Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus

Article abstract of DOI:10.1021/acs.jmedchem.8b01293

We recently reported the identification of a GroEL/ES inhibitor (1, N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an antibiotic of last resort. To follow up, we have synthesized 43 compound 1 analogs to determine the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria. Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects. Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA was not able to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay and that lead inhibitors were able to permeate through established S. aureus biofilms and maintain their bactericidal effects.

Full text of DOI:10.1021/acs.jmedchem.8b01293

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Article
Hydroxybiphenylamide GroEL/ES inhibitors are potent antibacterials
against planktonic and biofilm forms of Staphylococcus aureus
Trent Kunkle, Sanofar Abdeen, Nilshad Salim, Anne-Marie Ray, Mckayla Stevens, Andrew J
Ambrose, Jose Victorino, Yangshin Park, Quyen Q. Hoang, Eli Chapman, and Steven M Johnson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01293 • Publication Date (Web): 04 Nov 2018
Downloaded from http://pubs.acs.org on November 7, 2018
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Hydroxybiphenylamide GroEL/ES inhibitors are
potent antibacterials against planktonic and biofilm
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forms of Staphylococcus aureus.
AUTHOR NAMES
Trent Kunkle,a,1 Sanofar Abdeen,a,1 Nilshad Salim,a Anne-Marie Ray,a Mckayla Stevens,a
Andrew J. Ambrose,b José Victorino,a Yangshin Park,a,c,d Quyen Q. Hoang,a,c,d Eli Chapman,b
and Steven M. Johnsona,*
1 Co-first author
AUTHOR ADDRESSES
a Indiana University School of Medicine, Department of Biochemistry and Molecular Biology,
635 Barnhill Dr., Indianapolis, IN 46202
b The University of Arizona, College of Pharmacy, Department of Pharmacology and
Toxicology, 1703 E. Mabel St., PO Box 210207, Tucson, AZ 85721
c Stark Neurosciences Research Institute, Indiana University School of Medicine. 320 W. 15th
Street, Suite 414, Indianapolis, IN 46202
d Department of Neurology, Indiana University School of Medicine. 635 Barnhill Drive,
Indianapolis, IN 46202
KEYWORDS.
GroEL, GroES, HSP60, HSP10, molecular chaperone, chaperonin, proteostasis, small molecule
inhibitors, ESKAPE pathogens, MRSA, antibiotics.
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ABSTRACT
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We recently reported the identification of a GroEL/ES inhibitor (1: N-(4-
(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in
vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an
antibiotic of last resort. To follow-up, we have synthesized 43 compound 1 analogs to determine
the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria.
Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting
GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects.
Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and
cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA were not able
to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay, and that lead
inhibitors were able to permeate through established S. aureus biofilms and maintain their
bactericidal effects.
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INTRODUCTION:
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In 2013, the Centers for Disease Control and Prevention (CDC) released a report
highlighting the dangers posed by multi-drug resistant bacteria, in particular a group of six that
have been termed the ESKAPE pathogens: Enterococcus faecium (Gram-positive),
Staphylococcus aureus (Gram-positive), Klebsiella pneumoniae (Gram-negative), Acinetobacter
baumannii (Gram-negative), Pseudomonas aeruginosa (Gram-negative), and the Enterobacter
species (Gram-negative).1 The report estimated that these bacteria infect over two-million
people in the U.S. annually, leading to ~23,000 deaths. Of the ESKAPE pathogens, methicillin-
resistant strains of S. aureus (MRSA) were found to be the deadliest, causing ~80,000 infections
and ~11,000 deaths annually. Unfortunately, the development of new antibiotics over the past
four decades has continually declined, and continuing research programs tend to focus on re-
derivatizing already known antibacterial classes.2-4 In addition, many bacteria are intrinsically
able to evade antibiotic effects by forming biofilms, which are often hallmarks of chronic
infections.5 While vancomycin is effective at treating planktonic S. aureus, it is not able to
penetrate and kill bacteria within biofilms.6, 7 Thus, to circumvent these resistance mechanisms,
there is an urgent need for antibacterials that function through new mechanisms of action and
previously unexploited pathways.
