GroEL/ES inhibitors as potential antibiotics

Article abstract of DOI:10.1016/j.bmcl.2016.04.089

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth unde

Full text of DOI:10.1016/j.bmcl.2016.04.089

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3127–3134
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
GroEL/ES inhibitors as potential antibiotics
Sanofar Abdeen a, Nilshad Salim a, Najiba Mammadova a,y, Corey M. Summers a,à, Rochelle Frankson a,§
Andrew J. Ambrose b, Gregory G. Anderson c, Peter G. Schultz d, Arthur L. Horwich e, Eli Chapman b,
,
Steven M. Johnson a,
⇑
a Indiana University, School of Medicine, Department of Biochemistry and Molecular Biology, 635 Barnhill Dr., Indianapolis, IN 46202, United States
b The University of Arizona, College of Pharmacy, Department of Pharmacology and Toxicology, 1703 E. Mabel St., PO Box 210207, Tucson, AZ 85721, United States
c Indiana University-Purdue University Indianapolis, Department of Biology, 723 W. Michigan St., Indianapolis, IN 46202, United States
d The Scripps Research Institute, Department of Chemistry, 10550 North Torrey Pines Rd., La Jolla, CA 92037, United States
e HHMI, Department of Genetics, Yale School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06510, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the
Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES
chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with
small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we
report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and
Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.
GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in partic-
Received 28 March 2016
Revised 28 April 2016
Accepted 29 April 2016
Available online 4 May 2016
Keywords:
GroEL
GroES
HSP60
ular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range.
While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively
target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no
cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, com-
pounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kid-
ney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus
strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising can-
didates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.
Ó 2016 Elsevier Ltd. All rights reserved.
HSP10
Molecular chaperone
Chaperonin
Proteostasis
Small molecule inhibitors
ESKAPE pathogens
Antibiotics
The number of lives saved by antibiotics is a hallmark of the
success of this class of drugs. However, resistant bacterial strains
have been identified for every class of antibiotic, usually within a
few years of general therapeutic use.1–3 The threat of antibiotic
resistance is epitomized by the emergence of six multi-drug resis-
tant bacteria referred to as the ESKAPE pathogens: Enterococcus fae-
cium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species.4–8
The Centers for Disease Control (CDC) and Prevention Antibiotic
Resistance Threat Report lists these bacteria as serious threats
(level 4 out of 5) requiring prompt and sustained action.9 Most
alarming is that antibiotic resistance has mounted to the point
where therapeutics are severely limited or ineffective for once
easily treated infections. For example, ꢀ10,000 people per year
are estimated to die from methicillin-resistant S. aureus (MRSA)
infections in the United States.10 Moreover, the CDC estimates
the direct medical cost of treating antibiotic resistant bacterial
infections in the US is more than $20 billion per year.9 Clearly,
the rise of resistant bacterial strains requires enhanced research
efforts to ensure an ongoing antibiotic pipeline.
Current antibiotics primarily function by blocking cell wall con-
struction, structure and function of the cell membrane, protein
synthesis, DNA structure and function, or folic acid synthesis.11
Recently developed therapeutics for infections caused by drug-
resistant bacteria include the injectable carbapenem beta-lactam,
doripenem, which targets penicillin-binding proteins and inhibits
⇑
Corresponding author. Tel.: +1 317 274 2458; fax: +1 317 274 4686.
E-mail address: johnstm@iu.edu (S.M. Johnson).
Present address: Department of Genetics, Development and Cell Biology, Iowa
y
State University, 1210 Molecular Biology Building, Pannel Dr, Ames, IA 50011, United
States.
à
Present address: Department of Kinesiology, Iowa State University, 235 Barbara E.
Forker Building, Beach Rd, Ames, IA 50011, United States.
