Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity

Article abstract of DOI:10.1021/ja410385c

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.

Full text of DOI:10.1021/ja410385c

Subscriber access provided by DUESSELDORF LIBRARIES
Article
Restriction of the Conformational Dynamics of the Cyclic
Acyldepsipeptide Antibiotics Improves Their Antibacterial Ac-tivity
Daniel W. Carney, Karl R. Schmitz, Jonathan V. Truong, Robert T. Sauer, and Jason K. Sello
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja410385c • Publication Date (Web): 14 Jan 2014
Downloaded from http://pubs.acs.org on January 22, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Restriction of the Conformational Dynamics of the Cyclic
Acyldepsipeptide Antibiotics Improves Their Antibacterial Ac-
tivity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Daniel W. CarneyA‡, Karl R. SchmitzB‡, Jonathan V. TruongA, Robert T. SauerB, and Jason K. SelloA*
ADepartment of Chemistry, Brown University, 324 Brook Street, Providence, RI 02912; BDepartment of Biology,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
KEYWORDS: ClpP peptidase, antibacterial, macrocycle, dynamics, protein-ligand interaction, intramolecular hydrogen
bonding
ABSTRACT: The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mecha-
nism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The
potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through me-
dicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is forti-
fied by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the
peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule
into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be
engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations
in vitro. Remarkably, these compounds have up to 1,200-fold enhanced antibacterial activity when compared to those with the pep-
tidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation
of conformational dynamics may be used to improve the bioactivities of natural products.
Introduction
physiological roles of ClpP make it an attractive target for the
development of antibacterial agents.
Among the most interesting antibacterial drug targets to
emerge in the past decade is the proteolytic complex formed
by ClpP (caseinolytic peptidase) and its AAA+ partners
(ATPases associated with diverse cellular activities). ClpP is a
highly conserved peptidase that is involved in the turnover of a
wide variety of cellular proteins, including transcription fac-
tors that regulate virulence-factor production and stress re-
sponses.1-5 To form the catalytically active peptidase, ClpP
monomers self-assemble into heptameric rings that stack face-
to-face to form a barrel-shaped tetradecamer.6-8 The “barrel”
encloses a solvent-filled chamber that is decorated with four-
teen serine protease active sites and, in principle, is large
enough to accommodate a 50 kDa protein.6-8 However, narrow
axial pores at each end of the barrel prevent entry of folded
proteins into the proteolytic chamber.6-8 In fact, only small
peptides with 6 or fewer amino acids may freely diffuse into
the ClpP proteolytic chamber and be degraded. In the degrada-
tion of folded proteins, ClpP functions in conjunction with
AAA+ partners like ClpA, ClpX, and ClpC that recognize,
unfold, and coaxially translocate substrates into the proteolytic
chamber.9-12 These accessory ATPases play critical roles in
regulating the activity of ClpP. Genetic studies have estab-
lished that the clpP gene and genes encoding the AAA+ part-
ners are essential for virulence in some pathogenic bacteria
(e.g., Staphylococcus aureus, Listeria monocytogenes, and
Streptococcus pneumoniae) and for viability in others (e.g.,
Mycobacterium tuberculosis).13-20 Although no drugs that tar-
get ClpP have been introduced into clinic yet, the critical
Several molecules reported to perturb ClpP activity have been
discovered in unbiased screens for antibacterial agents or in
mechanistic investigations of natural products with antibacte-
rial activity.13,14 These compounds are classified as either acti-
vators or inhibitors of ClpP. Treatment of bacteria with inhibi-
tors of ClpP phenocopies the effects of clpP null mutations
(i.e., compromised virulence or viability);21,22 whereas bacteria
are killed upon exposure to ClpP activators.23 The first ClpP
activators to be reported were the cyclic acyldepsipeptide anti-
biotics (ADEPs).24,25 The representative members of this group
of antibiotics are “A54556A and B” produced by Streptomyces
hawaiiensis24 and enopeptins A and B produced by Streptomy-
ces sp. RK-1051 (Figure1).25 Collectively, the ADEPs have
been reported to exhibit potent activity against a broad range
of Gram-positive bacterial pathogens, including S. aureus, S.
pneumoniae, Enterococci, and M. tuberculosis.24-29 As reflect-
ed by the fact that no clinically used antibacterial drugs target
ClpP, the ADEPs’ have activity against multi-drug resistant,
pathogenic bacteria observed in clinical and community set-
tings.26,29
Structural studies indicate that the ADEPs bind at the subunit
interfaces of the ClpP tetradecamer, which also serve as dock-
ing sites for the accessory ATPases.30,31 A consequence of this
competitive binding is expansion of ClpP’s axial pores.26, 30-32
Remarkably, these ADEP-induced changes in ClpP quaternary
structure enable it to degrade oligopeptides and unstructured
or nascent proteins without the intervention of the accessory
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 9
ATPases. The indiscriminate degradation of cellular proteins tidolactone conformational dynamics. In addition, enzymatic
1
2
3
4
5
6
7
8
like the essential cell-division protein, FtsZ, by ADEP-
activated ClpP underlies the antibiotics’ toxicity.33 With re-
spect to mechanism, the ADEPs are unique because most anti-
biotics inhibit rather than activate their targets.
assays were used to measure the capacity of the ADEP analogs
to bind and activate ClpP. Finally, we used bioassays to assess
the toxicities of the compounds to three species of pathogenic
bacteria. It is noteworthy that some of the rigidified ADEPs
bind and activate ClpP at substantially lower concentrations in
vitro and have up to 1,200-fold enhanced antibacterial activity.
The efficacy of ADEPs at killing pathogenic bacteria and their
peculiar mode of action have prompted efforts to assess their
medicinal potential. Initially, the natural products were found
to be inactive in mouse models of systemic S. aureus infec-
tion, despite their potent antibacterial activity in vitro.29 Their
pharmacokinetic profiles were characterized by poor water
solubility, rapid systemic clearance, and chemical instability.
