A simple structural-based approach to prevent aminoglycoside inactivation by bacterial defense proteins. Conformational restriction provides effective protection against neomycin-B nucleotidylation by ANT4

Article abstract of DOI:10.1021/ja051722z

Herein, we describe how the conformational differences exhibited by aminoglycosides in the binding pockets of the ribosome and those enzymes involved in bacterial resistance can be exploited in the design of new antibiotic derivatives with improved activi

Full text of DOI:10.1021/ja051722z

Published on Web 05/17/2005
A Simple Structural-Based Approach to Prevent Aminoglycoside Inactivation
by Bacterial Defense Proteins. Conformational Restriction Provides Effective
Protection against Neomycin-B Nucleotidylation by ANT4
Juan Luis Asensio,*,† Ana Hidalgo,† Agatha Bastida,† Mario Torrado,† Francisco Corzana,†
Jose Luis Chiara,† Eduardo Garc´ıa-Junceda,† Javier Can˜ada,‡ and Jesu´s Jime´nez-Barbero‡
Instituto de Qu´ımica Orga´nica (CSIC), Juan de la CierVa 3, 28006 Madrid, Spain, and Centro de InVestigaciones
Biolo´gicas (CSIC), 28040 Madrid, Spain
Received March 18, 2005; E-mail: iqoa110@iqog.csic.es
The emergence of bacterial resistance to the major classes of
antibiotics has become a serious problem over recent years.1 For
aminoglycosides, the major biochemical mechanism for bacterial
resistance is the enzymatic modification of the antibiotic.1 The
search for new derivatives not susceptible to modification by
bacterial defense proteins constitutes an active field of research.2-5
Herein, we describe how the conformational differences exhibited
by these flexible ligands within the binding pockets of the ribosome
and of those enzymes involved in bacterial resistance can be
exploited for designing new antibiotic derivatives with improved
activity in resistant strains.
Staphylococcus aureus ANT4, an enzyme involved in the
bacterial defense against aminoglycosides, catalyzes the transfer
of one adenyl group from ATP to position O4 (see Figure 1) of
the glucose (Glc) unit present in most aminoglycosides. This transfer
leads to a sharp decrease in the drug affinity for its target RNA.
The 3D structure of ANT4 complexed to kanamycin has been
determined by using X-ray.6 In addition, the structures of related
analogues, such as gentamycin, tobramycin, and paromomycin,
bound to the target ribosomal RNA have also been determined
recently.7 Interestingly, the oligosaccharide conformation recognized
by the RNA and the enzyme are remarkably different (Figure 1).
In particular, the GlcR(1-4)-2-deoxy streptamine fragment adopts
a syn-Ψ conformation (Φ/Ψ ) -40/-30) for tobramycin and paro-
momycin in the RNA-bound state. This pseudodisaccharide moiety,
with different patterns of amination/hydroxylation at the Glc unit,
is present in all aminoglycosides. The syn-Ψ geometry also con-
stitutes the most populated minimum for the free antibiotics, accord-
ing to NMR data.8 In contrast, the analogous glycosidic linkage of
kanamycin bound to ANT4 is defined by an anti-ψ geometry (φ/ψ
) -21/152). This conformation represents a high-energy minimum
that must be stabilized by specific aminoglycoside-ANT4 interac-
tions. The different 3D shape adopted by these aminoglycosides in
the RNA- and enzyme-bound states suggests a possible structure-
based chemical strategy to overcome bacterial resistance. Assuming
that some degree of conformational distortion at the GlcR(1-4)-2-
deoxy streptamine fragment is required for enzymatic activity, it
should be possible to design a conformationally locked oligosac-
charide that still retains antibiotic activity, but that is not susceptible
to inactivation by ANT4.9 Following this strategy (Scheme 1), we
have designed and synthesized the conformationally constrained
neomycin-B derivative 5. In 5, a direct covalent bond has been
formed between positions O5Rib and N2Glc that, for the natural
antibiotic, are hydrogen bonded in the ribosome-bound state.7a,b
Compound 5 was prepared following the simple four-step
procedure shown in Scheme 1. Thus, hexa-N-(carbobenzyloxy)
derivative 2, readily prepared from neomycin-B 1, was treated with
Figure 1. (Bottom right) Schematic representation of the GlcR(1-4)-2-
deoxy streptamine fragment of tobramycin (X ) NH2, Y ) H, Z ) NH2,
R ) 3-amino-3-deoxy-R-Glc, R′ ) H) and paromomycin (X ) NH2, Y )
OH, Z ) OH, R ) H, R′ ) 2,6-diamino-2,6-dideoxy-L-Idoâ(1-3)Rib) in
their ribosome-bound states. (Top right) Schematic representation of the
same disaccharide fragment in kanamycin (X ) OH, Y ) OH, Z ) NH2,
R ) 3-amino-3-deoxy-R-Glc, R′ ) H) in the ANT4-bound state. The OH
group modified by the enzyme is marked with a gray arrow. The two
receptors recognize different conformations of the ligand. (Left) Our design
is based on locking this disaccharide fragment in its RNA-bound conforma-
tion by using a ribose (Rib) bridge (in red) to form the neomycin-B analogue
5. This modification might prevent inactivation of the antibiotic by ANT4,
while preserving antibiotic activity. The definition employed for the glyco-
sidic torsion angles (φ/ψ) is shown at the bottom left corner of the figure.
