Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases

Article abstract of DOI:10.1021/jacs.7b06935

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human

Full text of DOI:10.1021/jacs.7b06935

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES
Article
Discovery of a potent inhibitor class with high
selectivity towards clostridial collagenases
Esther Schönauer, Andreas Martin Kany, Jörg Haupenthal, Kristina Hüsecken, Isabel Josephin Hoppe,
Katrin Voos, Samir Yahiaoui, Brigitta Elsässer, Christian Ducho, Hans Brandstetter, and Rolf W. Hartmann
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06935 • Publication Date (Web): 18 Aug 2017
Downloaded from http://pubs.acs.org on August 18, 2017
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 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Discovery of a potent inhibitor class with high selectivity to-
wards clostridial collagenases
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
Esther Schönauer†⊥, Andreas M. Kany‡⊥, Jörg Haupenthal‡, Kristina Hüsecken‡, Isabel J.
Hoppe†, Katrin Voos§, Samir Yahiaoui‡, Brigitta Elsässer†, Christian Ducho§, Hans
Brandstetter†*, Rolf W. Hartmann‡§*
† Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Billrothstr. 11, 5020 Salz-
burg, Austria
‡ Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Design and Optimization,
Campus E8.1, 66123, Saarbrücken, Germany
§ Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123
Saarbrücken, Germany
ABSTRACT: Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial
infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve
selectivity against human metalloproteases such as MMPs. Using an SPR-based screening complemented with enzyme
inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed submicromolar affini-
ties towards collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also
efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani and ColQ1 from
the Bacillus cereus strain Q1, while showing negligible activity towards human MMPs-1, -2, -3, -7, -8 and -14. The most
active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by
the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The
here presented non-primed binding mode paves the way for the development of selective broad-spectrum bacterial colla-
genase inhibitors with potential therapeutic application in humans.
The physiological substrate of clostridial collagenases is
Introduction
collagen, the main component of the extracellular matrix
10,11
Clostridia represent a family of ubiquitously occurring
in mammals (up to 90%)
. Its defining characteristic is
gram-positive bacteria comprising perilous pathogens
causing diseases such as botulism (Clostridium botuli-
num), gas gangrene (C. perfringens), tetanus (C. tetani) or
pseudomembranous colitis (C. difficile) 1,2. These toxigenic
clostridia still represent a threat to public health, as teta-
nus and clostridial myonecrosis have maintained high
mortality rates and pseudomembranous colitis is a known
severe complication of antibiotic therapy 3–6. Furthermore,
substantial amounts of pathogenic clostridia were cul-
the collagen triple-helix, which is perpetuated by the tri-
plet repeat Gly-X-Y (X and Y positions are mostly occupied
by proline (28%) and hydroxyproline (38%))12. The natively
13,14
folded triple helix is highly resistant to proteolysis
.
Even the most prominent human collagenases, the matrix
metalloproteinases MMP-1, -2, -8, -13, -14, -18 can cleave
the triple helix only at a single site 15,16. In contrast to that,
clostridial collagenases can process collagen triple helices at
multiple sites, as the active site displays a remarkable selec-
7
tured in the last 60 years for the use as bio-weapons .
17
tivity for the Gly-Pro-Y triplets , and they can decompose
Consequently, massive efforts have been made aiming to
unravel the molecular basis of these life-threatening in-
fections. Nevertheless, such infections remain a major
challenge as this knowledge did not yet lead to satisfacto-
ry treatment options.
collagen completely into small peptides 18,19
.
The inhibition of these extracellular collagenases is con-
ceptually attractive, as it does not attack the pathogen
directly but rather blocks the colonization and infiltration
of the host by the clostridia. Thereby reducing the Dar-
winian selection pressure, targeting bacterial virulence is
considered a promising approach to combat the emerging
threat of drug resistant bacteria 20–22. To date, several anti-
virulence targets have been validated, demonstrating the
potential of this approach 23–28. Kassegne et al. showed for
example that a collagenase knock out strain from Lepto-
spira interrogans displayed reduced virulence in an in vivo
The high lethality of these bacteria is related to colla-
genases which are crucial for clostridial virulence, given
their critical role in colonization and evasion of host im-
mune defence, acquisition of nutrients, facilitation of
dissemination, or tissue damage during infection. Addi-
tionally, they might potentiate clostridial histotoxicity by
facilitating toxin diffusion 2,8,9
.
model 28
ACS Paragon Plus Environment
.

Journal of the American Chemical Society
Page 2 of 11
Targeting extracellular enzymes provides a substantial
In this study, we wanted to capitalize on the recent crys-
tal structures of the peptidase domains of three clostridial
1
2
3
4
5
6
7
8
benefit because inhibitors do not need to cross the bacte-
rial cell wall, which has turned out to be challenging in
many cases 29–31. Consequently, bacterial collagenases
represent prime targets for an effective therapy against
34,41
collagenases
with the aim to rationally develop small
organic molecules targeting collagenase ColH from C.
histolyticum. In the following we describe the discovery of
inhibitors which are highly active and selective for clos-
tridial collagenases over MMPs and have the potential to
be further optimized for a future therapeutic application
in humans. Their selectivity can be rationalized on basis
of a co-crystal structure of the peptidase domain of ColH
in complex with an inhibitor, revealing a distinct non-
primed binding mode of the inhibitor to the active site.
clostridial and bacillary infections 6,9,32,33
.