At Bayer Healthcare AG, a medicinal chemistry program was
established to optimize the structures of the ADEPs in ways
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
that would enhance their stability and biological activity.29
A
compound called ADEP-4, which had 160-fold greater poten-
cy and more chemical stability than the natural products,
emerged from their optimization program. It differs from eno-
peptin A and A54556A in three ways (Figure 1).29 First, it has
a more chemically stable heptenoyl moiety in place of the
conjugated polyenes. Second, rather than phenylalanine in its
appendant
side
chain,
ADEP-4
has
a
3,5-
difluorophenylalanine, which was credited with improving
compound bioavailability and binding to ClpP. Finally, the
natural ADEPs have a N-methylalanine residue within the core
macrocycle, whereas ADEP-4 has a cyclic amino acid, pipeco-
late (a six-membered ring), at the same position. The last fea-
ture was particularly important for enhancing potency. Ac-
cordingly, it was proposed that the incorporation of the pipe-
colate residue rigidifies the ADEP peptidolactone, thereby
reducing the entropic cost of ClpP binding.29 This rigidifying
structural feature must enforce a specific conformation that is
compatible with ClpP binding, as evidenced by the inactivity
of an ADEP analog with a proline residue (a five-membered
ring) in place of N-methyl alanine.34 In addition to potent ac-
tivity in vitro against S. aureus, S. pneumoniae, and Entero-
cocci,29 ADEP-4 and close analogs are reported to be toxic to
M. tuberculosis in vitro, particularly in combination with ef-
flux pump inhibitors.28 Importantly, ADEP-4 has impressive
activity in vivo. In fact, mice with potentially lethal infections
of S. aureus were cured by via intravenous administration of
ADEP-4.29 Recently, it has been reported to completely eradi-
cate S. aureus biofilms in vitro and in mouse models of chron-
ic infection when co-administered with the anti-bacterial drug
rifampicin.35
In a previous study,27 we investigated the consequences of
replacing amino acids in the ADEP macrocycle with more
conformationally constrained residues. One of the compounds
that we prepared was an analog of ADEP-4 with 4-methyl
pipecolate in place of the pipecolate. The methyl substituent
was predicted to further restrict the conformational flexibility
of the pipecolate and by extension that of the peptidolactone.
The compound with 4-methyl pipecolate was 2- and 4-fold
more potent than ADEP-4 against clinical isolates of methicil-
lin-resistant S. aureus and vancomycin-resistant E. faecalis,
respectively.27 Motivated by these initial findings, we sought
to study the phenomenon of restricting peptidolactone con-
formation and define its impact on the ADEPs’ binding to and
activation of ClpP and on their bioactivity. We utilized deuter-
ium exchange experiments with 1H-NMR to empirically
measure the effects of various structural modifications on pep-
Figure 1. Structures of ADEP natural products and optimized
synthetic analogs thereof.
Results and Discussion
Assessment of the Conformationally Biasing Hydrogen
Bonds in the ADEPs. Numerous non-covalent interactions
between the ADEPs and ClpP are observed in crystal struc-
tures of the complex.30,31 The ADEPs themselves adopt a
compact conformation that appears to be enforced by two
transannular hydrogen bonds between the peptidolactone and
the appendant side chain (Figure 2A, Table S1). Interestingly,
a similar conformation is observed in crystals of free ADEP,
where analogous hydrogen bonding between the peptidolac-
tone and the side chain has been predicted.29 The similarities
suggest that the free ADEPs may be pre-disposed to adopt a
conformation that is compatible with ClpP binding.
To test the prediction that free ADEPs exhibit intramolecular
hydrogen-bonding in solution, we performed experiments in
which 1H-NMR was used to measure deuterium exchange
rates of amide hydrogen atoms predicted to participate in the
bonds (Figure 2B). Hydrogen-deuterium exchange rates have
been shown to be dependent upon the presence and strength of
intramolecular hydrogen bonds in peptides.36 Accordingly, we
anticipated that deuterium exchange rates at the amide bonds
engaged in hydrogen bonds would be slower than at non-
hydrogen-bonded amides. Given the limited solubility of the
ADEPs in water, we selected deutero-methanol (CD3OD) as
the solvent for the deuterium exchange experiments; accepting
the possibility that the molecules’ conformations could differ
in organic and aqueous solvents. Immediately after preparation
of a dilute solution of ADEP 1a in CD3OD (Figure 2B, Figure
3), we monitored attenuation of the amide proton resonances
1
by H-NMR over a period of hours at 25 °C. As expected, the
hydrogen atoms of the three secondary amides in the ADEP
exchanged with deuterium at markedly different rates (Figure
2B, C). The amide hydrogen of the serine residue, which does
not participate in a transannular hydrogen bond, exchanged
completely in CD3OD within several seconds and could never
be observed in a 1H-NMR spectrum.
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
Figure 2. Trans-annular hydrogen bonding in an ADEP. A) Stereo-cartoon of an ADEP (gray ball-and-sticks) bound to E. coli ClpP
(adjacent subunits in green and orange), generated from crystal structure 3MT6.30 Two predicted hydrogen bonds are observed
within the ADEP (black; distances in Å), and several hydrogen bond networks (cyan) occur either directly between the ADEP and
ClpP or via ordered water molecules. B) Schematic representation of ADEP trans-annular hydrogen bonds C) Overlay of 1H-NMR
spectra of compound 1a over time in CD3OD. Amides participating in bonds are highlighted in blue and the non-bonding amide is
highlighted in red. The half-lives of the hydrogens of the alanine and difluorophenylalanine residues were 26.8 minutes and 3.87
minutes, respectively (see supporting information).