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While disrupting protein homeostasis has proven to be an effective antibacterial strategy
in the context of inhibiting the transcriptional and translational machineries, perturbing protein
folding pathways has gone largely unexplored.3 To facilitate newly synthesized polypeptides
folding into their active/native structural conformations, cells utilize a class of accessory proteins
termed molecular chaperones, also known as Heat Shock Proteins (HSPs).8 When molecular
chaperone functions are compromised, non-native polypeptides could misfold and aggregate,
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which is detrimental to cell viability.9-12 Thus, targeting molecular chaperones with small
molecule inhibitors should be an effective strategy for killing bacteria that is unique from the
mechanisms of current antibiotics. While significant efforts have been made to target HSP70
and HSP90 chaperones for developing anti-cancer agents, researchers are now beginning to
explore these chaperones as antibiotic targets.13-20 However, targeting HSP60 chaperonin
systems, called GroEL chaperonins in bacteria, has gone largely unexplored. GroEL is a homo-
tetradecameric protein complex that consists of two, seven-membered rings that stack back-to-
back with each other. GroEL functions to refold nascent polypeptides through a mechanism
unique from other molecular chaperones. To facilitate the folding of substrate polypeptides,
GroEL requires binding of ATP and a co-chaperone, called GroES. GroES binding to the GroEL
apical domains encapsulates the substrate polypeptides within the central cavity of GroEL, where
they can attempt to fold while being sequestered from the outside environment. Details of the
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GroEL/ES-mediated folding cycle have been extensively investigated and reported elsewhere.21-
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Since the GroEL/ES system is essential for bacterial viability under all conditions, we
hypothesize that blocking its functions with small molecule inhibitors should be an effective
antibacterial strategy.28, 29 A caveat to this strategy is that human HSP60 is moderately
conserved with the bacterial homologs (48% identity with the prototypical GroEL chaperonin
from Escherichia coli), which raises the question of potential off-target effects against human
cells. However, HSP60 is localized within the mitochondrial matrix of human cells, which is
highly impermeable to small molecules. Thus, even if compounds can inhibit HSP60 in vitro,
they may not reach and inhibit it in the mitochondrial matrix, permitting selective targeting of
bacteria over human cells.
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In a previous study, we performed high-throughput screening and identified 235
inhibitors of the GroEL/ES folding cycle.30 In a follow-up study evaluating a subset of these
GroEL/ES inhibitors for their antibacterial effects against the ESKAPE pathogens, we identified
compound 1 (Figure 1A) as a hit candidate for further antibacterial development.31 In particular,
compound 1 exhibited bactericidal effects against S. aureus that were comparable to vancomycin
(i.e. sub-M EC50). While compound 1 exhibited low-to-moderate cytotoxicity against human
liver (THLE-3) and kidney (HEK 293) cell lines, it still had >50-fold selectivity indices for
killing S. aureus bacteria. Intriguingly, compound 1 has been reported to have anthelmintic
activity against Trichocephalus muris, and two anthelmintic drugs used to treat parasitic
infections in livestock, closantel and rafoxanide (Figure 1B), bear striking resemblances to the
compound 1 scaffold.32, 33 In addition, Cheng et al. reported similar analogs that exhibited anti-
MRSA activities, although the reported structure-activity relationships (SAR) suggested that
their antibacterial effects were not entirely through targeting transglycosylase activity.34 Thus,
there is a high probability that targeting the GroEL/ES chaperonin system could be contributing
significantly to its antibacterial effects, which warrants further investigation.