Clostridial collagenases are zinc metalloproteinases of
~115 kDa with a multi-domain organization, homologues
of which are also found in many bacilli. The mature pro-
tein harbors an N-terminal collagenase unit of ~78 kDa,
which is the minimal collagenolytic entity, followed by a
varying composition of two to three accessory domains,
which are thought to be involved in collagen swelling and
binding to fibrillar collagen 34–38. The collagenase unit is
composed of the activator domain and the peptidase
domain 34. The peptidase domain harbors the catalytic
zinc ion, which is coordinated by the two histidines of the
canonical zinc-binding HEXXH motif, and a downstream
glutamate 4,34,35,39–41. The glutamate residue in the HEXXH
motif acts as the general acid/base, which polarizes the
catalytic water essential for catalysis. This polarized water
molecule performs the nucleophilic attack, while the zinc
ion serves as an oxyanion hole to the carbonyl oxygen of
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
Results and Discussion
Discovery of new inhibitory scaffold. To discover
new low-molecular-weight inhibitory compounds, a fo-
cused protease inhibitor library was screened with a sur-
face plasmon resonance (SPR)-based binding assay using
the amine-coupled peptidase domain of ColH (ColH-PD)
as ligand. To ensure the integrity of ColH-PD after immo-
bilization, the collagenase-specific peptidic substrate N-
(3-[2-furyl]acryloyl)-L-leucyl-glycyl-L-prolyl-L-alanine
62
(FALGPA) was used as positive control (Figure S1a). A
the scissile peptide bond 42
.
total of 1,520 structurally diverse small molecules with an
average MW of 389 ± 78 Da was screened at 100 μM.
Compounds showing a molecular weight-normalized
response higher than that of 500 µM FALGPA (i.e. 35
µRIU) were classified as hits. The SPR screen resulted in
Several groups have been working on the development
of clostridial collagenase inhibitors in the past, focusing
on the collagenases G (ColG) and H (ColH) from C. histo-
lyticum. In this context, besides the identification of ac-
202 primary hits. 19 compounds were excluded from the
tive compounds from Viola yedoensis 43, inhibitors based
63
subsequent testing as known promiscuous inhibitors
resulting in a hit rate of 12.0% (Figure S2).
,
44
on sulfonylated derivatives of
L-valine hydroxamate
have been synthesized as well as sulfonyl aminoacyl hy-
droxamates 45. Furthermore, compounds incorporating 5-
amino-2-mercapto-1,3,4-thiadiazole zinc binding func-
tions 46, arylsulfonyl-ureido and 5-dibenzo-suberenyl/
The secondary functional screening assessed the poten-
tial of the 183 SPR hits to inhibit the peptidolytic activity
of ColH-PD using a custom-made FRET substrate. Typi-
62
64
suberyl 47 or succinyl hydroxamate and iminodiacetic acid
cally, the FALGPA
and Wünsch
assays are used to
48
characterize the activity of clostridial collagenases due to
their easy setup and commercial availability next to their
specificity for these enzymes 62,65,66. However, the low
binding affinity of these peptides together with their low
signal-to-noise ratios severely limits the sensitivity of
these assays. Consequently, substantial amounts of en-
zyme and substrate are needed in characterization studies
(e.g. KM-values for FALPGA are in the mM range 62,67). To
facilitate our screening process, we designed and synthe-
sized a decapeptide (Mca-Ala-Gly-Pro-Pro-Gly-Pro-Dpa-
Gly-Arg-NH2) to be used as a substrate for a FRET-based
assay. Its sequence was based on the detailed profile of
the primed and non-primed cleavage site specificity of
hydroxamate moieties
have been described. These in-
hibitors follow the classic architecture of metalloprotease
inhibitors with a backbone that mimics the natural sub-
strate, which is connected via a linker to a zinc-binding
group that chelates the catalytic zinc ion and, thereby,
expels the essential catalytic water molecule from the
active site 49,50. These inhibitors were developed as sub-
strate analogues and/or designed based on inhibitors for
51
other metalloproteases that share the HEXXH motif
,
like thermolysin or MMPs 44,47,52–58. Unfortunately, the
synthetic clostridial collagenase inhibitors are not selec-
tive, inhibiting clostridial collagenases and MMPs alike
44,45,47,48,55–57,59–61. Therefore, they are not suitable for an
antibacterial therapy in humans. Consequently, novel and
more effective drug candidates are urgently needed.
clostridial collagenases as determined by Proteomic Iden-
17,68
tification of protease Cleavage Sites (PICS) recently
.
The assay sensitivity was increased by several orders of
magnitude compared to the FALGPA assay by the applica-
tion of the FRET technology 69,70. The KM value of this
substrate is 62 ± 8 µM for ColH-PD. The 183 SPR binders
were screened at a final concentration of 40 µM. The
inhibitor isoamylphosphonyl-Gly-Pro-Ala (Figure S1b)
was used as positive control in the assay 58. In sum, the
SPR-based and activity-based screenings led to six func-
tional hits (>25% inhibition) with molecular weights rang-
Efforts to design selective inhibitors were hampered by
the lack of high-resolution structural data on clostridial
collagenases until 2011. The first crystal structures re-
vealed that although there is no significant sequence
homology between the peptidolytic domains of clostridial
collagenases and MMPs, their active sites share a similar
catalytic zinc ion-binding geometry and the canonical
non-prime site substrate-recognition motif, the edge
strand 17.
ACS Paragon Plus Environment

Page 3 of 11
Journal of the American Chemical Society
25 ± 6 µM). Our further hits showed considerably weaker
ing from ~ 210 to ~ 385 Da (Figure S3). The two most ac-
1
2
3
4
5
6
7
8
tive inhibitors in this assay were mercaptoacetamides 1
and 2 (Table 1). Compound 2 led to a similar inhibition of
ColH-PD in vitro as isoamylphosphonyl-Gly-Pro-Ala both
at 40 µM, i.e. 82 ± 3 % and 81 ± 1%, respectively.
inhibition and, in one case, proved to be incompatible
with the FRET-assay at high concentration. The high
potency of the N-aryl mercaptoacetamides combined with
their relatively low molecular weight encouraged us to
investigate this promising compound class further in
order to improve the inhibitory activity.
Dose-response studies revealed an IC50 value of 1.9 ± 0.3
µM for this compound, while the non-substituted aniline
derivative 1 showed lower activity towards ColH-PD (IC50
9
Table 1. IC50 values of mercaptoacetamide compounds for ColH-PD.