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
By comparison, the amide hydrogen of the side chain difluor-
ophenylalanine residue required several minutes to completely
exchange with deuterium; whereas, the macrocycle alanine
amide hydrogen exchanged over the course of two hours (Fig-
ure 2C). These observations are consistent with the existence
of trans-annular hydrogen bonds that are analogous to those
inferred from the crystal structures of both free ADEP and
ADEP in complex with ClpP.29-31
Chemical Syntheses of ADEPs with Conformationally
Constrained Peptidolactones. We hypothesized that the bio-
active conformation of the ADEPs could be stabilized by judi-
cious replacement of particular amino acid constituents of the
peptidolactone with more conformationally constrained ana-
logs. First, we envisioned substituting the N-methylalanine
residue with pipecolate residues bearing C-4 substituents of
varying size. Cyclic amino acids are typically more conforma-
tionally constrained than their acyclic counterparts, and ring
substituents tend to limit ring conformational dynamics by
imposing high energetic penalties to certain conformations
(e.g., 1,3- diaxial strain). In a separate approach, we envi-
sioned replacing the serine residue of the macrocycle with
allo-threonine, a serine analog with a methyl substituent on the
β-carbon. This amino acid is more conformationally con-
strained because the methyl group confers additional torsional
strain about both the Cα-Cβ bond and Cβ-O bond. Important-
ly, we predicted that the methyl substituent of this diastere-
omer of threonine would not sterically clash with ClpP.
Figure 3. Library of ADEP analogs. The N-methylalanine, pipe-
colate, 4-methylpipecolate and 4-isopropylpipecolate residues are
highlighted in red. Serine and allo-threonine residues are high-
lighted in blue.
There are multiple precedents for the chemical syntheses of
1
Measurement of ADEP peptidolactone dynamics via H-
the cyclic acyldepsipeptide antibiotics and analogs thereof.27,
NMR Deuterium Exchange. With the desired ADEPs in
hand, we sought to examine the relative rigidities of the pepti-
dolactones empirically. We anticipated that the deuterium
exchange rates for the hydrogens of the amides engaged in the
hydrogen bonds would be dependent on the conformational
freedom of the ADEP peptidolactone. For all seven ADEPs,
the alanine amide hydrogens’ half-lives in CD3OD were
measured from the rates at which their resonances in 1H-NMR
spectra attenuated relative to those of a non-exchanging refer-
ence signal in the same spectra (Figure 4; Table 1). Compound
1a, a known molecule29 that is the closest analog of the natural
product enopeptin B with N-methylalanine and serine residues
in its peptidolactone, was expected to have the least rigid mac-
rocycle and thus served as a point of comparison for the other
ADEPs.
29, 37
The desired ADEP analogs were synthesized via a con-
vergent strategy that was previously developed in our labs (see
supporting information).27 The key tripeptide fragments con-
taining pipecolate or the substituted pipecolates were prepared
using Joullié-Ugi multicomponent reactions of dehydropiperi-
dines, a chiral isocyanoacetate derived from alanine, and Boc-
proline.27, 38 An ADEP with a natural product peptidolactone
and an additional six ADEPs with conformationally restricted
amino residues in the peptidolactone were chemically synthe-
sized (Figure 3).
ACS Paragon Plus Environment

Journal of the American Chemical Society
Using deuterium-exchange experiments, we systematically
Page 4 of 9
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
8
assessed the conformational consequences of replacing the N-
methylalanine and serine residues in the ADEP macrocycle
with conformationally constrained pipecolate and allo-
threonine residues, respectively (Figure 4). Interestingly, the
pipecolate moiety does not fortify both of the transannular
bonds that are apparent in compounds containing N-
methylalanine (compounds 1a and 1e). Indeed, we found that
the amide hydrogen of the difluorophenylalanine of compound
1b exchanged completely within seconds in CD3OD, whereas
the analogous hydrogen in compound 1a had a half-life of
3.87 minutes (see supporting information). Apparently, with
the pipecolate residue in the macrocycle, the potential donor
and acceptor atoms of the hydrogen bond are either too far
apart or do not have appropriate trajectories for bonding. In
contrast, the transannular hydrogen bond in which the alanine
residue is the donor is retained in all of the ADEPs and
strengthened by the presence of conformationally constrained
amino acids within the macrocycle. For instance, compound
1b harboring a pipecolate residue in the peptidolactone had a
slower rate of deuterium exchange rate than 1a. Further, we
found that the deuterium-exchange rate decreased as the steric
bulk of the C4 substituent on the pipecolate increased (see data
for compounds 1b, 1c, and 1d in Figure 4). Substitution of
allo-threonine for serine in the ADEP peptidolactone pro-
foundly slows the deuterium-exchange rate. For instance, the
half-lives of the alanine amide hydrogens in compound 1a,
which has serine, and compound 1e, which has allo-threonine,
are ~100-fold different. Likewise, the deuterium exchange rate
of the hydrogen atom of the difluorophenylalanine moieties in
compounds 1a and 1e differed by 2.8-fold. As expected, inclu-
sion of both pipecolate and allo-threonine (1f) into the pepti-
dolactone had a synergistic effect on deuterium exchange.
Interestingly, the apparent relationship between rigidifying
structural features and deuterium exchange rate was not ob-
served when 4-methylpipecolate and allo-threonine were pre-
sent together in the peptidolactone (1g). This compound had a
faster rate of deuterium exchange than compounds with allo-
threonine and either N-methylalanine (1e) or pipecolate (1f) in
the peptidolactone. A reasonable explanation for this observa-
tion is that substituted pipecolate residues and allo-threonine
each enforce slightly different low energy conformers. Ac-
cordingly, the opposing forces could prevent a single, low
energy conformer from being reached. As expected, the rate of
deuterium exchange increased for all compounds at an elevat-
ed temperature (i.e, 40°C), whereas the trend for relative rates
of deuterium exchange remained the same (see supporting
information), suggesting that the observed effects are the re-
sult of entropic factors. Overall, the general trend represented
by these data support our hypothesis that the incorporation of
conformationally constrained residues in the peptidolactone
has a rigidifying effect.
