cryoEM-Guided Development of Antibiotics for Drug-Resistant Bacteria

Article abstract of DOI:10.1002/cmdc.201900042

While the ribosome is a common target for antibiotics, challenges with crystallography can impede the development of new bioactives using structure-based drug design approaches. In this study we exploit common structural features present in linezolid-resistant forms of both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) to redesign the antibiotic. Enabled by rapid and facile cryoEM structures, this process has identified (S)-2,2-dichloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (LZD-5) and (S)-2-chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (LZD-6), which inhibit the ribosomal function and growth of linezolid-resistant MRSA and VRE. The strategy discussed highlights the potential for cryoEM to facilitate the development of novel bioactive materials.

Full text of DOI:10.1002/cmdc.201900042

DOI: 10.1002/cmdc.201900042
Communications
cryoEM-Guided Development of Antibiotics for
Drug-Resistant Bacteria
Matthew J. Belousoff,[a] Hari Venugopal,[b] Alexander Wright,[c] Samuel Seoner,[c]
Isabella Stuart,[a] Chris Stubenrauch,[a] Rebecca S. Bamert,[a] David W. Lupton,*[c] and
Trevor Lithgow*[a]
While the ribosome is a common target for antibiotics, chal-
lenges with crystallography can impede the development of
new bioactives using structure-based drug design approaches.
In this study we exploit common structural features present in
linezolid-resistant forms of both methicillin-resistant Staphylo-
coccus aureus (MRSA) and vancomycin-resistant Enterococcus
(VRE) to redesign the antibiotic. Enabled by rapid and facile
cryoEM structures, this process has identified (S)-2,2-dichloro-
N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)me-
thyl)acetamide (LZD-5) and (S)-2-chloro-N-((3-(3-fluoro-4-mor-
pholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (LZD-
6), which inhibit the ribosomal function and growth of linezol-
id-resistant MRSA and VRE. The strategy discussed highlights
the potential for cryoEM to facilitate the development of novel
bioactive materials.
dition to reaching resolution targets that allow unambiguous
placement of small molecules, cryoEM is a solution-phase tech-
nique which may prove advantageous. Although cryoEM-
guided design of novel ligands is underdeveloped, the interac-
tions of known drugs with their targets have been examined
with the Plasmodium 20S proteasome,[6] the GPCR family,[7]
pathogenic ribosomes,[8] receptor-bound insulin,[9] and impor-
tant HIV viral entry proteins,[10] to name a few examples. In
2017, Scheres, Baum, and co-workers exploited cryoEM to elu-
cidate the mode of action that mefloquine uses to inhibit the
Plasmodium falciparum ribosome, and then used this insight to
develop a next-generation molecule with enhanced antipara-
sitic activity.[11] As part of our studies into mechanisms of anti-
biotic resistance, we examined one of the escape routes that
Staphylococcus aureus uses to develop resistance to the riboso-
mal-interfering antibiotic, linezolid.[8a] These studies, and those
of others,[12] show that disparate mutations result in a common
structural rearrangement that lowers the affinity of linezolid
binding at the peptidyl transferase center (PTC), suggesting
that in order to preserve its enzymatic activity, the ribosome is
not infinitely malleable.[8a,12a,13] Taking advantage of these
common structural changes in linezolid-resistant (LinR) strains,
we postulated that simple modification of an amide group in
linezolid should reintroduce binding to the altered A-site.
Herein we report studies on this topic which have exploited
cryoEM to guide the development of new linezolid analogues
that target LinR strains of MRSA and VRE. Central to this work
was a cryoEM workflow that allowed rapid delivery of high-res-
olution structural information. This strategy should have po-
tential value in many areas of SBDD.
