
crobial origin. Annu. Rev. Microbiol. 1982, 36, 75-99.
(24) Parlow, J . J .; Dice, T. A.; Lachance, R. M.; Girard, T. J .; Stevens,
A. M.; Stegeman, R. A.; Stallings, W. C.; Kurumbail, R. G.;
South, M. S. Polymer-assisted solution-phase library synthesis
and crystal structure of R-ketothiazoles as tissue factor VIIa
inhibitors. J . Med. Chem. 2003, 46, 4043-4049.
(25) Costanzo, M. J .; Yabut, S. C.; Almond, H. R., J r.; Andrade-
Gordon, P.; Corcoran, T. W.; De Garavilla, L.; Kauffman, J . A.;
Abraham, W. M.; Recacha, R.; Chattopadhyay, D.; Maryanoff,
B. E. Potent, small-molecule inhibitors of human mast cell
tryptase. Antiasthmatic action of a dipeptide-based transition-
state analogue containing a benzothiazole ketone. J . Med. Chem.
2003, 46, 3865-3876.
(26) Cortes, A.; Cascante, M.; Cardenas, M. L.; Cornish-Bowden, A.
Relationships between inhibition constants, inhibitor concentra-
tions for 50% inhibition and types of inhibition: new ways of
analysing data. Biochem. J . 2001, 357, 263-268.
(27) Liu, S.; Widom, J .; Kemp, C. W.; Crews, C. M.; Clardy, J .
Structure of human methionine aminopeptidase-2 complexed
with fumagillin. Science 1998, 282, 1324-1327.
(1) Giglione, C.; Vallon, O.; Meinnel, T. Control of protein life-span
by N-terminal methionine excision. EMBO J . 2003, 22, 13-
23.
(2) Hirel, P. H.; Schmitter, M. J .; Dessen, P.; Fayat, G.; Blanquet,
S. Extent of N-terminal methionine excision from Escherichia
coli proteins is governed by the side-chain length of the penul-
timate amino acid. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8247-
8251.
(3) Moerschell, R. P.; Hosokawa, Y.; Tsunasawa, S.; Sherman, F.
The specificities of yeast methionine aminopeptidase and acetyl-
ation of amino-terminal methionine in vivo. Processing of altered
iso-1-cytochromes c created by oligonucleotide transformation.
J . Biol. Chem. 1990, 265, 19638-19643.
(4) Waller, J . P. The NH2 terminal residue of the proteins from cell-
free extract of E. coli. J . Mol. Biol. 1963, 7, 483-496.
(5) Chang, S. Y.; McGary, E. C.; Chang, S. Methionine aminopep-
tidase gene of Escherichia coli is essential for cell growth. J .
Bacteriol. 1989, 171, 4071-4072.
(6) Li, X.; Chang, Y. H. Amino-terminal protein processing in
Saccharomyces cerevisiae is an essential function that requires
two distinct methionine aminopeptidases. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12357-12361.

1328 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Letters
(28) Tahirov, T. H.; Oki, H.; Tsukihara, T.; Ogasahara, K.; Yutani,
K.; Ogata, K.; Izu, Y.; Tsunasawa, S.; Kato, I. Crystal structure
of methionine aminopeptidase from hyperthermophile, Pyrococ-
cus furiosus. J . Mol. Biol. 1998, 284, 101-124.
(32) Cosper, N. J .; D’souza, V. M.; Scott, R. A.; Holz, R. C. Structural
evidence that the methionyl aminopeptidase from Escherichia
coli is a mononuclear metalloprotease. Biochemistry 2001, 40,
13302-13309.
(33) Krishnan, R.; Tulinsky, A.; Vlasuk, G. P.; Pearson, D.; Vallar,
P.; Bergum, P.; Brunck, T. K.; Ripka, W. C. Synthesis, structure,
and structure-activity relationships of divalent thrombin inhibi-
tors containing an alpha-keto-amide transition-state mimetic.
Protein Sci. 1996, 5, 422-433.
(34) Copik, A. J .; Swierczek, S. I.; Lowther, W. T.; D’souza, V. M.;
Matthews, B. W.; Holz, R. C. Kinetic and spectroscopic charac-
terization of the H178A methionyl aminopeptidase from Es-
cherichia coli. Biochemistry 2003, 42, 6283-6292.
(29) Tahirov, T. H.; Oki, H.; Tsukihara, T.; Ogasahara, K.; Yutani,
K.; Libeu, C. P.; Izu, Y.; Tsunasawa, S.; Kato, I. High-resolution
crystals of methionine aminopeptidase from Pyrococcus furiosus
obtained by water-mediated transformation. J . Struct. Biol.
1998, 121, 68-72.
(30) DeLano, W. L. The PyMOL User’s Manual; DeLano Scientific:
San Carlos, CA, 2001.
(31) Tsutsumi, S.; Okonogi, T.; Shibahara, S.; Ohuchi, S.; Hatsushiba,
E.; Patchett, A. A.; Christensen, B. G. Synthesis and structure-
activity relationships of peptidyl R-keto heterocycles as novel
inhibitors of prolyl endopeptidase. J . Med. Chem. 1994, 37,
3492-3502.
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