1a
1b
1c
1d
1e
1f
9
1g
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
100
200
300
400
500
Time (minutes)
Figure 4. ADEP hydrogen-deuterium exchange in CD3OD. Deu-
terium exchange rates were measured for 2mM solutions of each
ADEP under pseudo-first order conditions in deuterated methanol
at 25 °C. The exchange rates for the hydrogen atoms of the ala-
nine residues within the peptidolactone are shown.
In vitro Assessment of ClpP Binding and Activation by the
Rigidified ADEPs. Binding of either the ADEPs or the regu-
latory ATPases to ClpP stabilizes an open conformation of the
peptidase pore and stimulates degradation of oligopeptides.10,
39,40
Based on predictions that the entropic of costs of ClpP
binding would be lower for the conformationally constrained
ADEP derivatives,27, 29 we expected that ADEP modifications
that enhance macrocycle rigidity would improve ClpP binding
and activation in a commensurate fashion. To test this hypoth-
esis, we assayed ClpP catalyzed hydrolysis of an internally
quenched fluorogenic decapeptide in the absence and presence
of the ADEP derivatives. Cleavage between an aminobenzoic
acid fluorophore and 2-nitrotyrosine quencher in this substrate
relieves quenching, resulting in increased fluorescence that
serves as a readout of peptidase activity. The capacities of
each of the compounds to activate ClpP were assessed across a
range of concentrations, and the resulting activities were fit to
yield apparent dissociation constants (Fig. 5A, Table 1). As
expected, we found a generally strong and positive correlation
between the potency of the compounds as activators of ClpP
and the deuterium exchange half-lives. Kapp values range from
7.5 µM for compound 1a, the parental compound having the
least rigid macrocycle, to 1.1 µM for compound 1g, which
possesses a significantly more rigid macrocycle. Interestingly,
compound 1d was a weaker activator of ClpP than compounds
1b and 1c, despite having a more rigid macrocycle. The bulky
C4-isopropyl substituent may be poorly accommodated by the
ClpP binding pocket. Nevertheless, structural modifications
that rigidify the ADEP peptidolactone can improve ClpP acti-
vation up to ~7-fold in vitro.
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
Gram-positive bacterial pathogens: S. aureus, S. pneumoniae,
1
and E. faecalis. Minimum inhibitory concentrations (MICs)
were determined by broth microdilution assays (Table 1). All
seven synthetic ADEPs exhibited strong antibacterial activity.
There were largely positive correlations between ADEP anti-
bacterial activity, peptidolactone rigidity, and the apparent
ClpP affinity. There were some exceptions. Compound 1d,
despite possessing a significantly more rigid peptidolactone,
exhibited ClpP affinity and antibacterial activity similar to that
of compound 1a. Again, the presence of the large isopropyl
substituent on the pipecolate residue most likely has a negative
effect on binding to ClpP and thus antibacterial activity. In
contrast, compound 1g, bearing both a 4-methylpipecolate
residue as well as an allo-threonine residue, was not the most
rigid compound (compound 1f in Table 1), yet it exhibited the
most potent antibacterial activity in the series. While these
exceptions cannot be completely explained, we do note that
ADEPs whose amide hydrogens of the alanine residues have
half-lives of more than 20 hours in the deuterium exchange
experiments have the highest ClpP affinities and most antibac-
terial activity against all three species of bacterial pathogens.
To the best of our knowledge, compound 1g has the lowest
MICs of any ADEP reported to date.27,29 The antibacterial
activity of 1g was 32-fold more potent against S. aureus, 600-
fold more potent against E. faecalis, and 1200-fold more po-
tent against S. pneumoniae than compound 1a, which has the
more flexible peptidolactone of the ADEP natural products.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Activation of ClpP and competition with ClpX by
ADEPs in vitro. (A) Rigidified ADEPs are more potent activa-
tors of ClpP peptide cleavage. Hydrolysis of a fluorogenic
decapeptide substrate (15 µM) by E. coli ClpP (25 nM) was
assayed in the presence of increasing concentrations of ADEP
compounds, and activity was fit to a non-cooperative binding
model (solid lines). Error bars represent standard deviation
among three replicates or standard error of the fit. Tighter
apparent affinities correlate with increased ADEP rigidity,
with the exception of compound 1d. See also Table 1. (B)
ADEPs with greater macrocycle rigidity compete more strong-
ly with ClpX for binding to ClpP. Fold change in ATPase ac-
tivity of E. coli ClpXΔN (10 nM) in the presence of E. coli
ClpP (50 nM) was assayed over increasing concentrations of
ADEPs, compared to the activity of ClpXΔN alone, and was fit
as above (no fit was obtained for 1d). More rigid ADEPs bet-
ter compete for binding to ClpP, and thus more effectively
relieve ClpP-mediated repression of ClpXΔN ATPase activity
(Table 1).