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
Compound
1
R
IC50 (µM)
25 ± 6
Compound
7
R
IC50 (µM)
0.19 ± 0.02
2
3
1.9 ± 0.3
8
9
0.19 ± 0.03
0.010 ± 0.002
19 ± 3
4
0.044 ± 0.007
10
23 ± 3
5
0.071 ± 0.009
0.12 ± 0.01
11
26 ± 4
31 ± 7
6
12
Characterization of mercaptoacetamide hits. 36 de-
rivatives of this compound class were purchased (Table 1,
3 and Table S1). Six derivatives showed improved inhibi-
tion compared to 2 (3-8). Generally, the introduction of
functional groups in para-position to the aniline turned
out to be favorable, considering the striking loss of activi-
ty of ortho-methoxy-substituted compound 11 compared
to its para-analogue 5 (ortho-effect), and ortho-chloro-
substituted compound 12 compared to its para-analog 7.
The superior performance of para-derivatives was also
evident regarding the 100-fold decrease in IC50 of 7 com-
pared to its meta-chloro-substituted counterpart 9. In
comparison to the unsubstituted compound 1, meta-
substituted compounds 9 and 10 showed no significant
improvement in IC50. Removal of the 3-methyl-group of
compound 2 even led to a 16-fold decreased IC50 (com-
pound 6), suggesting that the meta-substitution is not
beneficial for ColH inhibition.
Regarding electronic properties of our hits it becomes
apparent that the best compounds 3-5, displaying IC50
values in the two-digit nanomolar range, bear oxygen-
containing groups with hydrogen bond accepting proper-
ties.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 11
Selectivity against MMPs and broad-spectrum inhi- Crystal structure of the peptidase domain of ColH
1
2
3
4
5
6
7
8
bition of other bacterial collagenases. To determine
the selectivity of our compounds towards clostridial and
bacillial collagenases on the one hand, and MMPs on the
other, selected compounds (3 and 7, Figure 1) were tested
using in vitro inhibition assays with ColH-PD and the
peptidase domains of ColT (ColT-PD), the collagenase
units of ColG (ColG-CU) and of ColQ1 (ColQ1-CU) as well
as the catalytic domains of MMP-1, -2, -3, -7, -8 and –14.
The hydroxamate-based peptidomimetic batimastat (Fig-
ure S1c) is a highly potent and unselective inhibitor of
in complex with compound 3. To rationalize the bind-
ing mode of the N-aryl mercaptoacetamide-based inhibi-
tors, we aimed to solve the crystal structure of ColH-PD
in complex with compound 3. The structure was deter-
mined at 1.87 Å resolution with all residues being defined
in the electron density at excellent geometric and crystal-
lographic parameters (Table S3). The overall topology of
the peptidase domain showed the expected thermolysin-
like fold. The average root-mean-square displacement
(RMSD) of backbone atoms between the structure of the
apo-peptidase domain and the peptidase domain in com-
plex with isoamylphosphonyl-Gly-Pro-Ala were 0.133 Å
and 0.123 Å, respectively. The peptidase domain of ColH is
divided horizontally by the active-site cleft into an upper
N-terminal and a lower C-terminal subdomain. Substrates
can bind to the active-site cleft from the left (non-primed
side) to the right (primed side) when viewed in standard
orientation 76. Central elements of the N-terminal subdo-
main (NSD) are the active-site helix, and a mixed five-
stranded ß-sheet. The zinc-binding motif HEXXH, which
provides the two zinc-coordinating histidines and the
general acid/base glutamate, is located in the active-site
helix (Figure 2). The lowermost ß-strand of the mixed ß-
sheet shapes the upper perimeter of the active-site cleft,
the edge strand. The edge strand interacts in an antiparal-
lel manner with the substrate predominantly on the non-
primed side 34,77,78. The third zinc ligand is a glutamate
residue, located on the glutamate helix of the C-terminal
subdomain (CSD). The insertion of 30 residues between
the HEXXH motif and this glutamate residue shapes (i)
the non-primed side of the active-site cleft, and (ii) a
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
71
MMPs and was used as a positive control. MMPs are
highly similar to each other in their active-site topology,
which has made the development of selective active-site
directed MMP inhibitors a challenging task 72,73. The S1’
binding site is the major specificity determinant in MMPs.
Based on the S1’ site, the MMPs are typically divided into
deep, intermediate and shallow S1’ binding pocket groups
(e.g. deep: MMP-3, -12, -14; intermediate: MMP-2, -8 and -
9; shallow: MMP-1, and -7) 74. Therefore, we chose a panel
of MMPs to investigate the binding of our compounds to
71
all three S1’ pocket types. In line with published results ,
batimastat displayed IC50 values below 10 nM for all of
these MMPs (Table S2). As expected from this broad-
spectrum zinc metalloproteinase inhibitor, batimastat
also inhibited ColH-PD, ColT-PD, ColG-CU and ColQ1-
CU (Figure S4). Intriguingly, compounds 3 and 7 resulted
in no or negligible inhibition of the tested MMPs (Figure 1
and Figure S5). Only in case of MMP-2, we observed 25%
inhibition at 100 µM compound 3, while ColH-PD was
efficiently inhibited, showing less than 10% residual activ-
ity. Thus, we observed a more than 1,000-fold selectivity
of these two compounds for ColH over MMPs. Strikingly,
the clostridial collagenase homologues ColG and ColT,
and the bacillial collagenase ColQ1 were even more effi-
ciently inhibited, showing 5% or less residual activity
when treated with 100 µM of compound 3 or 7. A similar
compound scaffold had been reported by Zhu et al. to
inhibit LasB, an extracellular elastase from Pseudomonas
aeruginosa 75. In sum, these findings showed that the N-
aryl mercaptoacetamide-based inhibitors are not only
selective against MMPs, but are also potent broad-
spectrum inhibitors of bacterial collagenases.
calcium-binding site crucial for enzymatic activity 34,41,77
.