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While compound 1 itself is a promising GroEL/ES inhibitor to take forward as an anti-
bacterial candidate, there is room for further optimization before proceeding into a proof-of-
principle anti-bacterial efficacy models in animals (e.g. mice systemically infected with S.
aureus). As a first step in our optimization strategy, rather than adding various substituents and
substructures to the scaffold as is often done in drug development, we chose an opposite
approach and employed a molecular deconstruction strategy where we systematically removed
the various substituents and substructures (R1-R5 – Figure 1A) to evaluate their contributions to
inhibitor potency and selectivity. Thus, we synthesized a library of 43 analogs that contained all
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the different ± combinations of the R1-R5 groups. We then tested them in a series of assays to
achieve three primary objectives: 1) determine which groups are crucial to inhibit GroEL/ES and
HSP60/10 folding function in vitro; 2) identify groups that expand the therapeutic window
further between antibacterial efficacy and human cell cytotoxicity; and 3) determine if inhibitors
are effective against bacteria in biofilms, and whether or not bacteria will be able to generate
acute resistance to this molecular class. Results from these assays would then allow us to
identify the smallest effective inhibitor analog that maintains potency against bacteria while
reducing cytotoxicity to human cells. Knowing this information would then allow us to build
upon this base scaffold in a more rational approach to improve the pharmacological properties of
this antibacterial series.
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RESULTS AND DISCUSSION
Evaluating the effectiveness of compound 1 analogs for inhibiting the GroEL/ES-mediated
folding cycle.
As a first step in this study, we synthesized analogs 1-44 using well-established chemistry
as outlined in Scheme 1. A nucleophilic aromatic substitution reaction between 2-
mercaptobenzothiazole and 3,4-dichloronitrobenzene was employed to give the nitro-
intermediate 45 (containing the chloro-group in the R2 position), which was subsequently
reduced to the amine 46 using tin powder in a 10% mixture of HCl in AcOH.32, 35-38 The
intermediate amine 47, lacking the chloro-group at the R2 position, was synthesized in one step
by a nucleophilic aromatic substitution reaction between 2-chlorobenzothiazole and
aminothiophenol.39 Compounds 17-44 were prepared by reacting the respective aryl-acid
chlorides (where commercially available) with amines 46 or 47 with pyridine in anhydrous
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dicloromethane.36, 37 If only the respective aryl-carboxylic acids were commercially available,
we either converted them to their acid chloride counter-parts by reacting with thionyl chloride, or
coupled the acids directly to the amines using amide coupling procedures with DDC, DMAP,
and pyridine, or EDC, HOBt•H2O, and TEA in dichloromethane.37 The methoxy-bearing
analogs (29-44) were then further de-methylated to analogs 1-16 using BBr3 in anhydrous
dichloromethane.36-38, 40 Detailed synthetic protocols and compound characterizations (e.g. 1H-
NMR, MS, and RP-HPLC) are presented in the Experimental Section and Supporting
Information. We also purchased the two highly related anthelmintic drugs used in veterinary
medicine, closantel and rafoxanide, to determine whether they would also inhibit the GroEL/ES
chaperonin system. While their mode of action is reported to involve uncoupling of the proton
gradient of oxidative phosphorylation, which drives ATP production in the mitochondria of
parasites, it is possible that targeting GroEL/ES or HSP60/10 chaperonin systems could be
contributing to their antibiotic properties.41, 42
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After generating the compound library, we next employed a series of well-established
biochemical assays to evaluate compound inhibitory effects against the GroEL/ES chaperonin
system. As in previous studies, we used E. coli GroEL/ES as the surrogate chaperonin system
for refolding of the reporter enzymes malate dehydrogenase (MDH) and rhodanese (Rho).30, 31, 40
Briefly, binary complexes are formed between GroEL and denatured MDH or Rho, which are
then refolded to their native states upon addition of ATP and the GroES co-chaperonin.