Conclusion
The cyclic acyldepsipeptide antibiotics are a promising class
of antibacterial agents that act by binding and dysregulating
the activity of the ClpP peptidase. Reports by our group and
others state that the activities of these compounds can be dra-
matically improved by replacing certain amino acid constitu-
ents of the peptidolactone core structure with more conforma-
tionally constrained counterparts.27,29 It has been proposed that
improvements in bioactivity are a consequence of these amino
acids’ capacity to stabilize a bioactive conformation of the
ADEPs, which incurs a lower entropic cost upon binding to
ClpP.29 While compelling, this proposal had very little exper-
imental support. Herein, we present data indicating that re-
placement of selected constituent amino acids in the ADEP
peptidolactone core does indeed stabilize a bioactive confor-
mation. Specifically, analyses of ADEPs harboring conforma-
tionally constrained amino acids via deuterium exchange ex-
periments revealed that they exhibit the same trans-annular
hydrogen bonds in solution that are inferred from the crystal
structures of an ADEP in complex with ClpP. Our finding that
replacement of the N-methylalanine moiety of the ADEP natu-
ral products with a pipecolate attenuates deuterium exchange
of only one of the two donors (i.e., amide hydrogen of alanine)
in the trans-annular hydrogen bonds, whereas the substitution
of the natural serine with allo-threonine suppresses deuterium
exchange rates of both hydrogen bond donors indicates the
position of the conformationally constrained amino acid with-
in the macrocycle has important effects on molecular confor-
mation. In molecules with either one or two trans-annular hy-
drogen bonds, the inverse correlations between the number of
constrained amino acids constituting the peptidolactone and
the rates of deuterium exchange indicated that the amino acid
substitutions lock the ADEPs into a conformation that is com-
patible with ClpP binding. The apparent enhancements of the
conformationally constrained ADEPs’ capacities to both
In addition to modulating the quaternary structure of the ClpP
tetradecamer, ADEPs and the accessory ATPases share the
same binding sites and are known to compete for binding to
ClpP.30-32 As the rigidified ADEPs bound to ClpP more tightly,
we predicted that these compounds would be stronger compet-
itors for ATPase binding. We assayed binding competition by
exploiting the observation that E. coli ClpX ATPase activity is
depressed upon binding E. coli ClpP.39,40 Accordingly, we
inferred competition from the degree to which the ADEPs
relieved depression of ATP hydrolysis by ClpX (Fig. 5B, Ta-
ble. 1). As expected, IC50 values correlated with apparent af-
finities deduced from the peptidase activation experiments.
The increased competition with ClpX indicates that the more
rigid ADEPs bind more strongly to their original binding site
on ClpP, rather than to novel sites. Interestingly, compound 1d
did not effectively compete with ClpX, despite the observation
that it activated ClpP peptidase activity to the same extent as
compound 1a. Again, this weak suppression of ClpX ATPase
activity can most likely be ascribed to poor accommodation of
the isopropyl group in the ClpP binding pocket.
Assessment of the Bioactivities of the Conformationally
Constrained ADEPs in Antibacterial Assays. The antibacte-
rial activity of each compound was assessed against three
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
Table 1 - Comparison of in vitro and in vivo ADEP properties
1
MIC
2
3
4
5
6
7
D exchange t1/2
(min)
ClpP Activation
ClpX Competition
Compound
S. aureus
(µg/mL)
S. pneumoniae
E. faecalis
(µg/mL)
Kapp (µM)
IC50 (µM)
(µg/mL)
1a
1b
1c
26.1
61.6
115
7.5±0.34
2.9±0.077
3.0±0.10
53±39
33±6.5
18±4.2
0.78
0.39
0.39
0.024
0.006
0.012
0.012
0.015
0.003
8
9
1d
191
7.4±0.31
no fit
1.16
0.098
0.098
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1e
1f
2500
10000
1180
1.3±0.10
1.3±0.067
1.1±0.060
9.8±3.9
2.9±1.0
4.7±1.3
0.098
0.098
0.024
0.003
0.00076
<0.0001
<0.0001
<0.0001
<0.0001
1g
The bacterial concentrations (colony forming units/mL) in each well of the dilution antimicrobial susceptibility tests were as follows: S.
aureus (1.20 x 106), E. faecalis (8.5 x104), and S. pneumoniae (3.65 x 105). The deuterium exchange rates for the hydrogen atoms of the
alanine
residues
in
the
macrocycles
are
shown.
activate ClpP and compete with its binding to the accessory
ATPase ClpX corroborate the proposal that a bioactive con-
formation has been fortified. These improvements are also
consistent with the proposal that there is a lower entropic cost
in the binding of the rigidified ADEPs to ClpP. The latter
point is of particular interest because the commonly held view
that rigid ligands suffer a lower entropic cost in receptor bind-
ing than flexible ones41 has recently been challenged by cases
wherein there are entropic penalties for ligand preorganization
in receptor-ligand interactions.42 In any case, the finding that
ClpP activation by the ADEPs was enhanced by up to 7-fold
via the introduction of conformational constraints, while these
same changes enhanced antibacterial activity by up to 1,200-
fold indicates that there are other factors involved. A likely
explanation is that the constrained compounds are more cell-
permeable. Indeed, peptides with enforced transannular hy-
drogen bonds exhibit dramatically enhanced cell-permeability
and oral bioavailability because the bonding reduces the ener-
getic costs of desolvation that accompanies membrane pene-
tration in aqueous environments.43 Apparently, the conforma-
tional constraints that we have introduced enhance the
ADEPs’ intrinsic transannular hydrogen bonding interactions
that pre-dispose them for both ClpP binding and membrane
penetration.
which has a 4-methylproline residue in its macrocycle, has a
two-fold lower MIC against S. aureus, S. pneumoniae, E. fae-
calis, and E. faecium than enopeptin B which has an unsubsti-
tuted proline residue at the same position.29 In this study, we
found that the position of the methyl substituent on the ADEP
peptidolactone is very important. When comparing the ADEPs
lacking methyl substituents (compounds 1a and 1b) to analogs
harboring either 4-methyl pipecolate (compound 1c) or allo-
threonine (compound 1e), we find that the allo-threonine resi-
due exerts the strongest influence over conformational dynam-
ics, ClpP affinity, and bioactivity. Furthermore, it should be
noted that inclusion of allo-threonine in the ADEP peptidolac-
tone improves the MIC ten-fold, while inclusion of 4-
methylproline improves the antibacterial activity only two-fold
(as evidenced in the reported MICs of enopeptin A and eno-
peptin B).29
In this case, the unique characteristics of both the small mole-
cule ligands and their receptor facilitated in-depth studies of a
receptor-ligand interaction. Observations and modulations of
the conformational dynamics of ADEPs were accompanied by
measurements of their affinity for ClpP and antibacterial activ-
ity. A distinguishing feature of our multi-faceted study was the
use of deuterium-exchange 1H-NMR experiments to assess
relative differences in conformational rigidities of the ADEPs.