Scheme 1 a
a (a) CbzCl, Na2CO3, MeOH/H2O 3:1, 0 °C, 3 h, 92%; (b) 2,4,6-
triisopropylbenzenesulfonyl chloride (TIBSCl), Py, rt, 72 h, 44%; (c) H2
(1 atm), Pd/C, MeOH, trifluoroacetic acid, rt, 8 h, 90%; (d) H2O, pH 7.0,
60 °C, 7 days, 56% (see Supporting Information). In 1, 4, and 5, R′ )
2,6-diamino-2,6-dideoxy-â-L-idose. In 2 and 3, R′ ) 2,6-diamino-2,6-di-
N-(carbobenzyloxy)-2,6-dideoxy-â-L-idose.
2,4,6-triisopropylbenzenesulfonyl chloride in pyridine to yield the
primary sulfonate 3 regioselectively.10 Deprotection of the amino
groups by hydrogenation in MeOH/TFA gave crude sulfonate 4
† Instituto de Qu´ımica Orga´nica.
‡ Centro de Investigaciones Biolo´gicas.
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10.1021/ja051722z CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
mixture at 310 K was monitored by 1D NMR, and the final products
were characterized by 2D NMR and MALDI-TOF MS. As shown
in Figure 3, under these experimental conditions, adenylation of 1
is completed within 2 min. NMR analysis permitted the confirma-
tion that ANT4 selectively modifies OH4Glc. In fact, if only 1 equiv
of ATP is employed, this is the only product detected. Interestingly,
if 2 equiv of ATP is used, nonselective secondary adenylations are
detected, but at much longer reaction times. Indeed, under the
employed experimental conditions, the enzyme requires more than
12 h to consume the second ATP equivalent once the primary
adenylation is completed. The presence of diadenylated neomycin-B
derivatives was confirmed by MALDI-TOF.
A completely different behavior was exhibited by the confor-
mationally constrained analogue 5. In this case, under identical
experimental conditions, no reaction was detected even 30 min after
enzyme addition. At longer reaction times, slow nonselective
adenylation processes (similar to those described above for 1) were
observed. Nevertheless, NMR analysis of the reaction mixture
confirmed that 2 h after addition of the enzyme, more than 95% of
5 remained unmodified.
Figure 2. (Left) Average NMR conformation (see Supporting Information)
of neomycin-B analogue 5 superimposed onto the X-ray structure of
paromomycin complexed to ribosomal RNA.7a,b The 3D structure of the
constrained aminoglycoside closely resembles the paromomycin bioactive
conformation (φ and ψ values for both structures are also shown). (Right)
Experimental MIC values (µg/mL) measured for neomycin-B 1 and its
conformationally constrained mimic 5.
Finally, the in vivo activities of neomycin-B (1) and mimic 5
were tested employing the bacteria Escherichia coli DH5R
(pBBRIMCS-2), which expresses the resistance enzyme ANT4. As
expected, the MIC value for the natural antibiotic is significantly
increased (from 3 to 60 µg/mL). In contrast, the cyclic derivative
5 maintains the same activity observed for the nonresistant bacteria
(20 µg/mL). In conclusion, this simple modification leading to the
conformationally restricted 5 provides an effective protection against
aminoglycoside inactivation by S. aureus ANT4, both in vivo and
in vitro, while maintaining a significant antibiotic activity.
Thus, in our opinion, this example represents a test case of the
validity of a structure-based approach for designing and preparing
ligands that specifically interact with a given receptor and might
potentially be used as antibiotics.
Figure 3. Evolution of neomycin-B 1/ATP (left) and mimic 5/ATP (right)
mixtures in 20 mM phosphate buffer, pH 7.0, 10 mM MgCl2, 310 K, after
addition of ANT4 (1 µM). A is for ATP; 1-A and 1-A2 are for
monoadenylated and diadenylated derivatives of 1, respectively.