Combating antimicrobial resistance requires multiple therapeu-
tic strategies, including the discovery of new molecular scaf-
folds, re-engineering and repurposing of existing drugs, in ad-
dition to improvements in antimicrobial stewardship.[1] The dis-
covery of small molecules with antimicrobial activity can be fa-
cilitated by structure-based drug design (SBDD) strategies.[2]
Approaches of this type exploit structural information, ob-
tained using various biochemical assays, to drive the drug
design process. Although a number of techniques are em-
ployed in SBDD, the field remains heavily reliant on X-ray[3] and
NMR-based analysis,[4] techniques with orthogonal advantages
with regard to resolution, throughput, and analysis cost. With
the emergence of high-resolution cryoEM as a powerful tool
for determining structural information, its ability to be applied
in SBDD, particularly when examining biomolecules poorly
suited to X-ray analysis, has been discussed extensively.[5] In ad-
The bacterial ribosome is a common target for drug devel-
opment, with over 40% of antibiotics in clinical use targeting
its activity in protein synthesis.[14] The first-in-class oxazolidi-
none antibiotic[15] linezolid is one of the most recently intro-
duced drugs. Linezolid is widely used for the treatment of in-
fections caused by bacterial resistance to antibiotics such as
methicillin and vancomycin, particularly MRSA and VRE.[13a,16]
Linezolid binds to rRNA in the A-site of the peptidyl transferase
center of the ribosome (SI Figure S1), and inhibits protein syn-
thesis by sterically blocking recruitment of aminoacyl-tRNA. As
a first step in re-engineering a form of linezolid that could
bind and inhibit the altered A-site in LinR strains, we examined
the characteristics of the compacted drug-binding site in the
LinR ribosome. A large conformational change of the rRNA resi-
dues U2506 and G2505 (E. coli numbering used throughout) was
observed, which in turn remodels the linezolid-binding pocket
[a] Dr. M. J. Belousoff, I. Stuart, Dr. C. Stubenrauch, R. S. Bamert,
Prof. T. Lithgow
Infection and Immunity Program, Department of Microbiology, Monash
University, Clayton 3800 (Australia)
E-mail: trevor.lithgow@monash.edu
[b] H. Venugopal
Ramaciotti Centre for Electron Microscopy, Monash University, Clayton 3800
(Australia)
[c] A. Wright, S. Seoner, Prof. D. W. Lupton
School of Chemistry, Monash University, Clayton 3800 (Australia)
E-mail: david.lupton@monash.edu
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201900042.
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(Figure 1a, SI Figure S2a). Comparative analysis of these vari-
ous mutant ribosomes revealed a highly similar conformation
in most of the rRNA residues, raising the prospect that an in-
duced-fit mode of binding may be possible in the peptidyl
transferase pocket. The largest conformational change in the
LinR ribosome structure impacted the position of the oxazolidi-
none heterocycle (SI Figure S1a). In a linezolid:70S ribosome
rational analysis of the structural coordinates for the LinR ribo-
some (SI Figure S2) revealed binding opportunities for an
amide at the 5-position if it were redirected toward A2503
,
where a larger pocket is present that could potentially accom-
modate a bulkier amide than the acyl group present in linezol-
id (SI Figure S2). If such reorientation could be achieved, then
linezolid-based analogues should prove capable of evading
contraction-based drug resistance. To address this hypothesis,
a selection of amides at the 5-position of linezolid starting
with the common amine (S)-5-(aminomethyl)-3-(3-fluoro-4-
morpholinophenyl)oxazolidone-2 (2) itself,[17] were prepared
(Figure 1b).
complex, the amide carbonyl group points toward G2447
,
making a putative hydrogen bond with A2451 (SI Figure S2b). A
While the compounds displayed in Figure 1 would not nec-
essarily be viable clinical antibiotics due to undesirable phar-
macokinetics (solubility) and pharmacodynamics (potential tox-
icity; LZD-7 being a potential Michael acceptor and the halo-
genated compounds containing labile leaving groups), they
were selected to examine the viability of cryoEM as a tool of
value for targeting ligand biomolecule interactions.
Screening the compounds in a minimal inhibitory concentra-
tion (MIC) assay demonstrated that the compounds were
active as antibiotics in vitro (Table 1). The chloro (LZD-6) and
Table 1. Minimum inhibitory concentration for antibiotics against rele-
vant bacterial species: Staphylococcus aureus, Staphylococcus capitis,
Staphylococcus epidermidis, and vancomycin-resistant Enterococcus (VRE,
ATCC700221).
[a]
Compound
MIC [mgmLÀ1
]
S. aur.
MRSA (LinR)
S. cap.
S. epi.
VRE
linezolid
LZD-3
LZD-4
LZD-5
LZD-6
LZD-7
LZD-8
LZD-9
0.5
1
2
0.5
0.25
1
2
4
4
1
1
2
8
16
0.5
1
1
0.25
0.25
1
0.25
1
1
0.25
0.13
0.5
2
1
2
2
0.5
0.25
1
4
4
2
4
2
2
2
[a] MIC assays were carried out according to CLSI protocols.[18]
dichloro (LZD-5) derivatives were the most active in inhibiting
growth of LinR MRSA. These compounds bind to both the line-
zolid-sensitive (LinS) and LinR states of the drug-binding pocket
on the ribosome, as they inhibit both LinS and LinR strains of
S. aureus (Table 1). X-ray crystallographic analysis of LZD-5 and
LZD-6 (Figure 1c, SI Figure S3) showed a similar overall struc-
ture, with both crystallising in the same space group with very
similar unit cells (SI Table S1). Both showed a “linear” topology
with two molecules exhibiting a head-to-tail van der Waals in-
teraction, which formed the basis of the asymmetric unit of
the crystal lattice. The 2-oxazolidone moiety of both LZD-5
and LZD-6, lying in the same plane as the fluorinated aromatic
ring (although LZD-5 showed a greater offset between the
plane of the aromatic ring and the 2-oxazolidone ring which
was 308 compared to 88 in LZD-6). None of the other deriva-
tives showed improved activity, while some were significantly
worse. For example, the imide derivative LZD-9 showed rela-
tively poor MIC values for all bacterial strains (Table 1).
Figure 1. a) View of the drug-binding pocket from the cryoEM structures of
the 70S ribosomes from LinS (PDB ID: 5TCU and EMDB EMD-8402; left panel)
and LinR (PDB ID: 5T7V and EMDB EMD-8369; right panel) MRSA strains.
Overlaid is the binding position of linezolid. Green and red surfaces are the
solvent-accessible surface of the rRNA about the site of linezolid binding.
b) Synthetic scheme used to synthesise LZD-3–9.[17] c) Stick representation of
the X-ray crystal structures of LZD-5 and LZD-6.
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To assess whether the improved potency of LZD-5 and LZD-
6 was driven by more effective inhibition of ribosomal activity,
we assayed the compounds in a cell-free translation system. In
this system, synthesis of firefly luciferase is used to monitor ri-
bosomal activity using a luminescence readout. LZD-5 and
LZD-6 were more effective at inhibiting protein synthesis by
the ribosome than was the parent compound linezolid
(Table 2).
and accommodate the different acyl group position (Fig-
ure 2c). Relative to the crystal structure of LZD-6 alone, which
adopts a linear topology, the acyl tail rotates around the amide
bond to form more favorable interactions with the surround-
ing rRNA when in complex with the ribosome. This different
mode of binding shows that there is a degree of flexibility in
the binding of the oxazolidinone pharmacophore to the PTC,
and that the representative linezolid binding[19] is not the only
rigid option for this family of antibiotics.
In the ribosome, LZD-6 is assisted by additional interactions
with surrounding rRNA residues (Figure 2d). The 2-chloroacyl
group forms a hydrogen bond with the ribose of A2503, while a
hydrophobic interaction with the purine of A2503, and a p–p in-
teraction with the purine of G2061 are also gained (Figure 2e).
These interactions allow the 2-oxazolidone ring to form a fa-
vorable hydrophobic interaction with pyrimidine of U2504. All
other interactions with the morpholine and the fluorophenyl
ring are similar to linezolid, except that the morpholine ring in
LZD-5 and LZD-6 forms a hydrogen bond with the ribose of
A2451. These observations also provide a potential explanation
for why tedizolid, a new oxazolidinone antibiotic, effectively
binds the ribosome without having a morpholine, as it might
promote an induced fit at a similar location to interact with
A2451 via a hydrogen bond between tedizolid’s pyridine ring
and the ribose sugar.[21]
Table 2. Ribosome activity determined in a luciferase transcription/trans-
lation assay.
Compound
IC50 [mm][a]
linezolid
LZD-3
LZD-4
LZD-5
LZD-6
LZD-7
LZD-8
LZD-9
3.8Æ0.3
6Æ1
3.9Æ0.7
2.6Æ0.2
2.6Æ0.5
5Æ1
>40
>30
[a] IC50 plots are provided in Supporting Information Figure S8; values are
the meanÆSEM (n=3).