§
Present address: Department of Medicinal Chemistry and Molecular Pharmacol-
ogy, Purdue University, Hansen Life Sciences Research Building, 201 S. University
Street, West Lafayette, IN 47907, United States.
http://dx.doi.org/10.1016/j.bmcl.2016.04.089
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

3128
S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
Figure 1. General protocol for chaperonin-mediated biochemical assays. Compounds (I) are added at point A to a solution containing GroEL (or HSP60) with bound substrate
protein (e.g., malate dehydrogenase, MDH). Addition of GroES (or HSP10) and ATP initiates the refolding cycle, which is quenched with EDTA after a 60 min incubation.
Substrates (R) for the refolded reporter enzyme are added and after another 30–60 min incubation (until the DMSO control wells have reached ꢀ90% consumption of NADH),
absorbance is measured to evaluate the amount of refolded enzyme present, and by association the extent of chaperonin inhibition. Alternatively, addition of compounds at
point B enables determination of off-target inhibition of the reporter enzyme (i.e., native MDH enzyme activity). Chaperonin-mediated ATP hydrolysis is also evaluated using
a malachite green assay. Biochemical assays employing Rhodanese (Rho) are performed similarly (refer to Supporting information for detailed protocols).
cell wall synthesis;12 the cyclic lipopeptide, daptomycin, which
inserts into the bacterial membrane and leads to pore formation;13
quinupristin/dalfopristin, which bind to two different sites on the
50S ribosomal subunit and interfere with protein synthesis;14 the
oxazolidinone, linezolid, which also binds the 50S ribosomal sub-
unit;15 the tetracycline derivative, tigecycline, which targets pro-
tein synthesis via the 30S ribosomal subunit;16 and the
lipoglycopeptide, dalbavancin, which has the same mode of action
As these examples illustrate, most new antibiotics are derivatives
of existing drugs that also target the aforementioned pathways.
Unfortunately, bacterial resistance to these drugs is quick to
develop. These data argue for the continued pursuit of antibiotics
with entirely new modes of action, which may better avoid mech-
anisms of resistance and have longer effective life times.
An attractive strategy for the development of novel antibiotics
is to target bacterial protein homeostasis (proteostasis) mecha-
nisms, in particular molecular chaperones. Molecular chaperones
as vancomycin, binding to the
D
-Ala-
D
-Ala motif in the cell wall.17
O
S
O
S
S
Br
H
NO2
H
OH
OH
O
H
N
N
N
O
O
S
S
O
N
N
H
Cl
N
S
HO
N
H
N
HO
O
O
Br
O
1
5
8
9
S
O
N
O
HN
OH
OH
OH
O
N
N
N
H
N
O
NO2
OH
N
H
O2N
OH
N
H
N
H
OH
O
N
H2N
HO
O
O
10
11
14
23
15
18
O
O
N
O
O
O
O
O
N
N
N
O
S
O
O
S
O
N
N
N
N
H
HO
O
N
H
S
N
H
N
H
NO2
H
O
O
Cl
19
20
24
25
O
O
S
Br
S
H
N
H
N
H
N
O
H
N
N
N
O
S
N
O2N
N
O
O
O
O
O
O
Br
O
HO
O
OH
OH
27
28
29
31
O
O
HN
N
O
H
N
H
N
S
S
O2N
N
H2N
O
HN
O
O
N
O
N
O
N
S
O
N
H
O
O
F
Br
32
33
34
35
Figure 2. Structures of the 22 compounds under evaluation. For ease of comparison, compound numbering from 1 to 36 was maintained as presented in our previous high-
throughput screening study.37 Compounds 2–4, 6, 7, 12, 13, 16, 17, 21, 22, 26, 30, and 36 were omitted from evaluation as they were either not commercially available, or
purchased compounds were not readily identified by LC–MS and/or did not have acceptable purities confirmed by HPLC.