Acknowledgment. We thank DGES (CTQ2004-04494/BQU)
and Comunidad de Madrid for funding.
that smoothly cyclized in a highly regioselective way by mild
heating in water to give 5 in 56% yield.
Supporting Information Available: Extended experimental details
about the design, synthesis, NMR and computational analysis, and
enzymatic and biological testing of 5. This material is available free
of charge via the Internet at http://pubs.acs.org.
According to NMR and MD calculations, the conformational con-
straint imposed by the covalent bond of C5Rib to N2Glc in 5 is espec-
ially severe (see Supporting Information). In fact, only very minor
fluctuations around the glycosidic linkages and one unique pucker-
ing for the Rib ring are allowed. Moreover, the geometry of 5 close-
ly resembles the bioactive conformation of the natural antibiotic.
The average NMR structure of 5 superimposed on the X-ray struc-
ture of paromomycin in complex with A-site RNA7b is shown in
Figure 2. Deviations in φ/ψ values with respect to the bound struc-
ture are less than 11 and 21° for the Glc/Strp and Rib/Strp linkages,
respectively. In addition, the furanose ring is locked in a very similar
conformation to that present in the RNA-bound geometry, with an
identical orientation of the key polar groups, O3 and O4.
The biological activity of 5 was tested against different bacteria.
The obtained MIC values are shown in Figure 2. It is important to
bear in mind that OH5Rib is involved in RNA recognition,
participating in the hydrogen bond to G1491.7a,b Cyclization of
neomycin-B (1) to give 5 requires removal of this hydroxyl group.
Interestingly, despite this modification, 5 displays significant activity
against both Gram-positive and Gram-negative bacteria.
References
(1) (a) Magnet, S.; Blanchard, J. Chem. ReV. 2005, 105, 477-497. (b) Smith,
C. A.; Baker, E. N. Curr. Drug Targets: Infect. Disord. 2002, 2, 143.
(2) (a) Kondo, S.; Iinuma, K.; Yamamoto, H.; Maeda, K.; Umezawa, H. J.
Antibiot. 1975, 26, 412. (b) Kondo, S.; Hotta, K. J. Infect. Chemother.
1999, 5, 1.
(3) Inoue, M.; Nonoyama, M.; Okamoto, R.; Ida, T. Drugs Exp. Clin. Res.
1994, 20, 233.
(4) Fujimura, S.; Tokue, Y.; Takahashi, H.; Nukiwa, T.; Hisamichi, K.;
Mikami, T.; Watanabe, A. J. Antimicrob. Chemother. 1998, 41, 495.
(5) (a) Roestamadji, J.; Graspas, I.; Mobashery, S. J. Am. Chem. Soc. 1995,
117, 11060. (b) McKay, G. A.; Roestamadji, J.; Mobashery, S.; Wright,
G. D. Antimicrob. Agents Chemother. 1996, 40, 2648. (c) Haddad, J.;
Vakulenko, S.; Mobashery, S. J. Am. Chem. Soc. 1999, 121, 11922. (d)
Grapsas, I.; Lerner, S. A.; Mobashery, S. Arch. Pharm. 2001, 334, 295.
(6) Pedersen, L. C.; Benning, M. M.; Holden, H. M. Biochemistry 1995, 34,
13305.
(7) (a) Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Morgan-Warren, R.
J.; Wimberly, B. T.; Ramakrishnan, V. Nature 2000, 407, 340. (b) Vicens,
Q.; Westhof, E. Structure 2001, 9, 647. (c) Yoshizawa, S.; Fourmy, D.;
Puglisi, J. D. EMBO J. 1998, 17, 22, 6437. (d) Vicens, Q.; Westhof, E.
Chem. Biol. 2002, 9, 747.
(8) Asensio, J. L.; Hidalgo, A.; Cuesta, I.; Gonza´lez, C.; Can˜ada, J.; Vicent,
C.; Chiara, J. L.; Cuevas, G.; Jime´nez-Barbero, J. Chem.sEur. J. 2002,
8, 5228.
(9) A key example of how enzymatic activity can be modulated by small
conformational preferences of the substrates was reported recently: Galan,
M. C.; Venot, A. P.; Boons, G. J. Biochemistry 2003, 42, 8522.
(10) Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361.
In a second step, the activity of ANT4 toward neomycin-B (1)
and the locked derivative 5 as substrates was tested. Enzymatic
reactions were monitored in NMR tubes. ATP (1.5-3.0 mM) and
the aminoglycoside (1.5 mM) were dissolved in phosphate buffer.
After addition of the enzyme (1 µM), the evolution of the reaction
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