Figure 2. Peptidase domain of ColH in complex with the
hydrolysis product of compound 3. Close-up view of the
active site in ball-and-stick representation. The inhibitor
(blue) is shown in sticks with the maximum likelihood
weighted 2Fo − Fc electron density map contoured at 1 σ. The
catalytic zinc ion (dark grey), the calcium ion (green) and
water molecule (red) are shown as spheres. The S1’ site
formed by Gly425 and Gly426 in the edge strand (shown in
dark grey sticks) is indicated.
Figure 1. Inhibition of selected MMPs and bacterial
collagenases by N-aryl mercaptoacetamide compounds 3 and
7.
A well-defined electron density was observed for the
ligand bound in the active site. The structure of com-
ACS Paragon Plus Environment

Page 5 of 11
Journal of the American Chemical Society
Confirmation of thiol as active compound. To fur-
pound 3 could be clearly modelled into the density (Fig-
1
2
3
4
5
6
7
8
ure 2), except for the carbamoyl unit of the thiocarbamate
moiety. Instead, the electron density showed the sulfur
atom coordinating the catalytic zinc ion, suggesting that
the thioester group had been hydrolyzed in the co-
crystallization process. This result prompted an investiga-
tion of our newly discovered class of inhibitors with par-
ticular emphasis on the stability of the thiocarbamate
function in aqueous buffers such as the buffer system of
the functional assay and the crystallization buffer.
ther substantiate these findings, we followed two different
strategies. First, we studied the inhibitory activities of the
free thiols 13-15. Thus, we determined the IC50 values of
the free thiols 13-15 with ColH-PD in presence of the re-
ducing agent TCEP. The resulting IC50 values of 0.017, 0.21
and 40 µM corresponded well with 0.010, 0.19 and 31 µM
of the thiocarbamate analogues (Table 1 and Table 2).
9
Table 2. Inhibition of ColH-PD by thiol compounds in the
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
Stability of the thiocarbamate unit. Two inhibitors
with major differences in potency (7, 12) were selected
and the hydrolytic formation of the corresponding free
thiol was analyzed by liquid chromatography–mass spec-
trometry (LC-MS). Free thiols were synthesized as refer-
ences for the stability assay. The conversions of com-
pounds 7 and 12 into compounds 14 and 15, respectively,
proceeded rapidly in 10 mM HEPES, pH 7.5 at 22.5°C, with
thiocarbamate half-lives of 26.8 ± 1.4 min (7) and 20.6 ±
0.9 min (12, Figure 3). These results corroborated that the
inhibition of thiocarbamates 1-12 was predominantly due
to the respective free thiols. Considering the preparation
time and the pre-incubation time of one hour for each
compound with ColH-PD before the functional assay was
started by addition of the substrate the thiocarbamates
were quantitatively converted within the time frame of
the experiment. Thiol formation was also demonstrated at
pH 6.4, corresponding to the buffer used for co-
crystallization (Figure S6).
presence of 5 mM TCEP.
Compound
13
R
IC50 (µM)
0.017 ±
0.002
14
15
0.21 ± 0.01
40 ± 9
The results of the MMP and bacterial collagenase inhi-
bition assays could also be reproduced using the thiol
compounds, with 13 and 14 demonstrating a similarly high
selectivity against MMPs and a broad-spectrum inhibition
of bacterial collagenases (Figure 4).
Figure 3. Conversion of thiocarbamates 7 and 12 into the
respective corresponding free thiols. (a) Compound 7 into 14,
(b) Compound 12 into 15. Time course of hydrolysis in 10 mM
Hepes pH 7.4 (10% methanol) at 22.5°C was monitored by
LC-MS, showing conversion into corresponding thiol and to
minor extent into another compound which is most likely
Figure 4. Inhibition of selected MMPs and bacterial
collagenases by thiol compounds 13 and 14.
As a second strategy, we aimed to synthesize a structur-
al analogue of compound 1 lacking the hydrolytically
instable thioester motif. The formal replacement of the
sulfur atom with a methylene group led to the carbox-
amide analogue 16, which was prepared and tested for its
the disulfide oxidation product 79
.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 11
inhibitory activity towards ColH-PD (Figure 5). Com- frame of our assay, explaining the inactivity of these de-
1
2
3
4
5
6
7
8
pound 16 was devoid of any activity even at 1000 µM. This
demonstrated that the carbamoyl moiety in 1 does not
contribute to target binding, but is just part of a prodrug-
like structure which furnishes the corresponding bioac-
tive thiol by chemical hydrolysis.
rivatives by stability towards hydrolysis (Figure S7).
In addition, the thermodynamic profile of the interac-
tion between compounds 7 and 14 and ColH-PD was de-
termined. As expected, isothermal titration calorimetry
(ITC) measurements resulted in very similar affinities and
free energy values, resulting from the hydrolysis of 7 to
furnish thiol 14 (Table 4). Compound binding to ColH-PD
turned out to be enthalpy-driven. In sum, the findings
from the stability assay, the in vitro assay and the ITC
data confirmed the thiols as active compounds in our
enzyme inhibition assay.
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
Table 4. ITC and IC50 results of the thiocarbamate-thiol pair
7 and 14.
Figure 5. Structure and inhibitory activity of the non-
hydrolyzable carboxamide analogue 16.
Compound
IC50 (µM)a
KD (µM)b
7
14
0.19 ± 0.02
0.309 ± 0.045
-8.9 ± 0.1
-12.7 ± 1.2
3.8 ± 1.3
0.21 ± 0.01
0.360 ± 0.038
-8.8 ± 0.1
Table 3. Structure and activity of dithiocarbamates.