We have shown that in such cases, trans-annular hydrogen
bonding can be exploited to study the effects of structural
modification on conformational rigidity. We anticipate that
this approach to small-molecule dynamics could be applied to
studies of many ligand-receptor interactions because many
small molecules that interact with biological macromolecules
exhibit transannular hydrogen bonds (especially peptides). It is
a much simpler alternative to sophisticated multi-dimensional
NMR experiments wherein 15N- and 13C-labeled compounds
are used to assess the dynamics of small molecules.
It is well-known that the conformational constraints of macro-
cylic molecules can be further enhanced by judicious introduc-
tion of substituents on the ring.44 In this case, it is notable that
installation of small methyl substituents profoundly enhances
the affinity of a large macrocycle for its biomolecular receptor
and the molecules’ bioactivities. Although replacement of
hydrogen atoms with methyl groups is common in structure-
activity relationship (SAR) studies and medicinal chemistry
optimization programs,45 the inclusion of a methyl group on a
ligand typically is deleterious or minimally improves receptor
binding. Indeed, a recent analysis of published SAR studies by
Jorgensen and co-workers states that in 8% of cases the inclu-
sion of a methyl group enhances bioactivity 10-fold or better.46
In only 0.4% of cases did molecules with an additional methyl
group have 100-fold enhanced bioactivity.46 Their analysis
also revealed that significant improvements in bioactivity are
usually the result of the methyl group’s capacity to fill a hy-
drophobic environment in the receptor and to influence the
conformation of the ligand. Interestingly, the substituent effect
strategy exploited in medicinal chemistry is mirrored in the
ADEP natural products themselves. Specifically, enopeptin A,
The ADEP analogs reported herein constituted by the confor-
mationally constrained amino acids allo-threonine and 4-
methylpipecolate have some of the lowest MICs ever reported
for antibacterial agents. The most potent ADEP reported prior
to this work, ADEP-4, was reported to cure S. aureus infec-
tions in mice and S. pneumoniae infections in rats with even
greater efficacy than linezolid, a clinically used drug.29 Given
that our optimized analogs have MICs against S. pneumoniae
and E. faecalis that are 200-fold lower than those reported for
ADEP-4, it is tempting to speculate that a dramatically lower
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
and potentially safer dose of our most potent compound could MIC determinations: MIC determinations were performed
1
2
3
4
5
6
7
8
be efficacious in the treatment of infections caused by Strepto-
cocci, Enterococci, and potentially other Gram-positive patho-
gens. An added advantage of the optimized compounds re-
ported here with respect to drug development is that the key
allo-threonine residue is much less expensive and easier to
prepare than the 4-methylproline constituent of ADEP-4.29 The
promise of these molecules is further enhanced by the obser-
vations that peptides with strong transannular hydrogen bonds
have enhanced oral bioavailability.43, 44 Testing of these com-
pounds in animal models of infection is currently underway in
our laboratories. In total, our findings provide a compelling
illustration of how the pharmacological properties of natural
products can be improved by rational design.
BSL2+ conditions at the New England Center for Research
Excellence (NERCE) in biodefense at Harvard Medical
School following standard dilution antimicrobial susceptibility
testing protocols.50 Following incubation with the bacteria and
the compound, each well was visually examined for growth
with the unaided eye. The MIC is determined to be the first set
of replicate wells of the dilution series exhibiting no growth
when compared to the growth control wells.
9
ASSOCIATED CONTENT
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information. Details regarding elevated tempera-
ture deuterium exchange experiments, intramolecular hydro-
gen bond distances in ADEP-ClpP complex structure, and
compound synthesis and characterization (including tripeptide
crystal structures with the CIF files) can be found in the sup-
porting information. This information is available free of
charge via the internet at http://pubs.acs.org.
Experimental Section
H-D exchange kinetics. NMR samples were prepared by dis-
solving thoroughly dried ADEP in ampule sealed CD3OD at a
concentration of 2 mM. The ADEP in CD3OD was promptly
transferred to a clean NMR tube, purged with an argon atmos-
phere, then capped and sealed with parafilm before being
placed into the NMR spectrometer. Standard proton NMR
spectra were acquired periodically over the course of several
hours. The integration of the exchanging amide signal of inter-
est was calibrated to a non-exchanging reference peak. Each
data set was normalized such that the integral of the amide
signal of interest in the first spectrum acquired was equal to
1.00 and designated as t0. Data sets were plotted in Microsoft
excel as normalized integrals vs. time. Plotted data sets were
fit with exponential curves with Y intercepts set to 1. Ex-
change half-lives were calculated from the exponential func-
tions.
AUTHOR INFORMATION
Corresponding Author
*To whom correspondence may be addressed. E-mail:
jason_sello@brown.edu; Phone:(401) 863-1194.
Author Contributions
‡These authors contributed equally to this work.
ACKNOWLEDGMENTS
JKS and DCW gratefully acknowledge Brown University for
funding. JKS is the recipient of a NSF CAREER Award. The
work was supported in part by NIH grant GM-101988 to RTS.
Expert technical support on NMR and mass spectrometry were
provided by R. Hopson and T. Shen in the Brown University De-
partment of Chemistry. P. Williard at Brown determined crystal
structures of the multicomponent reaction products. C. Andersen
and A. Onderdonk from the New England Regional Centers of
Excellence in Biodefense at Harvard Medical School for perform-
ing the antibacterial assays with pathogenic strains.