Enzymatic activities of refolded native MDH or Rho act as coupled reporters of GroEL/ES
refolding functions since in the presence of chaperonin inhibitors, no functional reporter
enzymes are generated. Inhibition IC50 results for testing of compounds in these two refolding
assays are compiled in Table 1, and visually presented in the correlation plot in Figure 2A. The
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log-transformations of IC50 results and standard deviations are presented in Table S1 in the
Supporting Information. As seen in Figure 2A, there is a strong correlation between compounds
inhibiting in the GroEL/ES-dMDH and dRho refolding assays (Spearman correlation coefficient
comparing log(IC50) values in each assay is 0.9663, p < 0.0001), suggesting they are acting on-
target against the chaperonin system. As we do not know where the binding sites are for this
series of inhibitors, precise structure-function interpretation of the results remains elusive. In
general, though, the R1-benzothiazole coupled with the R3-hydroxyl are required for potent
inhibition. Halogenation at the R2-position (Cl) and R4/R5 positions (Br) further increases
inhibitor potency, likely through a combination of increased hydrophobic interactions of the
halides themselves, coupled with the electron-withdrawing capability of the bromines lowering
the pKa of the salicylate hydroxyl, which could enhance polar interactions within the binding
sites. Perhaps not surprisingly, we found that closantel and rafoxanide were, indeed, potent
GroEL/ES inhibitors; thus, an intriguing question to address in future studies will be how much
does targeting chaperonin systems contribute to the anthelmintic effects of these two veterinary
antibiotics?
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To further support on-target effects, we evaluated inhibitors against the native MDH and
Rho enzymes to identify false-positives that simply inhibit the reporter reactions of the coupled
refolding assays (detailed procedures are presented in the Supporting Information). While some
compounds were found to inhibit native MDH (e.g. 1, 5, closantel, and to lesser extents, 9, 13,
and rafoxanide), none of the analogs were found to inhibit native Rho enzymatic activity (Table
1 and Figure 2B). While these results further support that inhibitors are targeting the
chaperonin-mediated folding cycle, they also indicate that selectivity issues may be a liability
that future studies would need to address. Hearteningly, though, a few analogs (e.g. compound
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2, 3, 6, and 7) are moderate to potent inhibitors in the GroEL/ES-mediated refolding assays yet
remained inactive against the reporter enzymes, suggesting selectivity concerns are not
insurmountable.
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Determining the antibacterial effects of compounds against the ESKAPE pathogens.
We next evaluated compounds for antibacterial efficacy against the ESKAPE pathogens
in liquid media culture as per previously reported procedures.38 Inhibition EC50 results for
testing of compounds in these bacterial proliferation assays are shown in Table 2. In general,
this series of analogs was ineffective against the Gram-negative bacteria (K. pneumonia, A.
baumannii, P. aeruginosa, and E. cloacae), likely owing to drug efflux and/or impermeability to
the lipopolysaccharide (LPS) outer membranes of Gram-negative bacteria.31 However, with
regards to A. baumannii, notable exceptions are compounds 2 and 6, which exhibit EC50 values
of 2.9 and 12 µM, respectively. This suggests that drug efflux and LPS impermeability issues
may not be insurmountable with further inhibitor optimization. As previously observed with
compound 1, several analogs retained antibacterial efficacy against the Gram-positive bacteria,
E. faecium and S. aureus, although they were strikingly more effective against S. aureus. The
presence of the hydroxyl at the R3 position appears to be integral for potent inhibition of S.
aureus bacteria, almost regardless of substituents and substructures at the other positions.
However, it is noted that incorporation of the benzothiazole substructure at the R1 position leads
to the most potent inhibitors (and generally required to inhibit E. faecium), which may support
on-target effects since these analogs are able to inhibit GroEL/ES-mediated folding functions.
Importantly, these analogs are all equipotent against the MRSA strain that we evaluated
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compounds against (ATCC #BAA-44, HPV107 designation, isolated from a hospital in Lisbon,
Portugal).