S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
3129
Table 1
intermediate states associated with the GroEL/ES refolding
cycle.32–34 Thus, it should be possible to develop inhibitors that
selectively target the double-ring GroEL/ES cycle with its addi-
tional allosteric signals and conformational intermediates. Further-
more, HSP60/10 is in the mitochondrial matrix, which is often
protected from small molecule penetration; thus, even if com-
pounds are found that inhibit HSP60/10 in biochemical assays,
Biochemical IC50 results for E. coli GroEL/ES inhibitors
Biochemical assay IC50 values (lM)
#
Native Rho
reporter
activity
Native MDH GroEL/ES-
GroEL/ES-
dMDH
refolding
GroEL/ES-
dMDH
ATPase
reporter
activity
dRho
refolding
1
5
8
9
>100
14
>100
>100
0.25
12
2.5
23
>100
8.1
>100
2.1
>100
1.4
12
5.3
>100
52
>62.5
>62.5
7.1
>62.5
54
30
0.58
1.4
1.4
0.47
0.83
2.5
1.7
6.8
3.0
22
2.4
2.3
2.6
9.6
0.89
28
⁄
7.5
0.69
1.4
0.93
0.80
1.2
3.0
2.7
5.7
4.8
5.4
4.7
3.6
6.5
4.7
2.6
24
119
>250
>250
80
174
216
10
11
14
15
18
19
20
23
24
25
27
28
29
31
32
33
34
35
(A)
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
187
>250
217
>250
>250
107
100
⁄
⁄
10
8
18
11
>250
25
79
31
42
>100
24
>100
18
10
⁄
>100
>100
>100
1
#
Statistical analyses (two-tailed t-tests) were performed for compound log(IC50
)
values determined from the GroEL/ES-dRho and GroEL/ES-dMDH refolding assays.
Compounds for which there is a statistically significant difference between inhi-
bition results have been marked with a ‘*’ between the two assay results being
compared (p <0.05). P-Values could not be calculated for compounds marked with a
‘#’ as one IC50 is greater than the maximum compound concentration tested. For
most compounds, IC50 values are not statistically different (17/22 compounds),
suggesting they are ‘on-target’ for inhibiting the refolding of the dRho and dMDH
substrates. IC50 correlations are represented graphically in Figure 3.
0.1
0.1
1
10
100
GroEL/ES-dMDH Refolding IC50 (μM)
IC50 = inhibitor concentration resulting in 50% reduction of biochemical activity.
(B)
are a specialized class of proteins that help other proteins to prop-
erly fold to their native states. Among the molecular chaperones in
E. coli, the GroEL/ES chaperonin system is the only one essential for
growth under all conditions.18,19 GroEL is a central processing
machine that maintains the structural and functional integrity of
many other proteins (Fig. 1); for a review, see Refs. 19,20.19–22
Thus, targeting this one functional node results in a cascading
effect that leads to the dysfunction of numerous key cellular path-
ways, which is lethal to bacteria.18 Because no other drugs function
by targeting chaperonin systems, this strategy should be effective
against bacteria that are resistant to current antibiotics.
100
10
1
18
8
The central tenet of this antibiotic strategy raises the question
of whether bacterial GroEL/ES can be targeted specifically without
interfering with the metazoan counterpart in the mitochondria,
HSP60/10, whose partial deficiency in humans leads to disease.23
Human HSP60 shares 48% sequence identity with E. coli GroEL,
and thus there is the possibility of inhibitor cross-talk between
the two chaperonins. However, structural and functional differ-
ences between the two systems suggest that it should be possible
to develop inhibitors that selectively target bacterial GroEL/ES over
human HSP60/10. GroEL is a homo-tetradecameric protein consist-
ing of two, seven-membered rings that stack back-to-back.24–26
Through a series of events driven by ATP binding and hydrolysis,
unfolded substrate proteins are bound within the central cavity
of one GroEL ring and encapsulated by the heptameric GroES co-
chaperonin lid structure, triggering protein folding in a seques-
tered chamber.25–31 GroEL/ES works as an allosterically-controlled,
double-ring system, which is regulated through positive intra-ring
ATP-binding and negative inter-ring binding. In contrast, studies
have indicated that human HSP60/10 operates as a single-ring
species, through at least part of the cycle, removing many of the
0.1
0.1
1
10
100
Native MDH Activity IC50 (μM)
Figure 3. (A) Compounds inhibit nearly equipotently in the E. coli GroEL/ES-dMDH
and the GroEL/ES-dRho refolding assays. Data plotted in the grey zones represent
results beyond the assay detection limits (i.e., >100
assay, and >250 M for the dRho refolding assay). Compounds indicated by the
white squares are those with statistically significant differences between their IC50
values (p <0.05). (B) While some compounds inhibit either native MDH or Rho
individually, only compound 10 inhibits in both counter-screens, and only to a
minor extent against native MDH. Data plotted in the grey zones represent results
beyond the assay detection limits (i.e., >62.5
reporter assay, and >100 M for the native Rho enzymatic reporter assay).
lM for the dMDH refolding
l
lM for the native MDH enzymatic
l

3130
S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
Table 2
For most of the compounds, we found a direct correlation between
inhibition of both the GroEL/ES-mediated dMDH and dRho refolding
reactions (Table 1 and Fig. 3A). While some compounds inhibited
either the native MDH or Rho reporter reactions, only compound
E. coli and B. subtilis bacterial proliferation EC50 results for GroEL/ES inhibitors
Bacterial proliferation EC50 values ( M)
l
#
DH5
a
E. coli
MC4100
D
acrB E. coli
SM101 E. coli
B. subtilis
1
5
8
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
92
2.3
>100
27
0.33
76
>100
48
>100
84
>100
25
0.10
>100
>100
>100
>100
>100
0.47
16
(A)
>100
>100
>100
>100
>100
21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
11
14
15
18
19
20
23
24
25
27
28
29
31
32
33
34
35
100
3.3
7.6
>100
>100
>100
>100
>100
>100
>100
19
2.8
>100
>100
>100
>100
43
>100
83
72
10
18
1
68
>100
25
8
>100
13
>100
>100
EC50 = effective concentration of compound resulting in 50% reduction of bacterial
proliferation.
0.1
0.1
1
10
100
they may not display cytotoxicity if they failed to reach the mito-
chondrial matrix.
GroEL/ES-dMDH Refolding IC50 (μM)
We previously developed a series of compounds that bind to the
ATP sites of E. coli GroEL and inhibit the chaperonin refolding
cycle.35,36 We also conducted a high-throughput screen to discover
inhibitors of the E. coli GroEL/ES chaperonin system that target
sites other than the ATP pockets, as we had concerns that ATP-
competitive inhibitors may have off-target effects against other
ATP-dependent proteins in cells.37 We chose the E. coli GroEL/ES
homolog for screening because it is the best characterized chaper-
onin and has been a model system for studying this class of
proteins.30,31,38–40 It shares high homology with GroEL/ES systems
from other bacteria, with 56–97% identical amino acids for GroEL
and 44–94% identical amino acids for GroES among the ESKAPE
pathogens (refer to Table S1 in Supporting information). Thus,
E. coli GroEL/ES serves as an excellent surrogate to discover chap-
eronin inhibitors to treat bacterial infections. The assays we devel-
oped analyzed the full refolding cycle and used the substrate
reporter enzymes b-arylsulfotransferase IV (AST-IV) and malate
dehydrogenase (MDH), which require GroEL, GroES, and ATP in
order to return to their native, active states (refer to Fig. 1 for a
general overview of the assay protocols). From ꢀ700,000 mole-
cules, our high-throughput screening narrowed the number of
GroEL/ES inhibitors to 235. We investigated a subset of these hits
in greater detail to identify IC50 values for inhibiting GroEL/ES-
mediated substrate refolding and ATPase activity. While only a
few compounds inhibited GroEL/ES-mediated ATPase activity,
most were potent inhibitors of the refolding cycle and exhibited
minimal to no off-target effects against the reporter enzyme.37
Extending from our high-throughput screening studies, we have
been investigating the antibiotic potential of 22 of our initial GroEL/
ES inhibitor hits (Fig. 2). We first tested the GroEL/ES inhibitors in
two additional biochemical counter-screens to further support that
they are acting ‘on-target’ and are not simply artifacts or false-
positives. The first counter-screen evaluates for inhibition of
GroEL/ES-mediated refolding of a third stringent substrate, Rho-
danese (Rho). The second counter-screen evaluates for inhibition
of the native Rho enzymatic reporter reaction. Detailed protocols
for these two assays are presented in Supporting information).41–44
SM101 lpxA2
MC4100 ΔacrB
(B)
100
10
1
18
8
0.1
0.1
1
10
100
GroEL/ES-dMDH Refolding IC50 (μM)
B. subtilis
E. faecium
S. aureus
Figure 4. (A) Inhibitors of the E. coli GroEL/ES-dMDH refolding cycle are inactive
against parent E. coli bacteria; however, several exhibit antibacterial effects against
mutant E. coli strains with compromised efflux pumps (MC4100
DacrB) and
lipopolysaccharide (LPS) outer membranes (SM101 lpxA2). (B) Parent B. subtilis, E.
faecium, and S. aureus Gram-positive bacteria are more susceptible to the antibiotic
effects of GroEL/ES inhibitors. Compound 8 potently inhibits the growth of all three
strains, while compound 18 is potent against B. subtilis and S. aureus, but inactive
against E. faecium. Data plotted in the grey zones represent results beyond the assay
detection limits (i.e., >100 lM).

S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
3131
Table 3
EC50 results for GroEL/ES inhibitors against the ESKAPE pathogens
Bacterial proliferation EC50 values (lM)
#
E. faecium
S. aureus
MRSA
K. pneumoniae
A. baumannii
P. aeruginosa
E. cloacae
1
5
8
9
10
11
14
15
18
19
20
23
24
25
27
28
29
>100
90
>100
14
0.20
>100
82
>100
9.1
0.13
>100
>100
40
94
>100
1.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
95
>100
>100
>100
>100
89
0.70
5.4
1.4
>100
58
30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
85
>100
>100
>100
>100
>100
27
2.4
5.2
16
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.5
0.15
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15
>100
>100
>100
0.63
<0.05
1.2
2.1
>100
50
>100
>100
>100
>100
81
>100
>100
>100
>100
>100
>100
>100
>100
>100
32
>100
>100
>100
>100
2.5
<0.05
0.47
65
23
>100
>100
1.8
13
1.5
21
>100
>100
>100
>100
80
>100
61
>100
>100
74
15
56
54
>100
>100
>100
76
0.35
0.15
2.3
>100
>100
0.17
5.7
45
>100
>100
84
>100
>100
>100
0.059
<0.05
<0.05
2.2
31
32
33
34
35
Ampicillin
Minocycline
Rifampicin
Chloramphenicol
Kanamycin
Streptomycin
Vancomycin
Daptomycin
6.2
2.2
37
>100
>100
>100
8.2
3.6
0.20
5.7
45
3.4
>100
>100
16
>100
17
2.9
74
>100
0.30
33
>100
EC50 = effective concentration of compound resulting in 50% reduction of bacterial proliferation.
10 inhibited in both counter-screens, and only to
a
minor
(see Supporting information for a detailed protocol) was employed
in liquid media using DH5 E. coli cells as the initial test strain
(EC50 values are summarized in Table 2 and graphically in Fig. 4A).
Unfortunately, no significant inhibition of bacterial growth was
observed for any of the compounds up to 100 lM. Reasoning this
extent against native MDH (Table 1 and Fig. 3B). Thus, we are confi-
dent that compounds are on-target inhibitors of dMDH and dRho
refolding. We next evaluated the 22 compounds for their antibiotic
effects on E. coli cells. A general bacterial proliferation assay
a
Table 4
Human HSP60/10 biochemical IC50 and liver and kidney cytotoxicity EC50 results for the GroEL/ES inhibitors
Biochemical assay IC50 values ( M)
l
Human cell viability EC50 values (lM)
#
GroEL/ES-dMDH refolding
HSP60/10-dMDH refolding
HSP60/10-dMDH ATPase
THLE-3 (liver)
HEK 293 (kidney)
1
5
8
9
7.5
0.69
1.4
0.93
0.80
1.2
3.0
2.7
5.7
4.8
5.4
4.7
3.6
6.5
4.7
2.6
24
⁄
⁄
⁄
89
4.9
5.5
1.6
3.3
5.2
7.4
10
>100
16
2.8
13
8.2
15
13
1.8
38
>100
>100
>100
26
106
>250
3.6
>250
140
29
16
12
>100
>100
9.9
>100
>100
>100
41
3.6
>100
34
>100
>100
>100
>100
15
34
23
78
>100
>100
12
>100
98
>100
44
17
>100
55
10
11
14
15
18
19
20
23
24
25
27
28
29
31
32
33
34
35
⁄
⁄
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
#
>100
>100
>100
>100
7.7
⁄
⁄
#
#
31
42
>100
24
>100
61
62
>100
>100
>100
>100
>100
>100
>100
Statistical analyses (two-tailed t-tests) were performed for compound log(IC50) values determined from the GroEL/ES-dMDH and HSP60/10-dMDH refolding assays. Com-
pounds for which there is a statistically significant difference between inhibition results have been marked with a ‘⁄’ between the two assay results being compared (p <0.05).
P-Values could not be calculated for compounds marked with a ‘#’ as one IC50 is greater than the maximum compound concentration tested. IC50 correlations are represented
graphically in Figure 5A.
IC50 = inhibitor concentration resulting in 50% reduction of biochemical activity.
EC50 = effective concentration of compound resulting in 50% reduction in cell viability.

3132
S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
might be due to efflux of the molecules, we tested against an
That many of the most potent GroEL/ES biochemical inhibitors
were ineffective against the MC4100 acrB E. coli cells could be
MC4100
D
acrB E. coli strain, which has one of the central compo-
D
nents of the AcrA/AcrB/TolC efflux pump removed.45,46 Compounds
due to the presence of other efflux pumps that were still functional,
as it is known that E. coli contains several classes of efflux
pumps.47–50 However, another possibility is that the molecules
were not able to traverse the highly impermeable lipopolysaccha-
ride (LPS) outer membrane characteristic of Gram-negative bacte-
ria. To probe this, we tested the compounds against a mutant
SM101 lpxA2 E. coli strain, which has a temperature sensitive lpxA
allele leading to compromised LPS biosynthesis at non-permissive
8 and 18 were the most potent inhibitors of this efflux-compromised
E. coli strain (EC50 = 2.3 and 21 lM, respectively), with the remainder
of the compounds being inactive.
(A)
temperatures, and consequently
a greater permeability to
molecules.51,52 We found that 10 compounds inhibited the growth
100
18
of this E. coli strain, with compounds 8 and 18 still proving to be
the most effective (EC50 values of 0.33 and 3.3 lM, respectively).
These results were further supported by the ability of many com-
pounds to inhibit the growth of a Gram-positive bacterium, Bacillus
subtilis (Table 2 and Fig. 4B), which does not contain an LPS outer
membrane. That more compounds failed to inhibit either the
SM101 lpxA2 E. coli or B. subtilis bacteria is putatively because of
the presence of efflux pumps that were still intact for these strains,
and/or the continued impermeability of compounds across the cell
membranes.
While E. coli and B. subtilis were good model systems for initial
proof-of-principle studies, we wanted to elucidate the antibiotic
potential of our GroEL/ES inhibitors against a panel of more clini-
cally relevant bacteria, in particular the ESKAPE pathogens. We
adapted the general bacterial proliferation assay to test molecules
against E. faecium, S. aureus (plus an MRSA strain), K. pneumoniae,
A. baumannii, P. aeruginosa, and E. cloacae (see Supporting informa-
tion for detailed protocols). A summary of the EC50 values for com-
pounds against these bacteria is presented in Table 3 (and
graphically in Fig. 4B), with a comparison against several common
antibiotics. As four of the ESKAPE pathogens are Gram-negative (K.
pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae), it is not
surprising that the GroEL/ES inhibitors were largely ineffective
against them. The remaining two ESKAPE pathogens, E. faecium
and S. aureus, are Gram-positive bacteria. Compound 8 was very
8
10
1
0.1
0.1
1
10
100
GroEL/ES-dMDH Refolding IC50 (μM)
(B)
100
18
potent at inhibiting E. faecium growth (EC50 = 0.15
pound 32 was moderately potent (EC50 = 15 M). Somewhat sur-
prisingly, compound 18, which emerged as lead inhibitor
lM), and com-
l
a
against the E. coli and B. subtilis cells, was inactive against E. fae-
cium. The remaining compounds were ineffective against E. fae-
cium, which again supports the notion that the presence of an
LPS membrane is not the sole determinant leading to compound
inactivity. While compounds 8 and 18 emerged as the lead inhibi-
8
10
1
tors of S. aureus growth (EC50 = 0.20
four other compounds were also moderately effective with EC50
values between 10 and 50 M (5, 11, 19, and 28). To determine if
lM and 1.8 lM, respectively),
l
they were bactericidal or bacteriostatic, we analyzed lead com-
pounds 8 and 18 against the methicillin susceptible S. aureus strain
(ATCC 25923) and found that both were acting as bactericidal inhi-
bitors (refer to Fig. S1 in Supporting information). We further
tested compounds against an MRSA strain (ATCC #BAA-44:
HPV107 strain, SCCmec Type I, Iberian PFGE Type) and found a cor-
relation with the methicillin susceptible S. aureus strain (Table 3).
While we have identified numerous inhibitors of GroEL/ES bio-
chemical function, several of which we now know inhibit the
growth of pathogenic bacteria (in particular S. aureus and MRSA),
there remained the possibility that these compounds could be
toxic to host cells owing to targeting of HSP60/10. To account for
this possibility, in vitro counter-screens were carried out in analo-
gous chaperonin-mediated dMDH refolding and ATPase biochemi-
cal assays employing HSP60/10 (Table 4). As indicated in Figure 5A,
there was a correlation observed for inhibiting both E. coli GroEL/ES
and human HSP60/10; however, compounds were generally more
potent at inhibiting E. coli GroEL/ES. Notably, compounds 1 and
0.1
0.1
1
10
100
HSP60/10-dMDH RefoldingIC50 (μM)
Liver (THLE-3)
Kidney (HEK-293)
Figure 5. (A) Compounds inhibit both E. coli GroEL/ES and human HSP60/10
chaperonin systems, but are generally more selective for the bacterial homolog.
Compound 18 is inactive against human HSP60/10, whereas compound 8 exhibits
low selectivity for GroEL/ES. (B) Even though compounds can inhibit HSP60/10
biochemical function, many exhibit low or no cytotoxicity to human liver and
kidney cells. Compound 18 exhibits no cytotoxicity, whereas compound 8 exhibits
moderate or low toxicity. Data plotted in the grey zones represent results beyond
the assay detection limits (i.e., >100 lM).

S. Abdeen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3127–3134
3133
inhibitors that exhibit >50-fold selectivity for blocking the growth
of S. aureus bacteria versus human liver and kidney cytotoxicities.
Their antibiotic efficacies against S. aureus are comparable to
vancomycin, daptomycin, and streptomycin (Fig. 6). Furthermore,
they are effective against the MRSA strain evaluated here. We are
pursuing further medicinal chemistry derivatization of these
GroEL/ES inhibitors to develop lead analogs with more potent
antibiotic effects against S. aureus (and ideally other bacteria) that
remain non-toxic to mammalian cells. Since these inhibitors were
discovered in a targeted GroEL/ES screen, we consider this the
putative target, but we cannot rule out the possibility of off-target
effects contributing to antibacterial potency. We are designing
experiments to delineate the mechanism of action at the protein
level and the mode of action at the whole cell level for these
GroEL/ES inhibitors. The results presented here are encouraging,
leading us to believe we can selectively target bacterial GroEL/ES
chaperonin systems as an antibiotic strategy.
