Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA

Article abstract of DOI:10.1016/j.bmcl.2004.09.088

We report the identification of a novel compound that binds to the Escherichia coli ribosomal A-site. We observed binding using NMR, mass spectrometry, and docking techniques and demonstrate that the compound binds in the same position as occupied by amin

Full text of DOI:10.1016/j.bmcl.2004.09.088

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5987–5990
Identification of a novel non-carbohydrate molecule that binds to the
ribosomal A-site RNA
Shawn P. Maddaford,a,* Mina Motamed,b Kevin B. Turner,c Min Soo K. Choi,b
Jailall Ramnauth,a, Suman Rakhit,a Robert R. Hudgins,b Daniele Fabrisc and
Philip E. Johnsonb,*
aMCR Research Inc., Toronto, Ontario, Canada, M3J 1P3
bDepartment of Chemistry, York University, 4700 Keele St. Toronto, Ontario, Canada, M3J 1P3
cDepartment of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA
Received 30 August 2004; revised 29 September 2004; accepted 30 September 2004
Available online 22 October 2004
Abstract—We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site. Bindingby the
compound was observed usingnuclear magnetic resonance and mass spectrometry techniques. We show that the compound binds in
the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics.
Ó 2004 Elsevier Ltd. All rights reserved.
The aminoglycoside antibiotics such as ribostamycin,
neomycin B, paromomycin, tobramycin, gentamycin,
and kanamycin A are the most commonly used broad-
spectrum anti-infective agents because of their high effi-
cacy and low cost.1 In addition to beingbactericidal,
aminoglycosides have predictable pharmacokinetics
and often act synergistically with other antibiotics.
However, these agents have the potential for harmful
side effects that include deafness (ototoxicity) and kid-
ney toxicity (nephrotoxicity).2–4 Finally, aminoglycoside
antibiotics are also prone to the development of bacte-
rial resistance due to the action of bacterial aminoglyco-
5
side modifyingenzymes.
Aminoglycoside antibiotics act by binding to the ami-
noacyl-acceptor site (A-site) in helix 44 of the 16S ribo-
somal (Fig. 1) RNA and interferingwith the fidelity of
mRNA translation. Protein miscodingand truncation
results, with this ultimately leadingto bacterial cell
Figure 1. (a) Sequence and secondary structure of the E. coli ribosomal
A-site RNA model used in this study. Nucleotides not boxed
correspond to residues not present in the A-site but added for this
study. The numberingscheme corresponds to that of the E. coli rRNA
sequence. (b) Structure of the aminoglycoside paromomycin. The rings
of paromomycin are numbered with Roman numerals.
Keywords: rRNA; RNA–ligand interactions; NMR spectroscopy;
Mass spectrometry; Docking.
*
Correspondingauthors. Tel.: +1 416 736 2100x33119; fax: +1 416 736
5936 (P.E.J.). Present address: Neuraxon Inc., 2240 Speakman Drive,
Sheridan, Science and Technology Park, Mississauga, Ontario,
Canada, L5K 1A9. Tel.: +1 905 823 3110; fax: +1 905 823 3610
(S.P.M.); e-mail addresses: pjohnson@yorku.ca; shawnm@nrxn.com
Present address: Affinium Pharmaceuticals, 100 University Ave., 12th
Floor, Toronto, ON, Canada, MSJ 1V6.
death.6 Chemical footprintingstudies of E. coli rRNA
has shown that neomycin, paromomycin, gentamicin,
and kanamycin protect bases A1408 and G1494 in
the A-site.7 The interaction between aminoglycosides
and the A-site RNA has been extensively studied by
both nuclear magnetic resonance (NMR) and X-ray
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.088

5988
S. P. Maddaford et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5987–5990
crystallographic methods using short RNA models
8–13
of a compound (Scheme 1) with a chemical structure dis-
tinct from the aminoglycoside family that binds to the
ribosomal A-site.
containingthe A-site nucleotides
and by a crystal
structures of the intact 30S ribosome bound to paromo-
mycin.14 Additionally, NMR methods have been used as
a screeningmethod to discover new compounds that
bind to the A-site RNA.15 Both crystallography and
NMR spectroscopy have been used to probe the interac-
tion between RNA and a variety of non-aminoglycoside
The synthesis of 4 (MCR13) is outlined in Scheme 1.
The acid chloride 1 was reacted with 2-aminophenol
under basic conditions followed by base-mediated cycli-
zation to the tricyclic benzoxazepinone 2. The amide
nitrogen was alkylated with 1-(2-chloroethyl)pyrrolidine
to give 3. Nitro group reduction with tin(II) chloride
16,17
A-site bindingmolecules.
Together these high-resolution structures provide a
wealth of information about the interaction between
aminoglycosides and their A-site RNA target. At the
A-site the RNA backbone is distorted by the bulged
nucleotides A1492 and A1493 in the paromomycin-A-
site complexes. This has the effect of wideningthe major
groove and providing a distinct binding pocket for
aminoglycosides. The major interactions between the
aminoglycosides and the binding pocket of the RNA
include numerous electrostatic, hydrogen bonding and
hydrophobic interactions. The crystal structures of
paromomycin bound to the ribosomal A-site reveals sev-
eral close charge–charge interactions between the amino
groups of the aminoglycoside and the phosphates of the
RNA backbone.9,14 Notable interactions include those
between ringII with the phosphates of A1493 and
G1494 and between ringIV and the phosphate of
G1405. These interactions seemed particularly note-
worthy to us. With this in mind, we felt non-carbo-
hydrate scaffolds with two basic nitrogen-containing
appendages would be suitable candidates for binding
to the A-site. Havingexamined an in-house library, we
decided upon MCR13 (Scheme 1; 4) as a potential can-
didate. In addition to possessingtwo charged sites, the
two aromatic rings of the tricyclic core offered the pos-
sibility of hydrophobic or pi-stackinginteractions with
the RNA base pairs.
dihydrate in refluxingethanol and conversion of the cor-
18
respondinganiline to the acetamidine group
the dibasic compound 4.
provided
We have used NMR spectroscopy and mass spectrome-
try techniques to show that MCR13 binds to a model
of the E. coli 16S ribosomal A-site. The A-site RNA
for NMR studies was produced by in vitro transcription
from a DNA template.19 This sequence (Fig. 1) is identi-
cal to the molecule used by Fourmy et al.8 for the NMR
structure determination of the paromomycin–RNA
1
complex. The one-dimensional H NMR spectrum of a
0.5mM RNA sample in 10mM sodium phosphate
(pH6.4) acquired at 25°C is shown in Figure 2. This plot
shows the downfield imino proton region and is very
similar to spectra reported previously8 for the same
sequence in the same buffer conditions. To the RNA
we titrated in a solution of MCR13 in identical buffer
(Fig. 2) to a final concentration that is a twofold molar
excess of MCR13 over RNA. We observed line broaden-
ingfor a subset of the imino resonances for nucleotides
primarily located near the aminoglycoside binding site
(G1405, G1497, G1491, U1406, and U1495).
This selective line broadeningprovides two pieces of
information: that bindingis occurrin,g and that the
Given the inherent problems with aminoglycoside anti-
biotics, an unmet need exists for the development of a
new class of antibiotics, which bind to the bacterial
16S ribosomal RNA but do not possess toxic side effects
and that are not susceptible to bacterial resistance by
enzymatic modification. Here we report the discovery
Scheme 1. Reagents and conditions: (i) 2-Aminophenol, NaHCO3,
ether, water; 0°C to rt, 77%; (ii) NaOH, H2O, 90°C, 6h, 96%; (iii) 2-
Chloroethylpyrrolidine hydrochloride, aq NaOH, acetone, 60°C; (iv)
SnCl2, ethanol, reflux, 1h, 52%; (v) Thioacetimidic acid naphthalene-2-
yl methyl ester hydrobromide, ethanol, rt.
Figure 2. Bindingof MCR13 to the A-site RNA determined by NMR
spectroscopy. Shown are the NMR spectra of the A-site RNA in the
absence and presence of a 1:1 and 2:1 molar ratio of MCR13 to RNA.
The identity of the imino peaks that show line broadeningupon
bindingare labeled in the unbound spectrum.

S. P. Maddaford et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5987–5990
5989
bindingsite is near those residues whose NMR spectrum
changes. This binding-induced line broadening is indica-
tive of either chemical exchange, such as hydrogen ex-
change with water, or conformational exchange taking
place. The residues that experience line broadeningare
the same as those identified previously that show a
change in chemical shift upon paromomycin binding
to the A-site.10,20 Similar NMR line-broadeningeffects
were observed for A-site bindingcompounds identified
by Fesik and co-workers15 and Mayer and James.16
fey et al. studyingweak bindingligands to the A-site
RNA by ESI-FTMS23 and from estimates of the practi-
cal limit of observingnon-covalent complexes by ESI-
FTMS methods we estimate that MCR13 binds to the
A-site RNA in the low millimolar range, with a Kd of
less than about 15mM.
A competitive bindingexperiment between compound
MCR13 and paromomycin was then performed to see
if the two molecules share a common bindingsite in
the A-site RNA. Equimolar amounts of MCR13,
paromomycin, and A-site RNA were combined and
analyzed usingESI-FTMS (data not shown). The result-
ingmass spectrum showed only the presence of paromo-
mycin bound to the RNA. This confirms that
paromomycin binds with a higher affinity to the ribo-
somal A-site than MCR13. Additionally, the absence
of an observable MCR13-A-site RNA complex or ter-
nary MCR13-paromomycin-A-site RNA complex sug-
gests that MCR13 and paromomycin share a common
bindingsite on the A-site RNA.
The bindingof MCR13 to the A-site RNA was also
tested by electrospray ionization (ESI) Fourier trans-
form mass spectrometry (FTMS) usingan ApexIII
FTMS (Bruker Daltonics) equipped with a thermally as-
sisted Apollo ESI source and an active shielded 7T
superconductingmagnet. The non-covalent complexes
formed by different aminoglycosides with the A-site
RNA have been previously investigated using this tech-
nique, which allowed for the observation of these inter-
actions intact in the gas phase.21,22 The mass spectra
provided by a 10lM sample of the A-site RNA (Dhar-
macon Inc.) in 50mM ammonium acetate/10% isoprop-
yl alcohol in the absence and presence of a twofold
molar excess of MCR13 are shown in Figure 3a and b.
A non-covalent complex formed between the RNA
and MCR13 can be observed with a 1:1 stoichiometry,
although with low abundance. The presence of a large
percentage of unbound RNA indicates that the ligand
binds weakly to the A-site RNA. On the contrary, a con-
trol experiment carried out by mixingA-site RNA with
an equimolar amount of the aminoglycoside paromomy-
cin (Fig. 3c) provided an intense signal for the 1:1 com-
plex. A much lower percentage of unbound RNA
indicates that the bindingaffinity is much stronger for
paromomycin than for MCR13. From the work of Grif-
To gain structural insight as to how MCR13 interacts
with RNA we used Autodock 3.0524 to model the bind-
ingof MCR13 to the A-site RNA. For the docking
experiments we used both the structure of the RNA
bound to paromomycin as determined by NMR8
(PDB code: 1PBR) and X-ray crystallographic methods9
(PDB code: 1J7T). Dockingsimulations were carried
out on a dual 2GHz PowerPC Macintosh G5. The ini-
tial structure of the MCR13 ligand was modeled with
ab initio (6-31G*) geometry optimization using the soft-
ware Gaussian 98.25 Dockingof MCR13 to both RNA
structures produced similar results, the lowest energy
docked structures always had MCR13 docked to the
A-site in a similar position as occupied by paromomycin
in the RNA–aminoglycoside complex. The lowest bind-
ingenergy at 273.15K usingthe NMR-derived RNA
structure was À17.51kcalmolÀ1. When the crystal struc-
ture was used as the docking target a binding energy of
À15.44kcalmolÀ1 resulted. Figure 4 shows the lowest
energy docked structure obtained for the MCR13-
RNA complex.
Figure 3. Bindingof MCR13 to the A-site RNA observed by
electrospray ionization Fourier transform mass spectrometry (ESI-
FTMS). Shown are the mass spectra of the A-site RNA in the absence
(a) and presence of a twofold molar excess of MCR13 (b). A control
experiment includingthe aminoglycoside paromomycin is shown in (c).
Figure 4. The lowest energy structure of MCR13 docked to the E. coli
ribosomal A-site RNA (PDB code: 1PBR). MCR13 is shown in red
while the RNA is in gray.

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shape about the seven-member central ringand has the
correct geometry to fit into the major groove pocket
formed by the A-site nucleotides. MCR13 is approxi-
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A-site. Bindingwas demonstrated usingNMR spectro-
scopy and ESI-FTMS methods and we showed that
MCR13 binds at the same site as the aminoglycoside
family of antibiotics. Future work will focus on improv-
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Acknowledgements
25. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.,
Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.;
Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.;
Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.;
Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J.
J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain,
M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.;
Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez,
C.; Pople, J. A., Gaussian, Pittsburgh, PA, 2003.
This work was supported by fundingfrom the Natural
Sciences and Engineering Research Council of Canada
(NSERC) to P.E.J. and R.R.H., and from the National
Institutes of Health (R01-GM643208) to K.B.T. and D.F.
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