ΔG (kcal mol-1)b
ΔH (kcal mol-1)b
-TΔS (kcal mol-1)b
Nb,c
S
H
-15.4 ± 0.3
6.6 ± 0.4
N
R
S
N
O
0.54 ± 0.05
0.48 ± 0.03
a IC50 refers to the functional FRET-assay.
Inhibition
at 100 µM
(%)
Compound
R
b Results are from at least two independent measurements.
c
The low stoichiometry could be explained by incomplete
zinc occupation of the active sites 41
.
no
inhibition
17
Cytotoxicity test. Regarding the potential therapeutic
use of our compounds in humans we investigated the
cytotoxic properties of selected N-aryl mercaptoacetam-
ides. Cytotoxicity tests using HEP G2 cells showed com-
pounds 13 and 14 to display low cytotoxicity, comparable
to the marketed antibiotic rifampicin (Table 5), while
doxorubicin as control showed the expected cytotoxic
effect. These findings underline the potential of our com-
pounds for the development of novel anti-infectives.
no
inhibition
18
19
20
21
Table 5. Cytotoxicity of 13, 14 and three reference com-
pounds in HEP G2 cells.
no
inhibition
Concentration
(µM)
Reduction of
viability (%)
Compound
13
100
100
100
1
17 ± 12
28 ± 12
29 ± 5
50 ± 5
13 ± 7
no
inhibition
14
Rifampicin
Doxorubicin
Batimastat
100
12 ± 3
Zinc coordination by a thiolate. The identification of
the thiol as active compound in our functional assays was
in excellent agreement with the crystal structure analysis
which demonstrated that only a sulfur atom, to be precise
a thiolate, was coordinating the catalytic zinc ion. To
validate our conclusions on the protonation state of the
sulfur atom, we calculated the pKa values for the thiol
Further in line with our findings, dithiocarbamates 17-21
(Table 3) were inactive towards ColH-PD. LC-MS experi-
ments with the dithiocarbamate analogue of our best hit 3
showed no formation of free thiol 13 within the time
ACS Paragon Plus Environment

Page 7 of 11
Journal of the American Chemical Society
group resulting from the hydrolysis of thiocarbamate 3 in
solution and when bound to the active site using the
their non-primed substrate pockets, and that (ii) the resi-
1
2
3
4
5
6
7
8
dues on the edge strand and the zinc ion are positioned as
such as to allow productive interactions with the thiolate.
Yet, the MMPs, lacking the zinc-binding glutamate, can-
not provide the hydrogen-bonding partner for the amide
nitrogen of the mercaptoacetamide inhibitor. Hence, is
the interaction with the gluzincin-specific Glu487 crucial
for selectivity? To test this hypothesis, we mimicked the
zinc-liganding sphere of MMPs in ColH-PD by mutating
Glu487 into a histidine. A comparison of the apparent
inhibition constant Ki(app) of compound 11 towards wild-
type ColH-PD and the mutant E487H, 92 ± 8 µM and 166
± 23 µM, respectively, showed that the mutation did not
result in a drastic change in the inhibitory potency. This
suggests that the interaction with the edge strand on the
non-primed site, mediated via main-chain contacts, is the
main structural selectivity determinant. This hypothesis is
further supported by a structural analysis of the edge-
strand conformations in MMPs and clostridial collagenas-
es in the ligand-bound state. Within each family, the
ligand-bound edge-strand conformation is highly con-
served (Figure 6). Compared to the clostridial situation,
the edge strand in MMPs is tilted by 27-29°. This tilted
orientation could explain the inefficient binding of the N-
aryl mercaptoacetamide compounds to the MMPs, sug-
gesting that the interactions with the non-primed edge
strand (S1 – S3) are the crucial selectivity determinants.
80
Molecular Operating Environment (MOE) software
.
The pKa of the thiol group was strongly lowered from 9.0
in the solvent to 3.1 by the direct coordination to the zinc
ion. This suggests that the thiol is fully ionized to the
thiolate form in both the activity assay and the crystalliza-
tion experiment upon binding to the active site.
Binding mode of the N-aryl mercaptoacetamide
compound to the active site of ColH-PD. The hydroly-
sis product of compound 3 binds to the S3 to S1 substrate
binding pockets (Figure 2). The thiolate coordinates the
zinc ion with a sulfur-to-zinc distance of 2.27 Å. In the S1
pocket, the amide oxygen of compound 3 forms a hydro-
gen bond with the main-chain amide nitrogen of Tyr428
(3.11 Å) of the NSD, while the amide nitrogen of com-
pound 3 hydrogen-bonds with the carbonyl oxygen (OE2)
of Glu487 (2.97 Å) of the CSD. In addition, the benzene
ring of the ligand is involved in a π-π-stacking interaction
with the imidazole ring of His459 (centroid-centroid
distance of 3.80 Å). The oxygen of the acetyl group of 3
interacts via a bridging water molecule (3.07 Å) with the
main-chain oxygen of Tyr428 in S1 (3.11 Å) and with the
main-chain nitrogen of Glu430 in S3 (2.84 Å). Thus, the
inhibitor is well-braced in-between the NSD and the CSD
of the peptidase domain.
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
Importantly, the binding mode of the inhibitor is not
directed towards the primed substrate-binding sites, but
towards the non-primed recognition sites in-between the
calcium-binding site and the catalytic zinc ion. Thus, this
complex of ColH-PD with the thiol derived from com-
pound 3 is the first to describe non-primed interactions
between a clostridial collagenase and an active site-
directed ligand.
It was also interesting to see that related mercaptoa-
cetamide derivatives were shown to inhibit LasB from P.
aeruginosa 75. Analysis of our best compound 13 in an in
vitro LasB inhibition assay revealed a more than 1000-fold
lower activity compared to ColH (data not shown). This is
likely due to the distinct binding mode of compound 13 to
the non-primed binding site of ColH (Figure 2) in con-
trast to the proposed primed binding mode of the related
Identification of selectivity determinant. The thiol
derived from compound 3 interacts with two central ele-
ments of the active site: (i) the zinc ion and its liganding
sphere (His455, Glu456, His459, and Glu487), and (ii) the
edge strand (Gly425-Glu430). These two central elements
are also present in MMPs 17: A structurally, but not se-
quentially, homologous edge strand frames the upper rim
of the active site in MMPs, and the zinc-liganding sphere
composed of the HEXXH motif and a third proteinaceous
ligand is nearly identical between the MMPs and the
clostridial collagenases. The geometry of the zinc-
liganding sphere is almost perfectly superimposable in
clostridial collagenases and MMPs (RMSD= 0.060 Å be-
tween ColH and MMP-1). Only the third zinc-binding
residue differs. While in MMPs, this position is occupied
by a histidine; in clostridial collagenases, this ligand is a
glutamate provided by the gluzincin-specific glutamate
helix. Given this high similarity in the active site between
clostridial collagenases and MMPs, this triggered the
question of how we can rationalize the observed differ-
ences in selectivity of the N-aryl mercaptoacetamide
compounds towards the two enzyme families. A first in-
silico structural analysis of the active sites of MMP-1, -2, -
3, -8, -12 and -13 suggested that (i) these enzymes could
accommodate the mercaptoacetamide compounds in
compounds in LasB by Zhu et al. 75
.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
Figure 6. Close-up on the superpositioned active sites of
Corresponding Author
1
2
3
4
5
6
three MMPs (a) and of three clostridial collagenases (b) in
the ligand bound state. The ligands have been removed for
better visualization. The HEXXH motif is shown in sticks, the
zinc ions (grey) and calcium ions (green) are shown as
spheres. The edge strand on top of the catalytic zinc is
highlighted in color.
Hans Brandstetter (hans.brandstetter@sbg.ac.at)
Rolf W. Hartmann (Rolf.Hartmann@helmholtz-hzi.de)
Author Contributions
The manuscript was written through contributions of all authors. / All
authors have given approval to the final version of the manuscript. /
⊥ These authors contributed equally.
7
8
9
With regard to future inhibitor design, these findings
suggest that by (i) amplification of the interactions with
the edge strand and (ii) extension of the inhibitor scaffold
in para, i.e. by developing the compound further into the
non-primed substrate recognition pockets, even more
potent and selective compounds could be developed. In
compounds with optimized affinity to the edge strand
and the non-primed substrate binding pockets, we plan to
investigate the replacement of the thiol moiety by a less
reactive ZBG. Such lead compounds hold the promise of
higher efficacy and therefore higher safety in potential
therapeutic applications in humans.
Funding Sources
The authors declare no competing financial interest.
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
ACKNOWLEDGMENT
This work was supported by grants from the Austrian Science
Fund (project W_01213). We thank Dr. Elfriede Dall for X-ray
data collection, Dr. Werner Tegge for peptide synthesis, Dr.
Christine Maurer and Jeannine Jung for technical support.
ABBREVIATIONS
ColH, collagenase H; ColG, collagenase G; CSD, C-terminal
subdomain; CU, collagenase unit; MMP, human metallopro-
teinase; FS1-1, Mca-Ala-Gly-Pro-Pro-Gly-Pro-Dpa-Gly-Arg-
NH2; LC-MS, liquid chromatography–mass spectrometry;
NSD, N-terminal subdomain; PD, peptidase domain; RIU,
refractive index units; RMSD, root-mean-square displace-
ment; SPR, surface plasmon resonance.
Conclusion
We identified a novel compound scaffold for the selec-
tive inhibition of clostridial collagenases. Starting with an
SPR-based primary screening of a focused library, we
validated the SPR-hits in a secondary enzyme inhibition
assay using a custom-tailored FRET peptide substrate for
clostridial collagenases. Two mercaptoacetamide deriva-
tives were the most potent functional hits in this assay.
Further derivatization of these initials hits, in particular
the introduction of oxygen-containing groups in para-
position to the aniline, led to the generation of highly
potent N-aryl mercaptoacetamide-based clostridial colla-
genase inhibitors with IC50 values in the two-digit nano-
molar range. These compounds showed unprecedented
selectivity against MMPs, while at the same time they
displayed a broad-spectrum inhibition of bacterial colla-
genases. The selectivity of these compounds could be
rationalized on basis of a co-crystal structure of ColH-PD
with the most active compound, revealing a distinct non-
primed binding mode of the inhibitor to the active site.
The mercaptoacetamides were also shown to display no
cytotoxicity towards human cells. These insights pave the
way for the development of selective broad-spectrum
bacterial collagenase inhibitors with potential therapeutic
application in humans.
REFERENCES
(1)
Cato, E.; George, W.; Finegold, S. In Bergey’s
Manual of Systematic Bacteriology; Williams &
Wilkins: Baltimore 1986; pp 1141–1200.
(2)
(3)
(4)
Hatheway, C. L. Clin. Microbiol. Rev. 1990, 3,
66–98.
Burke, M. P.; Opeskin, K. Am. J. Forensic Med.
Pathol. 1999, 20, 158–162.
Bruggemann, H.; Baumer, S.; Fricke, W. F.;
Wiezer, A.; Liesegang, H.; Decker, I.; Herzberg,
C.; Martinez-Arias, R.; Merkl, R.; Henne, A.;
Gottschalk, G. Proc. Natl. Acad. Sci. U. S. A.
2003, 100, 1316–1321.
(5)
(6)
(7)
Taubes, G. Science 2008, 321, 360.
Taubes, G. Science 2008, 321, 356–361.
Arnon, S. S.; Schechter, R.; Inglesby, T. V;
Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.;
Eitzen, E.; Fine, A. D.; Hauer, J.; Layton, M.;
Lillibridge, S.; Osterholm, M. T.; O’Toole, T.;
Parker, G.; Perl, T. M.; Russell, P. K.; Swerdlow,
D. L.; Tonat, K.; Working Group on Civilian
Biodefense. JAMA 2001, 285, 1059–1070.