Protein expression and purification. E. coli ClpP bearing a
C-terminal His6 tag and single-chain pseudo-hexameric E. coli
ClpXΔN (amino acids 62-424) were expressed and purified by
metal affinity, anion exchange, and gel-filtration chromatog-
raphy as described.39,47
Activity and competition assays. In vitro assays were per-
formed at 30 °C in PD buffer (25 mM HEPES, pH 7.5, 100
mM KCl, 5 mM MgCl2, 1 mM DTT, 10% (wt/vol) glycerol,
10% (vol/vol) DMSO) using a SpectraMax M5 microplate
reader (Molecular Devices). Peptidase activation was meas-
ured by incubating 25 nM of ClpP tetradecamer and ADEP
analog with 15 µM of an internally quenched fluorogenic pep-
tide substrate, Abz-KASPVSLGYNO2D,48 incorporating a 2-
aminobenzoic acid (Abz) fluorophore and 3-nitrotyrosine
(YNO2) quencher. Peptide hydrolysis by ClpP was monitored
by following the increase in 420 nm fluorescence upon 320
nm excitation. Initial analysis of peptidase data showed negli-
gible cooperativity, thus data were fit to a quadratic form of a
non-cooperative binding equation, assuming 14 equivalent
ADEP binding sites per ClpP tetradecamer.
REFERENCES
1. Maurizi, M. R.; Thompson, M. W.; Singh, S. K.; Kim, S. Meth.
Enzymol. 1993, 244, 314-331.
2. Gominet, M.; Seghezzi, N.; Mazodier, P.. Microbiology 2011,
157, 2226-2234.
3. Gottesman, S.; Wickner, S.; Maurizi, M. R. Genes Dev. 1997,
11, 815-823.
4. Sauer, R. T.; Baker, T. A. Annu. Rev. Biochem. 2011, 80, 587-
612.
5. Yu, A. Y. H.; Houry, W. A. FEBS Lett. 2007, 581, 3749-3757.
6. Wang, J.; Hartling, J. A.; Flanagan, J. M. Cell 1997, 91, 447-
456.
7. Wang, J.; Hartling, J. A.; Flanagan, J. M. Crystal Structure De-
termination of Escherichia coli ClpP Starting from an EM-Derived
Mask, J. Struct. Biol. 1998, 124, 151-163.
8. Szyk, A.; Maurizi, M. R. J. Struct. Biol. 2006, 156, 165-174.
9. Gottesman, S.; Roche, E.; Zhou, Y.; Sauer, R. T. Genes Dev.
1998, 12, 1338-1347.
10. Singh, S. K.; Grimaud, R.; Hoskins, J. R.; Wickner, S.; Mau-
rizi, M. R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8898-8903.
11. Baker, T. A.; Sauer, R. T. Trends Biochem. Sci. 2006, 31, 647-
653.
To assay ADEP competition for ClpX binding to ClpP, 50 nM
of ClpP tetradecamer, 10 nM of ClpXΔN pseudo-hexamer, 0 -
100 µM of ADEP and 2.5 of mM ATP were incubated with an
NADH-coupled ATP regeneration system.49 ATP hydrolysis
was monitored by following the coupled disappearance of
NADH, via decrease in 340 nm absorbance. Pseudo-
hexameric ClpXΔN is functionally identical to monomerically
12. Alexopoulos, J. A.; Guarné, A.; Ortega, J. J. Struct. Biol. 2012.
179, 202-210.
encoded ClpXΔN 39, 44a
and was used to ensure hexamer stabil-
,
13. Raju, R. M.; Goldberg, A. L.; Rubin, E. J. Nat. Rev. Drug Dis-
cov. 2012, 11, 777-789.
ity at low ClpX concentrations. ATPase data were fit as above,
assuming two ClpX binding sties per ClpP tetradecamer.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
14. Roberts, D. M.; Personne, Y.; Ollinger, J.; Parish, T. Future
microbial. 2013, 8, 621-631.
15. Frees, D.; Qazi, S. N. A.; Hill, P. J.; Ingmer, H. Mol. Microbiol.
2003, 48, 1565-1578.
16. Frees, D.; Sorensen, K.; Ingmer, H. Infect. Immun. 2005, 73,
8100-8108.
17. Kwon, H.; Kim, S.; Choi, M.; Ogunniyi, A. D.; Paton, J. C.;
Park, S.; Pyo, S.; Rhee, D. Infect. Immun. 2003, 71, 3757-3765.
18. Kwon, H.; Ogunniyi, A. D.; Choi, M.; Pyo, S.; Rhee, D.; Paton,
J. C. Infect. Immun. 2004, 72, 5646-5653.
19. Robertson, G. T.; Ng, W.; Foley, J.; Gilmour, R.; Winkler, M.
E. J. Bacteriol. 2002, 184, 3508-3520.
20. Gaillot, O.; Pellegrini, E.; Bregenholt, S.; Nair, S.; Berche, P.
Mol. Microbiol. 2000, 35, 1286-1294.
21. a) Böttcher, T.; Sieber, S. A. J. Am. Chem. Soc. 2008, 130,
14400-14401. b) Böttcher, T.; Sieber, S. A. ChemBioChem 2009, 10,
663-666.
34. Mann, A. The Practice of Medicinal Chemistry 2003, 2, 233-
250.
35. Conlon, B.; Nakayasu, E.; Fleck, L.; LaFleur, M.; Isabella, V.;
Coleman, K.; Leonard, S.; Smith, R.; Adkins, J.; Lewis, K. Na-
ture 2013, 503, 365-370.
36. Steffel, L. R.; Cashman, T. J.; Reutershan, M. H.; Linton, B. R.
J. Am. Chem. Soc. 2007, 129, 12956-12957.
37. Schmidt, U.; Neumann, K.; Schumacher, A.; Weinbrenner, S.
Angew. Chemie Int. Ed. 1997, 36, 1110-1112.
38. Carney, D.; Truong, J.; Sello, J. K. J. Org. Chem. 2011, 76,
10279-10285.
39. Martin, A.; Baker, T. A.; Sauer, R. T. Mol. Cell 2007, 27, 41-
52.
40. Joshi, S. A.; Hersch, G. L.; Baker, T. A.; Sauer, R. T. Nat.
Struct. Mol. Biol. 2004, 11, 404-411.
