Vancomycin binding to low-affinity ligands: Delineating a minimum set of interactions necessary for high-affinity binding

Article abstract of DOI:10.1021/jm990361t

Bacterial resistance to vancomycin has been attributed to the loss of an intermolecular hydrogen bond between vancomycin and its peptidoglycan target when cell wall biosynthesis proceeds via depsipeptide intermediates rather than the usual polypeptide int

Full text of DOI:10.1021/jm990361t

4714
J . Med. Chem. 1999, 42, 4714-4719
Va n com ycin Bin d in g to Low -Affin ity Liga n d s: Delin ea tin g a Min im u m Set of
In ter a ction s Necessa r y for High -Affin ity Bin d in g§
Patrick J . Loll,*,‡ J effrey Kaplan,‡ Barry S. Selinsky,† and Paul H. Axelsen‡
Department of Pharmacology, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, and
Chemistry Department, Villanova University, Villanova, Pennsylvania 19085
Received J uly 14, 1999
Bacterial resistance to vancomycin has been attributed to the loss of an intermolecular hydrogen
bond between vancomycin and its peptidoglycan target when cell wall biosynthesis proceeds
via depsipeptide intermediates rather than the usual polypeptide intermediates. To investigate
the relative importance of this hydrogen bond to vancomycin binding, we have determined
crystal structures at 1.0 Å resolution for the vancomycin complexes with three ligands that
mimic peptides and depsipeptides found in vancomycin-sensitive and vancomycin-resistant
bacteria: N-acetylglycine, D-lactic acid, and 2-acetoxy-D-propanoic acid. These, in conjunction
with structures that have been reported previously, indicate higher-affinity ligands elicit a
structural change in the drug not seen with these low-affinity ligands. They also enable us to
define a minimal set of drug-ligand interactions necessary to confer higher-affinity binding
on a ligand. Most importantly, these structures point to factors in addition to the loss of an
intermolecular hydrogen bond that must be invoked to explain the weaker affinity of vancomycin
for depsipeptide ligands. These factors are important considerations for the design of vancomycin
analogues to overcome vancomycin resistance.
In tr od u ction
X-ray crystal structures have been previously re-
ported for complexes of vancomycin with the ligands
acetate (Ac)3,4 and N-acetyl-D-alanine (AcDA).5 We now
report three new crystal structures of vancomycin
complexes with N-acetylglycine (AcG), D-lactate (DLac),
and 2-acetoxy-D-propanoic acid (AcDLac), the latter
being a mimic of the depsipeptide found in resistant
bacteria (see Figure 1). These structures suggest that
additional factors beyond the loss of an intermolecular
hydrogen bond are required to explain the reduced
affinity of vancomycin for depsipeptide ligands. Insights
derived from the new structures have important impli-
cations for the rational development of novel agents
active against resistant bacteria.
Vancomycin is a glycopeptide antibiotic derived from
the soil microorganism Nocardia orientalis. While the
class of glycopeptide antibiotics has many members,
vancomycin is the only one in common clinical use, and
it occupies an important medical niche in the treatment
of infections due to broadly resistant Gram-positive
organisms. However, its effectiveness is threatened by
the emergence of resistant pathogens that are spreading
rapidly and becoming a major public health menace. The
need for new therapeutic agents to combat resistance
is prompting detailed examinations of the structure and
mechanism of action of vancomycin and related com-
pounds.
Glycopeptide antibiotics act by inhibiting biosynthesis
of the bacterial cell wall. The specific targets of vanco-
mycin are peptides terminating with the sequence -L-
Lys-D-Ala-D-Ala-COOH. The binding of vancomycin to
these targets prevents the transpeptidation reaction
necessary for the synthesis of peptidoglycan. The most
common form of bacterial resistance substitutes D-
lactate for the C-terminal D-alanine residue, producing
a depsipeptide. This depsipeptide still functions as a
substrate in the transpeptidation reaction, allowing
resistant bacteria to synthesize normal peptidoglycan.
However, the affinity of vancomycin for its target is
reduced by 1000-fold. This reduction in affinity has been
attributed chiefly to the loss of a hydrogen bond donor
that would otherwise interact with an acceptor on the
vancomycin backbone.1,2
Resu lts
Nom en cla tu r e. The asymmetric unit of this crystal
form contains two vancomycin molecules, referred to
herein as V1 and V2. The seven amino acid residues of
the vancomycin monomer are denoted V1:1, V1:2, ...V1:7
and V2:1, V2:2, ...V2:7. The glucose and vancosamine
sugars on each monomer are indicated as “G” or “V”,
instead of a residue number. The asymmetric unit also
contains one ligand molecule, bound to V2.
An tibiotic Str u ctu r es. The vancomycin:AcG, van-
comycin:DLac, and vancomycin:AcDLac structures are
isomorphous with the vancomycin:Ac complex previ-
ously described.3,4 In each case, two antibiotic molecules
form an unsymmetric homodimer in which the peptide
portions of the dimer are related by an almost perfect
2-fold noncrystallographic symmetry axis. Hydrogen
bonds between the two halves of the dimer follow the
pattern of a two-stranded antiparallel â-sheet. The N-
and C-terminal ends of the peptide bend outward away
from the dimer interface, giving each monomer a
concave shape and forming the ligand recognition
* Corresponding author. E-mail: loll@pharm.med.upenn.edu. Tel:
(215) 898-1294. Fax: (215) 573-2236.
‡ University of Pennsylvania.
† Villanova University.
§ Abbreviations: Ac, acetate; AcG, N-acetylglycine; AcDA, N-acetyl-
D-alanine; DLac, D-lactate; AcDLac, 2-acetoxy-D-propanoic acid; NMR,
nuclear magnetic resonance; rms, root-mean-square.
10.1021/jm990361t CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/07/1999

Vancomycin Binding to Low-Affinity Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4715
F igu r e 1. Structures of vancomycin and certain of its ligands. 1 and 2 represent the physiological ligands from vancomycin-
sensitive and vancomycin-resistant bacteria, respectively; the site of the peptide f depsipeptide change is marked with a star.
Structures of ligands for which vancomycin complex structures are available are shown at right.
Ta ble 1. Rms Differences (in Å) in Atomic Positions between
refinements converged to essentially identical results
for the antibiotic structure. In contrast, rms differences
Various Vancomycin:Ligand Complexesa
AcG
DLac AcDLac
Ac
AcDA #1b AcDA #2b
between these four structures and that of the previously
described vancomycin:AcDA complex5 are significantly
greater, indicating that vancomycin adopts a somewhat
different structure in the latter complex. This difference
may be a consequence of the higher affinity that
vancomycin has for AcDA and/or differences in crystal
packing.
AcG
0.089
0.124
0.067
0.091
0.035
0.064
0.711
0.699
0.714
0.692
0.737
0.724
0.730
0.717
0.224
DLac
AcDLac
Ac
AcDA #1 0.996 0.972
AcDA #2 0.996 0.972
0.108
0.147 0.105
0.103 0.072
0.102
0.987
0.983
0.986
0.985
0.325
a
Superpositions were carried out with all 202 non-hydrogen
atoms of the vancomycin dimer (bottom half of matrix, plain
typeface) and with the 134 core atoms remaining after carbohy-
drate and side chains are removed (upper half of matrix, italics).
Liga n d Bin d in g. There are significant differences
in the manner by which Ac, AcG, AcDA, DLac, and
AcDLac bind to vancomycin (see Figure 2). Although the
two carboxylate oxygens of each ligand form the same
three hydrogen bonds to the vancomycin backbone, the
positions of the other ligand atoms are different in each
complex. The amide proton of AcG makes a hydrogen
bond interaction with the carbonyl oxygen of V2:4. The
carbonyl oxygen of the acetyl group points away from
vancomycin into solution and does not participate in any
hydrogen bonds with vancomycin or any symmetry-
related complexes. Although the binding mode of AcG
is similar to that of AcDA, AcG sits higher in the binding
pocket (i.e., closer to V2:G) because there are no bulky
substituents at the CR position.
b
There are two crystallographically independent dimers in the
asymmetric unit of the N-Ac-D-Ala crystals.
pocket. The carbohydrate portions of the dimer project
outward above these concave surfaces and form part of
the ligand recognition pockets. The carbohydrate does
not obey the 2-fold symmetry of the rest of the dimer,
rendering the two ligand recognition surfaces non-
equivalent. The ligand binding site in V1 is occupied
by a symmetry-related copy of V1 and the Asn side chain
of V1:3, while V2 is occupied by ligand.