Experimental Section
ASSOCIATED CONTENT
Supporting Figures and Tables (Molecular structures of posi-
tive controls; SPR screening hits; functional screening hits;
inhibition of the selected MMPs and bacterial collagenases
by batimastat; MMP inhibition assay; LC-MS analysis of
compound 3 and of dithiocarbamate 21; structure and activity
of additional thiocarbamates and related compounds; data
collection and refinement statistics); Experimental Section.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(8)
(9)
(10)
(11)
Matsushita, O.; Okabe, A. Toxicon 2001, 39,
1769–1780.
Popoff, M. R.; Bouvet, P. Future Microbiol.
2009, 4, 1021–1064.
Burgeson, R. E.; Nimni, M. E. Clin. Orthop.
Relat. Res. 1992, 282, 250–272.
Brozek, J.; Grande, F.; Anderson, J. T.; Keys, A.
Ann. N. Y. Acad. Sci. 1963, 110, 113–140.
AUTHOR INFORMATION
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
(12)
Ramshaw, J. A.; Shah, N. K.; Brodsky, B. J.
Struct. Biol. 1998, 122, 86–91.
(33)
Supuran, C. T.; Scozzafava, A.; Mastrolorenzo,
A. Expert Opin. Ther. Pat. 2001, 11, 221–259.
1
2
3
4
5
6
7
8
(13)
Bruckner, P.; Prockop, D. J. Anal. Biochem. 1981,
110, 360–368.
(34) Eckhard, U.; Schönauer, E.; Nüss, D.;
Brandstetter, H. Nat. Struct. Mol. Biol. 2011, 18,
1109–1114.
(14)
(15)
Bächinger, H. P.; Bruckner, P.; Timpl, R.;
Prockop, D. J.; Engel, J. Eur. J. Biochem. 1980,
106, 619–632.
(35)
Matsushita, O.; Jung, C. M.; Katayama, S.;
Minami, J.; Takahashi, Y.; Okabe, A. J. Bacteriol.
1999, 181, 923–933.
Nagase, H.; Visse, R.; Murphy, G. Cardiovasc.
Res. 2006, 69, 562–573.
9
(36) Matsushita, O.; Jung, C. M.; Minami, J.;
Katayama, S.; Nishi, N.; Okabe, A. J. Biol. Chem.
1998, 273, 3643–3648.
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
(16)
(17)
Fields, G. B. J. Biol. Chem. 2013, 288, 8785–8793.
Eckhard, U.; Huesgen, P. F.; Brandstetter, H.;
Overall, C. M. J. Proteomics 2014, 100, 102–114.
(37)
Matsushita, O.; Koide, T.; Kobayashi, R.;
Nagata, K.; Okabe, A. J. Biol. Chem. 2001, 276,
8761–8770.
(18)
(19)
Seifter, S.; Harper, E. Academic Press: New
York, 1971; pp 649–697.
(38) Wang, Y.-K.; Zhao, G.-Y.; Li, Y.; Chen, X.-L.;
Xie, B.-B.; Su, H.-N.; Lv, Y.-H.; He, H.-L.; Liu,
H.; Hu, J.; Zhou, B.-C.; Zhang, Y.-Z. J. Biol.
Chem. 2010, 285, 14285–14291.
Mookhtiar, K. A.; Van Wart, H. E. Matrix Suppl.
1992, 1, 116–126.
(20) Rasko, D. A.; Sperandio, V. Nat. Rev. Drug
Discov. 2010, 9, 117–128.
(39) Jung, C. M.; Matsushita, O.; Katayama, S.;
Minami, J.; Sakurai, J.; Okabe, A. J. Bacteriol.
1999, 181, 2816–2822.
(21)
(22)
(23)
Heras, B.; Scanlon, M. J.; Martin, J. L. Br. J. Clin.
Pharmacol. 2015, 79, 208–215.
Clatworthy, A. E.; Pierson, E.; Hung, D. T. Nat.
Chem. Biol. 2007, 3, 541–548.
(40) Bond, M. D.; Van Wart, H. E. Biochemistry
1984, 23, 3085–3091.
Storz, M. P.; Maurer, C. K.; Zimmer, C.;
Wagner, N.; Brengel, C.; Jong, C. De; Lucas, S.;
Müsken, M.; Häussler, S.; Steinbach, A.;
Hartmann, R. W.; Storz, M. P.; Jong, J. C. De. J.
Am. Chem. Soc 2012, 134, 16143–16146.
(41)
Eckhard, U.; Schönauer, E.; Brandstetter, H. J.
Biol. Chem. 2013, 288, 20184–20194.
(42) Matthews, B. W. Acc. Chem. Res. 1988, 21, 333–
340.
(43) Oshima, N.; Narukawa, Y.; Takeda, T.; Kiuchi,
(24) Lu, C.; Maurer, C. K.; Kirsch, B.; Steinbach, A.;
Hartmann, R. W. Angew. Chemie - Int. Ed. 2014,
53, 1109–1112.
F. J. Nat. Med. 2013, 67, 240–245.
(44) Supuran, C. T.; Scozzafava, A. Eur. J. Pharm. Sci.
2000, 10, 67–76.
(25)
Hung, D. T.; Shakhnovich, E. A.; Pierson, E.;
Mekalanos, J. J. Science 2005, 310, 670–674.
(45) Clare, B. W.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2001, 44, 2253–2258.
(26) Wagner, S.; Sommer, R.; Hinsberger, S.; Lu, C.;
Hartmann, R. W.; Empting, M.; Titz, A. J. Med.
Chem. 2016, 59, 5929–5969.
(46) Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2002, 12, 2667–2672.
(47) Ilies, M. A. M.; Banciu, M. D.; Scozzafava, A.;
Ilies, M. A. M.; Caproiu, M. T.; Supuran, C. T.