100
10
1
0.1
8
18
Van
Dap
Strep
S. aureus
Liver (THLE-3)
Kidney (HEK 293)
Acknowledgments
MRSA
Figure 6. Compounds 8 and 18 are potent antibacterials against S. aureus & MRSA,
with moderate/low to no toxicity to human liver and kidney cells. Results for
Vancomycin (Van), Daptomycin (Dap), and Streptomycin (Strep) are shown for
We are grateful to the Genomics Institute of the Novartis
Research Foundation for working with us on the previous high-
throughput screening that initially identified these GroEL/ES bio-
chemical inhibitors. We also thank Dr. Leendert Hamoen from
the University of Amsterdam, Swammerdam Institute for Life
Sciences (SILS) for the B. subtilis 168 cells. The human HSP60
expression plasmid (lacking the 26 amino acid N-terminal mito-
chondrial signal peptide) was generously donated by Dr. Abdus-
salam Azem from Tel Aviv University, Faculty of Life Sciences,
Department of Biochemistry, Israel. We thank Dr. Quyen Hoang
from the IU School of Medicine, Department of Biochemistry and
Molecular Biology for helpful discussions and the use of various
lab equipment. This work was supported in part by an IU Biomed-
ical Research Grant, an IU Collaborative Research Grant, the
Showalter Trust Foundation, and startup funds from the IU School
of Medicine (S.M.J.) and the University of Arizona (E.C.).
comparison. Arrows (") indicate EC50 results are >100
lM (the maximum concen-
trations tested).
18 displayed 12-fold and >17-fold selectivities, respectively, for
inhibiting E. coli GroEL/ES over human HSP60/10. Unfortunately,
the other lead inhibitor against S. aureus and MRSA bacteria, com-
pound 8, was only 4-fold selective. Thus, we were concerned about
the cytotoxicity against human cells of 8 and other compounds
that inhibited the HSP60/10 refolding cycle.
To gauge for potential cytotoxicity of chaperonin inhibitors to
host tissues, we next evaluated compounds in viability assays
using cultured human liver (THLE-3) and kidney (HEK 293) cells.
These two stable cell lines were chosen because they are derived
from the two main organs responsible for drug metabolism and
excretion. An Alamar Blue-based cell viability assay was employed
to probe for cytotoxicity.53,54 We observed that inhibition of
HSP60/10 biochemical activity did not directly translate into cell
toxicity and that many compounds were only moderately toxic
or non-toxic to both cell lines (Table 4 and Fig. 5B). This could be
due to the fact that the inner mitochondrial membrane is highly
impermeable to compounds, and thus inhibitors may not be able
to penetrate to the mitochondrial matrix to reach HSP60/10. Com-
pounds 8 and 18 were the most potent inhibitors of S. aureus pro-
liferation, with the greatest therapeutic windows compared to
liver and kidney cell cytotoxicity (Fig. 6). In particular, compound
Supplementary data
Supplementary data (comparison of ESKAPE pathogen GroEL
and GroES sequence homology; tabulations of log(IC50) and log
(EC50) results with standard deviations; bacterial inhibition curves
to determine bacteriostatic vs. bactericidal mechanisms of action;
experimental protocols for biochemical and cell-based assays; syn-
thetic protocols and characterization data for compounds 10, 15,
23, 24, and 25; and HPLC purity and MS characterization of test
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2016.04.089.
8, which exhibits an EC50 of 0.20 lM against S. aureus proliferation,
has a therapeutic window of 60-fold with THLE-3 cells, and 390-
fold with HEK 293 cells. Compound 18, which exhibits an EC50 of
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