Rms differences in atomic positions after pairwise
least-squares superposition of the non-hydrogen van-
comycin atoms in these four complexes are approxi-
mately 0.1 Å (Table 1). Rms differences for atoms in the
polypeptide ‘backbone’ are about one-half as large,
approaching the level of uncertainty expected in the
coordinate determination. Thus, these four independent
The crystals of the DLac complex were grown from a
racemic mixture of D- and L-lactate, but only one isomer
appears in the crystal. The bound ligand is identified
as D-lactate from (a) the relative intensities of the
hydroxyl oxygen and methyl carbon peaks in the

4716 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Loll et al.
F igu r e 2. (a) Stereoillustration of ligand binding by vancomycin. Shown is the AcG ligand bound by the vancomycin monomer
V2. AcG is shown in bold, and hydrogen bonds between the ligand and drug are shown as dashed lines. (b) Schematic representation
of how ligand recognition by vancomycin varies for five different ligands.
electron density map and (b) the geometry of the
hydrogen bond interactions available to the hydroxyl
oxygen (see below). The methyl group of DLac points
toward V2:G causing the CR of DLac to be 0.6 Å further
away from V2:G than the CR of AcG. The hydroxyl group
of DLac is well-positioned to donate a hydrogen bond to
the carbonyl oxygen on residue V2:3 (oxygen-oxygen
distance ) 2.8 Å).
The most surprising result is seen in the AcDLac
complex. Strong density is only seen for the lactate
portion of the ligand molecule. Diffuse density can be
seen near the ester oxygen of the ligand, but the acetyl
group cannot be positioned with confidence into the
electron density map, and we presume it to be present
but disordered. The portion of the ligand that can be
positioned shows the æ torsion angle of the ligand is
rotated 180° relative to that in the DLac complex, with
the ester oxygen pointing toward V2:G, rather than
toward V2:3. It is unlikely that the bound ligand is
hydrolyzed AcDLac because the products of hydrolysis
would be acetate and DLac, and the electron density we
observe in the vancomycin:AcDLac complex is clearly
different from the density observed for the vancomycin:
Ac and vancomycin:DLac complexes. In addition, after
the DLac complex crystals were harvested, the reservoir
buffer from the crystallization experiment was exam-
ined by reverse-phase HPLC, and no evidence for
hydrolysis was observed.
DLa c Bin d in g Affin ity. Binding affinities have been
obtained previously for vancomycin and Ac, AcG, DLac,
and AcDLac using a magnetic resonance.6 We have
repeated this measurement of the affinity of Ac for
vancomycin, obtaining a value of 15 M-1, in reasonable
agreement with the value previously reported (see Table
2). When the method was applied to AcDLac, minimal
changes in 13C NMR chemical shifts were observed. Due
to limited solubility of the AcDLac:vancomycin complex
at high AcDLac concentrations, some uncertainty exists

Vancomycin Binding to Low-Affinity Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4717
Ta ble 2. Affinities of Ligands for Vancomycin
V2:4 aromatic ring, and they both donate hydrogen
bonds to the carbonyl of V2:4. Yet, the affinity of AcDA
is almost 4-fold higher even despite the entropic cost of
restricting rotation about the N-C (ψ) bond in AcDA
upon binding. The acetate methyl group of AcDA does
interact with the aromatic rings of V2:6 and V2:7, but
as mentioned in the previous paragraph, these interac-
tions are unlikely to be responsible for significantly
higher affinity. It is more likely that the acetate
carbonyl forms a hydrogen bond with the charged
N-methylleucyl residue of another vancomycin monomer
in a face-face dimer. This hydrogen bond is clearly
formed in the crystal structure of AcDA. Inferences
drawn here from the crystal structure of the DLac
complex and ligand affinity measurements now support
its energetic significance in solution.
ligand
abbreviation
affinity (M-1
)
acetate
Ac
30a; 15b
300a
80a
N-acetyl-D-alanine
N-acetylglycine
AcDA
AcG
2-acetoxy-D-propanoic acid
D-lactate
AcDLac
DLac
40b
80a
a
b
Reference 6. This work.