41. a) Böhm, H.; Klebe, G. Angew. Chemie Int. Ed.1996, 35, 2588-
2614. b) Khan, A. R.; Parrish, J. C.; Fraser, M. E.; Smith, W. W.;
Bartlett, P. A.; James, M. N. Biochemistry. 1998, 37, 16839-16845. c)
Nakanishi, H.; Kahn, M. The Practice of Medicinal Chemistry 2nd
Ed. Academic Press. 2003, 477-500. d) Searle, M. S.; Williams, D.
H. J. Am. Chem. Soc. 1992, 114, 10690-10697. e) Vajda, S.; Wheng,
Z.; Rosenfeld, R.; DeLisi, C. Biochemistry. 1994, 33, 13977-13988.
42. a) Benfield, A. P.; Teresk, M. G.; Plake, H. R.; DeLorbe, J. E.;
Millspaugh, L. E.; Martin, S. F. Angew. Chemie Int. Ed. 2006, 45,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22. Compton, C. L.; Schmitz, K. R.; Sauer, R. T.; Sello, J. K. ACS
Chem. Biol. 2013, in press.
23. Leung, E.; Datti, A.; Cossette, M.; Goodreid, J.; McCaw, S. E.;
Mah, M.; Nakhamchik, A.; Ogata, K.; El Bakkouri, M.; Cheng, Y.
Chem. Biol. 2011, 18, 1167-1178.
24. Michel, K. H.; Kastner, R. E. A54556 antibiotics and process
for production thereof. 1985. Patent
25. Osada, H.; Yano, T.; Koshino, H.; Isono, K. J. Antibiot. 1991,
44, 1463-1466.
26. Brotz-Oesterhelt, H.; Beyer, D.; Kroll, H.; Endermann, R.;
Ladel, C.; Schroeder, W.; Hinzen, B.; Raddatz, S.; Paulsen, H.; Hen-
ninger, K.; Bandow, J. E.; Sahl, H.; Labischinski, H. Nat. Med. 2005,
11, 1082-1087.
27. Socha, A. M.; Tan, N. Y.; LaPlante, K. L.; Sello, J. K. Bioorg.
Med. Chem. 2010, 18, 7193-7202.
28. Ollinger, J.; O'Malley, T.; Kesicki, E. A.; Odingo, J.; Parish, T.
J. Bacteriol. 2012, 194, 663-668.
29. Hinzen, B.; Raddatz, S.; Paulsen, H.; Lampe, T.; Schumacher,
A.; Häbich, D.; Hellwig, V.; Benet-Buchholz, J.; Endermann, R.;
Labischinski, H.; Brötz-Oesterhelt, H. ChemMedChem 2006, 1, 689-
693.
30. Li, D. H. S.; Chung, Y. S.; Gloyd, M.; Joseph, E.; Ghirlando,
R.; Wright, G. D.; Cheng, Y.; Maurizi, M. R.; Guarne, A.; Ortega, J.
Chem. Biol. 2010, 17, 959-969.
31. Lee, B.; Park, E. Y.; Lee, K.; Jeon, H.; Sung, K. H.; Paulsen,
H.; Rubsamen-Schaeff, H.; Brotz-Oesterhelt, H.; Song, H. K. Nat.
Struct. Mol. Biol. 2010, 17, 471-478.
32. Kirstein, J.; Hoffmann, A.; Lilie, H.; Schmidt, R.; Rübsamen-
Waigmann, H.; Brötz-Oesterhelt, H.; Mogk, A.; Turgay, K. EMBO
Molecular Medicine 2009, 1, 37-49.
6830-6835.
R. Biopolymers 2008, 89, 653-667.
b)
Udugamasooriya,
D.
G.;
Spaller,
M.
43. a) Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lok-
ey, R. S.; Jacobson, M. P. J. Am. Chem. Soc. 2006, 128, 14073-14080.
b) Rezai, T.; Yu, B.; Millhauser, G. L.; Jacobson, M. P.; Lokey, R.
S. J. Am. Chem. Soc. 2006, 128, 2510-2511. c) Veber, D. F.; Johnson,
S. R.; Cheng, H.; Smith, B. R.; Ward, K. W.; Kopple, K. D. J. Med.
Chem. 2002, 45, 2615-2623. d) Bock, J. E.; Gavenonis, J.; Kritzer, J.
A. ACS Chem. Biol. 2012, 8, 488-499.
44. a) Marsault, E.; Peterson, M. L. J. Med. Chem. 2011, 54, 1961-
2004. b) Giordanetto, F.; Kihlberg, J. J. Med. Chem. 2013. in press
45. Barreiro, E. J.; Kümmerle, A. E.; Fraga, C. A. Chem. Rev.
2011, 111, 5215-5246.
46. Leung, C. S.; Leung, S. S.; Tirado-Rives, J.; Jorgensen, W. L.
J. Med. Chem. 2012, 55, 4489-4500.
47. a) Kim, Y.; Burton, R. E.; Burton, B. M.; Sauer, R. T.; Baker,
T. A. Mol. Cell 2000, 5, 639-648. b) Martin, A.; Baker, T. A.; Sauer,
R. T. Nature 2005, 437, 1115-1120.
48. Lee, M. E.; Baker, T. A.; Sauer, R. T. J. Mol. Biol. 2010, 399,
707-718.
49. Burton, R. E.; Siddiqui, S. M.; Kim, Y.; Baker, T. A.; Sauer, R.
T. EMBO J. 2001, 20, 3092-3100.
50. Willer, M.; Hindler, J.; Cockerill, F. CLSI 2009, 29, 12-15.
33. Sass, P.; Josten, M.; Famulla, K.; Schiffer, G.; Sahl, H.;
Hamoen, L.; Brötz-Oesterhelt, H. Proc. Natl. Acad. Sci. U.S.A. 2011,
108, 17474-17479.
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment