A R T I C L E S
Haddad et al.
bacteria were diluted 100 times in LB broth and subgrown for several
hours, and 10 µL of diluted cultures was transferred into the antibiotic-
containing microtiter plates to bring final inoculums to 105 CFU/mL
(CFU stands for colony forming units). Cultures were incubated
overnight at 35 °C, and microtiter plates were checked from below
with a reflective viewer. MICs were defined as the lowest concentrations
of the drug at which the microorganism did not demonstrate visible
growth.
Methods
Proton (1H) and carbon (13C) nuclear magnetic resonance spectra
were recorded on either a Varian 400 or a Varian unity-500 MHz
spectrometer. Chemical shifts are recorded in parts per million (δ)
relative to tetramethylsilane (δ 0.00). Infrared (IR) spectra were recorded
on a Nicolet 680 DSP FTIR spectrometer. Low-resolution mass spectra
(MS) were recorded on a Kratos MS 80RFA spectrometer. High-
resolution mass spectra were performed by the Michigan State
University Mass Spectrometry Facility. Melting points were obtained
on an Electrothermal melting point apparatus and are uncorrected. Thin-
layer chromatography (TLC) was performed with Whatman precoated
K6F silica gel 60A (0.25 mm thickness plates). The plates were
visualized by either ninhydrin spray or immersion in a p-anisaldehyde
solution and warming on a hot plate. All chromatography solvents were
reagent grade. Tetrahydrofuran (THF) was distilled from sodium
benzophenone ketyl, and dichloromethane was distilled from calcium
hydride. 1,1-Dimethoxycyclohexane was purchased from the TCI
America Co. N-Cbz-protected diaminoalkanes were either prepared33
or purchased from the Fluka chemical co. as hydrochloride salt. The
amine free bases were prepared by treatment of the HCl salts with
Amberlite IRA-400 (OH-) ion-exchange resin. 3-Amino and 4-amino
phenylacetic acids were purchased from Fluka chemical co. and
converted to the N-Cbz-protected derivatives prior to use. Pyruvate
kinase (PK), lactic dehydrogenase (LD), phospho(enol)pyruvate (PEP),
ATP, and NADH were purchased from the Sigma Chemical Co. All
other reagents were purchased from the Aldrich Chemical Co. Spec-
trophotometric studies were performed on a Hewlett-Packard 8453 diode
array instrument. All calculations were performed by the MS Excel
program.
Kinetic Determinations with Resistance Enzymes. Kinetic studies
were performed for phosphotransferase activities of the bifunctional
enzyme AAC(6′)/APH(2′′) and APH(3′)-Ia, as well as the acetyltrans-
ferase activity of AAC(6′)/APH(2′′), using the methods described by
Azucena et al.27 The assay mixture consisted of 66 mM PIPES, pH
7.5, 11 mM magnesium acetate, 22 mM potassium acetate, 1.76 mM
phosphoenol pyruvate, 0.1 mM NADH, 6.1 units of pyruvate kinase,
21 units of lactate dehydrogenase, 100 nM enzyme, the aminoglycoside
substrate (at various concentrations), and 150 µM ATP in 500 µL total
volume. The components of the assay mixture were mixed in a cuvette
in the absence of ATP and enzyme. The solution was allowed to
equilibrate at room temperature for 2 min. The reaction was started by
the addition of ATP and enzyme. The progress of the reaction was
monitored spectrophotometrically at 340 nm. Lineweaver-Burk plots
were obtained to determine the Km and kcat values.
For the acetyltransferase activity assay, the method of Haas and
Dowding was employed.34 The reaction mixture was composed of 58
mM citric acid, 124 mM tripotassium citrate, 18 mM magnesium
acetate, 6 mM dithiothreitol, the aminoglycoside substrate (at various
concentrations), and 120 µM acetylcoenzyme A (specific activity, 21
mCi/mmol) in a total volume of 30 µL. The reaction was started by
the addition of 5 µL of enzyme (final concentration of 100 nM) and
was stopped at 1, 2, 3, and 4 min by the addition of 10% tricholoroacetic
acid. Kinetic constants were determined from Lineweaver-Burk plots.
Preparation of F-AS RNA. A 5′-fluorescein-labeled A-site model
(5′-F-GGCGUCACACCUUCGGGUGAAGUCGCC-3′) (F-AS) was
obtained from Xeragon Oligoribonucleotides (Zurich, Switzerland)) or
synthesized by using standard silyl phosphoramidite chemistry with
reagents from Glen Research (Sterling, Virginia). The RNA was purified
by electrophoresis on denaturing (8 M urea) 15% polyacrylamide gels,
followed by electroelution with 1X TBE (90 mM Tris-HCl, 90 mM
boric acid, 2.5 mM Na2EDTA, pH 8.3) in an Amicon centrilutor and
Centricon 3’s (Amicon). The F-AS RNA was stored at -20 °C in 10
mM HEPES, pH 7.4. RNA concentrations were determined spectro-
photometrically using a molar extinction coefficient of 253 300 M-1
Docking and Molecular Modeling. The NMR structure of paro-
momycin bound to the rRNA A site was used as the starting template.2
Rings I and II (2-deoxystreptamine and 6′-deoxy-6′-aminoglucose,
respectively) in the NMR structure of paromomycin were retained, and
the remainder of the structure was removed. These two rings constitute
the aminoglycoside paromamine (a structurally similar compound to
neamine having a hydroxyl group in place of the amine at position 6′).
With this structure at hand, the Connolly surface of the complex (i.e.,
A-site RNA template bound by paromamine) was computed, which
defines the “receptor site”. Two ligand databases, the NCI-3D database
and Cambridge Structural Database, collectively containing a total of
273 000 compounds, were used to dock the individual compounds into
the “receptor site” using the program DOCK version 4.0. The
electrostatic and steric counterparts on the receptor site were matched
with the docked compounds. This data set was reduced to 40 compounds
on the basis of their best fit into the “receptor site”. Each compound in
the set of 40 was considered in the receptor site individually. These 40
compounds fit in the space near the aminoglycoside-binding site and
were scored by the program favorably for their ability to bind to the
depressions and niches of the surface of the rRNA structure. We then
envisioned neamine analogues that would be covalently tethered to these
entities individually. Many of these compounds were predicted to bind
the surface such that they were amenable to attachment to neamine at
position N1 and O6 (marked in the structure 1). The tethers were
designed such that they themselves would have potential favorable
electrostatic interactions with the rRNA A-site. The visualization and
structure editing were performed using the Sybyl molecular modeling
program.35 This complex was energy-minimized using the Amber 5.0
package.36 The point charges on compound 4 were obtained from ESP
charges calculated by MOPAC package (PM3 Hamiltonian), and the
cm-1
.
Fluorescence Measurements. Fluorescence experiments were per-
formed on a Spex Fluoromax luminescence spectrometer. To renature,
the F-AS RNA (∼150 µM) was placed in a heating block at 20 °C,
heated to 85 °C for 2 min, then slowly cooled back to 20 °C over a 2
h period. The RNA solutions contained 10 mM HEPES, pH 7.4, 150
mM NaCl, 3 mM Na2EDTA, and 1 µM F-AS. Fluorescence emission
spectra were obtained with an excitation wavelength of 490 nm with a
band-pass of 2.9 nm (0.7 mm slit width) over the range of λem ) 500-
600 nm. All measurements were taken at 37 °C. Samples were incubated
in a temperature-controlled cuvette holder in the Fluoromax for 2 min
before fluorescence intensities were measured. Aliquots of the antibiotic
were added sequentially, allowing 2 min of equilibration time before
each fluorescence measurement.
Fluorescence intensities were corrected for volume changes using
the following equation: Fi,corr ) Fi,obs*Vi/V0, where Fi,corr is the corrected
intensity for point i of the titration, Fi,obs is the measured intensity at
point i, Vi is the volume after the ith addition, and V0 is the initial volume
(typically 350 µL).
Determination of Minimum Inhibitory Concentration (MICs).
The MICs of all antibiotics, including our synthetic aminoglycosides,
were determined by microdilution broth procedure. We performed
sequential 2-fold dilutions of antibiotics in 100 µL of Luria-Bertani
(LB) broth in sterile 96-well microtiter plates. Overnight cultures of
(34) Haas, M.; Dowding, J. E. Methods Enzymol. 1972, 72, 248.
(35) Sybyl version 6.5, Tripos Inc., St. Louis, MO.
(33) Atwell, G. J.; Denny, W. A. Synthesis 1984, 1032-1033.
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3236 J. AM. CHEM. SOC. VOL. 124, NO. 13, 2002