Bioorg. Med. Chem. 2003, 11, 2227–2239.
(27)
Böttcher, T.; Sieber, S. A. J. Am. Chem. Soc.
2008, 130, 14400–14401.
(28) Kassegne, K.; Hu, W.; Ojcius, D. M.; Sun, D.;
Ge, Y.; Zhao, J.; Yang, X. F.; Li, L.; Yan, J. J.
Infect. Dis. 2014, 209, 1105–1115.
(48) Santos, M. A.; Marques, S.; Gil, M.; Tegoni, M.;
Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2003, 18, 233–242.
(29) Lewis, K. Nat. Rev. Drug Discov. 2013, 12, 371–
(49) Jacobsen, F. E.; Lewis, J. A.; Cohen, S. M.
387.
ChemMedChem 2007, 2, 152–171.
(30) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.;
Pompliano, D. L. Nat. Rev. Drug Discov. 2007,
6, 29–40.
(50) Rouffet, M.; Cohen, S. M. Dalt. Trans. 2011, 40,
3445–3454.
(51)
Hooper, N. M. FEBS Lett. 1994, 354, 1–6.
(31)
Graef, F.; Vukosavljevic, B.; Michel, J.-P.; Wirth,
M.; Ries, O.; De Rossi, C.; Windbergs, M.;
Rosilio, V.; Ducho, C.; Gordon, S.; Lehr, C.-M. J.
Control. Release 2016, 243, 214–224.
(52)
Grobelny, D.; Galardy, R. E. Biochemistry 1985,
24, 6145–6152.
(53)
Vencill, C. F.; Rasnick, D.; Crumley, K. V;
Nishino, N.; Powers, J. C. Biochemistry 1985, 24,
3149–3157.
(32)
Peterkofsky, B. Methods Enzymol. 1982, 82, 453–
471.
ACS Paragon Plus Environment

Journal of the American Chemical Society
(54) Dive, V.; Yiotakis, A.; Nicolaou, A.; Toma, F. 390, 11–18.
Page 10 of 11
1
2
3
4
Eur. J. Biochem. 1990, 191, 685–693.
(68) Schilling, O.; Overall, C. M. Nat. Biotechnol.
2008, 26, 685–694.
(55)
Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. 2000, 8, 637–645.
(69) Fields, G. B. Methods Mol. Biol. 2010, 622, 393–
(56) Scozzafava, A.; Ilies, M. A.; Manole, G.;
433.
5
6
7
8
9
Supuran, C. T. Eur. J. Pharm. Sci. 2000, 11, 69–
79.
(70) Knight, C. G. Methods Enzymol. 1995, 248, 18–
34.
(57)
Supuran, C. T.; Briganti, F.; Mincione, G.;
Scozzafava, A. J. Enzyme Inhib. 2000, 15, 111–128.
(71)
(72)
(73)
Rasmussen, H. S.; McCann, P. P. Pharmacol.
Ther. 1997, 75, 69–75.
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
(58) Galardy, R. E.; Grobelny, D. Biochemistry 1983,
22, 4556–4561.
Cathcart, J.; Pulkoski-Gross, A.; Cao, J. Genes
Dis. 2015, 2, 26–34.
(59) Scozzafava, A.; Supuran, C. T. J. Med. Chem.
2000, 43, 3677–3687.
Overall, C. M.; López-Otín, C. Nat. Rev. Cancer
2002, 2, 657–672.
(60) Scozzafava, A.; Supuran, C. T. Eur. J. Med.
Chem. 2000, 35, 299–307.
(74) Park, H. I.; Jin, Y.; Hurst, D. R.; Monroe, C. A.;
Lee, S.; Schwartz, M. A.; Sang, Q.-X. A. J. Biol.
Chem. 2003, 278, 51646–51653.
(61)
Supuran, C. T. In Drug Design of Zinc-Enzyme
Inhibitors; John Wiley & Sons, Inc.: Hoboken
(NJ), USA, 2009; pp 721–729.
(75)
Zhu, J.; Cai, X.; Harris, T. L.; Gooyit, M.; Wood,
M.; Lardy, M.; Janda, K. D. Chem. Biol. 2015, 22,
483–491.
(62) Van Wart, H. E.; Steinbrink, D. R. Anal.
Biochem. 1981, 113, 356–365.
(76) Gomis-Rüth, F. X.; Botelho, T. O.; Bode, W.
(63) Baell, J. B.; Holloway, G. A. J. Med. Chem. 2010,
53, 2719–2740.
Biochim. Biophys. Acta 2012, 1824, 157–163.
(77)
Cerdà-Costa, N.; Xavier Gomis-Rüth, F. Protein
Sci. 2014, 23, 123–144.
(64) Wünsch, E.; Heidrich, H. H.-G. Hoppe-Seyler´s
Zeitschrift für Physiol. Chemie 1963, 333, 149–151.
(78) Gomis-Rüth, F. X. J. Biol. Chem. 2009, 284,
(65) Komsa-Penkova, R. S.; Rashap, R. K.; Yomtova,
V. M. J. Biochem. Biophys. Methods 1997, 34,
237–249.
15353–15357.
(79) Pramar, Y.; Gupta, V. Das; Bethea, C. J. Clin.
Pharm. Ther. 1992, 17, 185–189.
(66) Watanabe, K. Appl. Microbiol. Biotechnol. 2004,
63, 520–526.
(80) Labute, P. Proteins 2009, 75, 187–205.
(67) Eckhard, U.; Schönauer, E.; Ducka, P.; Briza, P.;
Nüss, D.; Brandstetter, H. Biol. Chem. 2009,
ACS Paragon Plus Environment

Page 11 of 11
Journal of the American Chemical Society
Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the man-
uscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation,
concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for
TOC graphic specifications.
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
Insert Table of Contents artwork here
11
ACS Paragon Plus Environment