To determine if the improved activity was due to the fore-
shadowed conformational changes, single-particle cryoEM data
was collected yielding refined maps for LZD-5:70S and LZD-
6:70S to a global resolution of 3.1 and 2.8 ꢁ, respectively (SI
Figure S4a). As the molecules of interest bind in the 50S ribo-
somal subunit, Euler angle refinement was focused on the 50S
subunit. This allowed well-resolved maps around the peptidyl
transferase center, with the local resolutions around the area
of interest both being <2.9 ꢁ (SI Figure S4b,c). Given reports
that use of the Volta Phase Plate (VPP) may lead to more facile
visualisation of small bound ligands due to its higher contrast
micrographs (SI Figure S5a),[20] the LZD-5:70S structure was
collected using a VPP, while the LZD-6:70S was collected using
a standard varied defocus strategy. Both maps are strikingly
similar, with the atomic structures superimposable (SI Fig-
ure S3). Using the VPP collection we were able to collect high-
quality data (SI Figure S5b,c), which resulted in similar resolu-
tion (SI Figure S2a,b) but with significantly fewer micrographs
(SI Table S2). By periodic movement of the VPP, we were able
to keep the phase shift within suitable boundaries (SI Fig-
ure S5d,e) leading to high-quality reconstructions (SI Fig-
ure S5 f) and similar map quality (SI Figure S6a) to the varied
defocus collection. This means a substantial decrease in valua-
ble microscope time to achieve equally high-quality and inter-
pretable data for unambiguous drug placement in the ribo-
some.
While LZD-6 had two-fold greater ability to inhibit bacterial
growth, we set out to examine if the decreased MIC activity
correlated with any decrease in the acquisition of drug resist-
ance. The evolution of resistance toward LZD-6 by S. aureus
was determined by serially passaging S. aureus through broth
containing various concentrations of LZD-6. After 16 days of
serial passaging on 0.5 mgmLÀ1 LZD-6, a spontaneous mutant
was isolated. This mutant was cultured and shown to be drug-
resistant S. aureus by serial broth microdilution, yielding a MIC
value of 2 mgmLÀ1. The rate at which the drug-resistant
S. aureus evolved is ꢀ1 cell per <108 generations, which is
more rapid than the observed rate of ꢀ1 cell per <1010 gener-
ations for the parent compound, linezolid. However, it was
found that while the MIC value of the resistance mutant was
four-fold greater than the starting strain, the LZD-6 resistance
phenotype came with a mild fitness cost, as evidenced by a
slower growth phenotype (SI Figure S7a,b). This fitness versus
resistance cost had an even more profound effect when we
generated resistance mutants by long-period static growth at
sub-MIC concentrations of LZD-6 (SI Figure S7c,d).
Medicinal chemistry approaches to developing antibiotics,
particularly targeting the ribosome, can be hampered by chal-
lenges gaining structural information from which to design
novel compounds. Exploiting knowledge of structural changes
in the ribosomal drug-binding pocket that gives rise to linezol-
id resistance in MRSA, we have designed analogues of linezolid
that overcome these changes, thereby binding and inhibiting
the LinR ribosome. There is reason to believe that in vivo, the
evolution of resistance to LZD-5 and LZD-6 will be more chal-
lenging to bacteria, given that these compounds bind through
an induced fit into both states (i.e., LinS and LinR) of the drug-
binding pocket and given that the active site of the ribosome
The density map around LZD-6 was interpretable at 2.8 ꢁ
resolution, and the position and stereochemistry of the mole-
cule could be placed unambiguously (Figure 2a). LZD-6 adopts
a similar binding position to that of linezolid (Figure 2b); how-
ever, the 2-chloroacyl group is now directed toward rRNA resi-
due A2503 and the 2-oxazolidone ring is in a slightly different
orientation to maximise hydrophobic interactions with U2504
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Figure 2. a) Diagrammatic representation of the cryoEM structure of the peptidyl transferase center in the 50S ribosomal subunit from MRSA with LZD-6
bound at the peptidyl transferase center,[19] highlighting how LZD-6 would inhibit protein synthesis by blocking binding of incoming tRNA into the A-site.
Overlaid are the positions of the 3’ end of the tRNAs in the A-site and P-site (blue and green, respectively). b) CryoEM density map drawn at 4s around the
binding site of LZD-6 in the A-site of the peptidyl transferase center (PTC). Stick representations show the rRNA residues; thicker sticks show the oxazolidine
molecule. c) Cartoon representation of the structural superposition of the binding region of the LZD-6:70S complex (cyan) and the linezolid:70S complex (ma-
genta) structure (PDB ID: 4WFA).[19] d) Cartoon representation of the structural superposition of the binding region of both the LZD-6:70S complex (cyan) and
the same region of the LinR ribosome structure (red) from MRSA (PDB ID: 5T7V)[8a] e) Enlargement of the binding site of LZD-6 (grey and CPK coloring) in the
drug-binding pocket, showing in dotted lines interactions with surrounding rRNA residues (blue: hydrogen bond interactions; grey: van der Waals/p–p inter-
actions); left and right are 1808 rotations about the viewing plane.
where the compounds bind is not infinitely malleable and that
there is an observable growth fitness cost that coincides with
resistance. Structural analysis of the modified drug bound to
the ribosome showed how these modified structures induced
codes EMD-7867 for LZD-5–MRSA50S and EMD-7870 for LZD-6–
MRSA50S.
a fit to the ribosome through additional interactions with rRNA Acknowledgements
residues in the binding pocket. This work showcases the use of
cryoEM as a framework for the rational design of new antibiot-
ics. Although LZD-5 and 6 are unlikely to display drug-like fea-
tures, ongoing work in our laboratories is focused on the use
of cryoEM to provide facile access to structural information of
value to the pursuit of novel antibiotics.
We acknowledge the support staff and facilities at the Monash
Ramaciotti Centre for Cryo-Electron Microscopy. We thank Georg
Ramm for Titan Krios support, Anton Peleg for providing clinical
isolates of MRSA and VRE, and Ana Traven and Jamie Rossjohn
for comments on the manuscript. We acknowledge the NVIDIA
Corporation for donating GPUs to aid with calculations used in
this research. This work was supported by the Multi-modal Aus-
tralian ScienceS Imaging and Visualisation Environment (MAS-
SIVE) (www.massive.org.au). The work was funded by the Nation-
al Health & Medical Research Council of Australia (Program
Grant 1092262 to T.L.) and the Australia Research Council (DP
170103567 to D.W.L.). The funders had no role in study design,
data collection and interpretation, or the decision to submit the
work for publication.
Experimental Section
Detailed method descriptions are available in the Supporting Infor-
mation. All figures were generated with either PyMOL or UCSF Chi-
mera. The atomic structures have been deposited in the RCSB Pro-
tein Data Bank with accession codes 6DDD for LZD-5–MRSA50S
and 6DDG for LZD-6–MRSA50S. The cryoEM density maps were de-
posited in the Electron Microscopy Data Bank under accession
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Conflict of interest
The authors declare no conflict of interest.
Keywords: antibiotic resistance · cryoEM · linezolid · structure-
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Manuscript received: January 20, 2019
[9] G. Scapin, V. P. Dandey, Z. Zhang, W. Prosise, A. Hruza, T. Kelly, T. May-
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Accepted manuscript online: January 22, 2019
Version of record online: && &&, 0000
ChemMedChem 2019, 14, 1 – 6
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ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ

COMMUNICATIONS
M. J. Belousoff, H. Venugopal, A. Wright,
S. Seoner, I. Stuart, C. Stubenrauch,
R. S. Bamert, D. W. Lupton,* T. Lithgow*
The bacterial ribosome is a common
target for antibiotics. However, it is a
difficult drug target for structural study
by conventional X-ray crystallography.
We report the use of single-particle
cryo-electron microscopy to aid in the
structure-based drug design of new an-
tibiotics based on the oxazolidinone,
linezolid. We use this technique to show
by a rational design approach that it is
possible to synthesise new antibiotics
with antibacterial activity against line-
zolid-resistant forms of both methicillin-
resistant Staphylococcus aureus and van-
comycin-resistant Enterococcus.
&& – &&
cryoEM-Guided Development of
Antibiotics for Drug-Resistant Bacteria
&
ChemMedChem 2019, 14, 1 – 6
www.chemmedchem.org
6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!