in the limiting chemical shift values and hence in the
calculated binding constant. Nonetheless, we can con-
clude that AcDLac binds relatively weakly to vancomy-
cin and can place an upper estimate on the binding
constant of 40 M-1. Furthermore, the 13C NMR chemical
shifts indicate that AcDLac binds differently to vanco-
mycin than other ligands. For ligands such as Ac or
AcDA, the largest changes in 13C NMR chemical shift
(ca. 100 Hz) are observed for the carbonyl carbons of
residues 3, 4, and 5.6,7 However, when AcDLac is titrated
into vancomycin, these same carbonyl carbons exhibit
only small changes in chemical shift (less than 10 Hz).
This suggests that AcDLac binds differently to vanco-
mycin than do other ligands, in agreement with the
crystal structure described in this report.
Fourth, comparison of the Ac and AcDLac structures
suggests that both of these ligands depend primarily on
the binding of carboxylate for their affinity to vanco-
mycin.
These comparisons reveal that a variety of different
factors contribute to the recognition of ligands by
vancomycin, including the ligand’s flexibility and con-
formational preferences, its ability to participate in
specific nonbonded interactions, and its ability to sup-
port formation of face-face vancomycin dimers. Iden-
tification of these factors has important implications for
the design of vancomycin analogues with an affinity for
depsipeptide ligands and the potential to overcome
vancomycin resistance. For example, Stack et al.9 have
shown that vancomycin monomers, when covalently
linked with a tether segment of sufficient length, are
effective against bacteria exhibiting depsipeptide tar-
gets; Loll et al.5 have argued that this antibacterial
activity is likely due to the formation of face-face
dimers and have shown that the requisite length of the
tether corresponds precisely to that which is needed to
span the linkage sites in a face-face dimer. In this
context, the data reported herein suggest that the
favorable free energy associated with formation of a
face-face dimer more than compensates for the loss of
an intermolecular bond associated with binding a dep-
sipeptide ligand. The difference between the free ener-
gies of binding (∆∆G) of AcDA and DLac is roughly 3.3
kJ ‚mol-1, which can be attributed primarily to the
electrostatic interaction across the dimer interface
between the terminal acetate of the ligand and the
N-methylleucine group of the drug. In contrast, the ∆∆G
for Dlac versus AcDLac is only 2.4 kJ ‚mol-1, correspond-
ing to the loss of the hydrogen bond between ligand and
the carbonyl of V2:4.
Discu ssion
The structural data reported in this paper in combi-
nation with previously reported structures reveal that
a number of factors control high-affinity binding of
ligand to vancomycin. This is best demonstrated by
pairwise comparison of different complexes.
First, comparison of the vancomycin complexes with
AcDA and AcG confirms that the lower affinity of AcG
is not due to a drastically altered binding mode. An
earlier computational study of ligands terminating in
D-alanine and glycine identified three factors expected
to contribute to lowered affinity for AcG: a larger
entropic cost to the binding of AcG due to greater
freedom of rotation for its CR and carboxylate group
(hindered by the R-methyl group in AcDA), favorable van
der Waals interactions between the R-methyl group of
AcDA and the aromatic ring of V2:4, and bond angle
strain induced in the AcG ligand upon binding.8 The
resolution of these crystal structures does not permit
unrestrained refinement, so we cannot address the
question of bond angle strain, but by showing that AcG
and AcDA bind in the same general way, the structural
data help support the other two computational predic-
tions. When bound to AcDA, vancomycin forms face-
face and back-back dimers in the crystal;5 however, the
vancomycin:AcG complex forms only back-back dimers,
even though AcG appears to be properly positioned to
mediate face-face dimerization. This implies that higher
ligand affinity helps promote dimer formation.
Second, comparison of the AcG and DLac complexes
shows that AcG interacts with the aromatic rings of
V2:6 and V2:7, yet the affinity of AcG for vancomycin
is no greater than that of DLac. This indicates that any
enhancement of AcG affinity attributable to its acetate
group is either insignificant or negated by entropic,
electrostatic, and angle strain factors.
Third, comparison of the AcDA and DLac complexes
shows that the carboxylate groups in both ligands accept
hydrogen bonds from the same donors, their R-methyl
groups form the same van der Waals contacts with the
The structure of vancomycin in the AcDA complex
differs substantially from that seen in all of the other
complexes considered here; the drug is in a more closed
conformation, apparently gripping AcDA more tightly
than other ligands. The affinity of AcDA for vancomycin
is also higher than any other ligand considered here
(Table 2). This suggests that tightly bound ligands can
elicit a structural change not observed with lower-
affinity ligands, and the threshold for this change
corresponds to an affinity constant between 100 and 300
M-1. This is reflected in the crystal packing seen in low-
affinity complexes where one of the two ligand binding
pockets in each dimer is occupied by a crystal contact.

4718 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Loll et al.
repeatedly with diethyl ether. The organic phase was dried
over CaSO4 and filtered; its volume at this point was 150 mL.
Pyridine was added to the solution (4.9 g, 62 mmol), after
which acetyl chloride (3.9 g, 50 mmol) was added dropwise
with stirring. A white precipitate appeared immediately. After
40 min excess pyridine was precipitated by the addition of 1
mL of concentrated HCl; the solution was then filtered and
concentrated in a rotary evaporator. The remaining material
was dissolved in water, frozen, and lyophilized; 1.35 g of a clear
colorless oil remained after lyophilization (21%): 1H NMR (300
MHz, D2O) δ 5.04 (q, 1H, J ) 7.0 Hz), 2.16 (s, 3H), 1.50 (d,
3H, J ) 7.1 Hz); 13C NMR (75 MHz, D2O) δ 178.2, 176.2, 72.5,
22.8, 18.8. Anal. (C5H8O4) C, H, O.
Mea su r em en t of Liga n d Bin d in g Con sta n ts via 13C
NMR Sp ectr oscop y. Stock solutions of 400 mM ligand and
24 mM vancomycin were prepared in 50 mM potassium
phosphate buffer, pH 6.0, containing 10% D2O. The stock
solutions were mixed with buffer to yield a final solution of
12 mM vancomycin and 0-200 mM ligand. A solution of 400
mM AcDLac, 12 mM vancomycin was prepared by the addition
of solid vancomycin to the 400 mM AcDLac stock. NMR spectra
were acquired immediately after sample preparation. 13C NMR
spectra were acquired on a Varian XL-300 spectrometer at a
13C frequency of 75 MHz. Spectra were acquired with com-
posite pulse proton decoupling, using a 90° (15-ms) excitation
pulse and a 2-s interpulse delay. Spectra were acquired into
32K data points with a spectral width of 233 ppm. Ap-
proximately 12 000 acquisitions were averaged per spectrum.
Spectra were acquired at room temperature (297 ( 2 K).
Limiting chemical shift changes were estimated from plots of
13C chemical shift versus concentration of AcDLac. Binding
constants were calculated from plots of ∆δ/∆δlim versus (∆δ/
∆δlim) × (concentration).
Presumably, the ligands are so weakly bound that under
crystallization conditions they are unable to displace the
symmetry-related molecule. However, the addition of
AcDA to any of the tetragonal crystals immediately
causes them to crack and dissolve, indicating that the
more tightly bound ligand can displace a symmetry-
related vancomycin molecule from this binding pocket.
From the structures currently available, we can
identify a minimum set of drug-ligand interactions
necessary to trigger this structural change in vancomy-
cin. The set includes: (1) three hydrogen bonds formed
between the carboxylic acid of the ligand and amide
protons of the antibiotic, (2) interactions between the
R-methyl group of the ligand and the aromatic ring of
residue 4, (3) the hydrogen bond donated by a nitrogen
or oxygen of the ligand to the carbonyl oxygen of residue
4 of the antibiotic, and (4) various contacts between an
acyl group attached to the C-terminal residue and both
monomers in a face-face dimer. This acyl group is
acetyl in AcG and AcDA but would be D-alanyl in the
natural ligand. Each of the relatively low-affinity struc-
tures described herein exhibits only three of these four
interactions, implying that all four interactions must
be formed in order for vancomycin to adopt the high-
affinity conformation.
Con clu sion s
Binding of high-affinity ligands by vancomycin is
associated with a structural change in the drug and the
formation of ligand-mediated face-face dimers. The
structural change and dimerization may be coupled,
since while low-affinity ligands bind in a manner
compatible with face-face dimer formation, they elicit
neither the structural change nor face-face dimers. We
have identified a minimal set of interactions, all of
which must occur between the drug and its ligand in
order for high-affinity binding to be observed. These
results show that the loss of an intermolecular hydrogen
bond is only one of the factors needed to explain the
failure of vancomycin to recognize depsipeptide ligands
found in resistant bacteria, and it will be important to
compensate for these other factors when designing
agents targeted against vancomycin-resistant organ-
isms. A question that remains is whether the high-
affinity conformation of vancomycin seen with AcDA
reflects the full extent of the structural change that is
induced in vancomycin by ligands, or if tighter binding
by longer and more physiologically relevant ligands will
elicit further structural changes.
Cr ysta lliza tion . Crystals were prepared by the hanging
drop vapor diffusion method; 5.0-µL drops containing 30 mg/
mL vancomycin in water were mixed with equal volumes of
reservoir buffer and suspended from a siliconized cover slip
over 1.0 mL of reservoir buffer. The plates were sealed with
plastic tape and maintained at 291 K. The reservoir buffers
contained 100 mM ligand, which was used as a buffering agent
at pH 4.6, and 2.0-2.4 M NaCl. Racemic lactic acid was used
in the reservoir buffer for the DLac complex. Bipyramidal
crystals formed within 1 week, growing to maximum dimen-
sions of 0.2 × 0.3 × 0.5 mm. Shortly prior to data collection,
the crystals were transferred to a solution of 30% v/v glycerol
in reservoir buffer. After 1-2 min, the crystals were mounted
in nylon loops and flash-cooled by plunging into liquid N2.
Da ta Collection a n d P r ocessin g. Diffraction data were
collected at beamline A1 at the Cornell High Energy Synchro-
tron Source and beamline X-12B at the National Synchrotron
Light Source. Beamline A1 at CHESS was fitted with a binned
2K×2K CCD detector,10 while beamline X-12B utilized a 30-
cm MAR image plate detector. At CHESS, several data
collection passes were made, using the unattenuated beam for
high-resolution data and attenuation for the more intense low-
resolution data. A similar strategy was used at NSLS, except
that instead of attenuating the beam the exposure time was
drastically reduced. In addition, the crystal used at X-12B was
returned still frozen to the home laboratory, where additional
low-resolution data were collected using a 30-cm MAR image
plate detector mounted on a rotating anode source equipped
with focusing mirrors and a Ni foil. Crystals were maintained
at ca. 100 K in a stream of N2 gas at all times during data
collection. Images were processed using the programs DENZO
and SCALEPACK.11 Observations containing overloaded pixels
were not included in the data set.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Pharmaceutical grade vancomy-
cin hydrochloride (Vancocin, Eli Lilly) was used without
further purification. 2-Acetoxy-D-propanoic acid was synthe-
sized as described below; N-acetylglycine was obtained from
Sigma and lithium D-lactate from Aldrich. Other materials
were obtained commercially and were of the highest purity
available. TLC was carried out using silica-60 plates (EM
Science) and a mobile phase of 1% acetic acid in ethyl acetate.
HPLC analyses were conducted with an analytical Vydac C18
column, using 50 mM K2HPO4 as the mobile phase and
identifying analytes by absorbance at 220 nm. NMR spectra
were acquired on a Varian XL-300 spectrometer.
Refin em en t. Refinement was carried out against F2 using
the program SHELXL-93.12 All three of the complexes are
isomorphous with the vancomycin:Ac complex structure de-
termined previously.3,4 Thus, starting structures were obtained
by removing the carbohydrate groups, ligand, and residue 1
and 3 side chains from the vancomycin:Ac structure and
subjecting this fragment to rigid body refinement. R values
after rigid body refinement were typically about 0.45. Missing
Syn th esis of AcLa c. The sodium salt of D-lactic acid (5.5
g, 49 mmol) was dissolved in 25 mL of a 65:35 (v/v) mixture of
water and concentrated HCl. This solution was extracted

Vancomycin Binding to Low-Affinity Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4719
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