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When comparing the EC50 results of this series of compounds against E. faecium bacteria
with the IC50 values obtained in the GroEL/ES-dMDH refolding assay (Figure 3A), we note an
almost linear correlation (Spearman correlation coefficient comparing log(I/EC50) values in each
assay is 0.9628, p < 0.0001), which may indicate on-target effects against GroEL/ES driving
antibacterial activity. When performing the same comparison with MRSA bacteria (Figure 3B),
although a trend is evident between antibacterial efficacy and GroEL/ES inhibition MRSA
(Spearman correlation coefficient comparing log(I/EC50) values in each assay is 0.8042, p <
0.0001), several compounds that are not GroEL/ES inhibitors still remain effective against
bacteria (e.g. 10-16). This finding could be a result of S. aureus GroEL/ES functioning
differently than the E. coli GroEL/ES chaperonin system, which we use as a surrogate in these
studies; however, we cannot rule out potential off-target effects, especially since we know that
some analogs could inhibit MDH and also potentially be targeting transglycosylase as reported
by Cheng et al.34 Further studies are warranted to determine how E. faecium and S. aureus
GroEL/ES function compared to E. coli GroEL/ES, and to identify the specific mechanisms of
action of these inhibitors in both E. faecium and S. aureus bacteria.
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While some GroEL/ES inhibitors can target human HSP60/10 in vitro, many display
moderate to low cytotoxicity to human cells.
Knowing which compounds were effective GroEL/ES inhibitors with antibacterial
properties, we next evaluated whether they would 1) inhibit human HSP60/10, and 2) exhibit
cytotoxicity to two cell lines that we typically employ for cell viability testing in vitro: THLE-3
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liver cells and HEK 293 kidney cells. These assays were performed as previously reported, with
detailed protocols presented in the Supporting Information.31, 38, 40 Briefly, the HSP60/10-dMDH
folding assay was conducted analogously to the GroEL/ES-dMDH folding assay so IC50 results
could be directly compared. The human cell cytotoxicity assays used Alamar Blue reagents to
measure the viability of liver and kidney cells that had been incubated with test compounds over
a 48 h time period. Biochemical inhibition (IC50) and cell viability (cytotoxicity; CC50) results
for these assays are presented in Table 3.
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While some analogs selectively inhibit E. coli GroEL/ES over human HSP60/10 (e.g. 3,
4, 7, and 8), IC50 values between the GroEL/ES-dMDH and HSP60/10-dMDH folding assays
were nearly the same for most analogs (Figure 4A - Spearman correlation coefficient comparing
log(IC50) values in each assay is 0.8351, p < 0.0001). We note, though, that comparison of these
results is convoluted by the fact that some of these analogs also inhibit native MDH, and thus
could be false positives in the HSP60/10-dMDH folding assay owing to simply inhibiting the
MDH reporter reaction. While HSP60/10 inhibition could potentially be teased out by
employing rhodanese as the denatured reporter enzyme as we do in the case of the GroEL/ES-
dRho refolding assay, in our experience, the equivalent HSP60/10-dRho refolding is not as
robust, potentially owing to the lower stability of human HSP60 compared to E. coli GroEL.
When comparing the biochemical and cell-based results, there does not appear to be a
noticeable correlation between HSP60/10-dMDH refolding assay IC50 values and liver and
kidney cell viability assay CC50 values (Figure 4B - (Spearman correlation coefficient values are
0.4791 (p < 0.0008) and 0.3286 (p < 0.0258) when comparing HSP60/10-dMDH refolding assay
log(IC50) values with liver and kidney cell viability log(CC50) values, respectively).
Interestingly, compounds that bear the R1 benzothiazole and R3 hydroxyl substructures (1-8) are
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generally less cytotoxic against the HEK 293 cells than their counterparts without the R1
benzothiazole (9-16), and also less cytotoxic to the THLE-3 liver cells. Since inclusion of these
two substructures generally afforded more potent chaperonin inhibitors, the differences between
these results may suggest that compound cytotoxicities are predominantly a result of off-target
effects and not from targeting HSP60/10 itself. This would not be surprising since some analogs
are also able to inhibit native MDH (e.g. 1, 5, 9, 13, closantel, and rafoxanide). When comparing
EC50 values of compounds inhibiting the proliferation of susceptible and methicillin-resistant S.
aureus with cell viability CC50 values against the human liver and kidney cells (Figure 4C), we
note that many analogs exhibit >50-fold selectivity indices. Considering we have only been
looking at the effects that removing substituents and substructures have on the potency and
selectivity of this series of analogs, these are exciting initial results to move forward from in
future med-chem efforts where we begin to append and optimize the various substituents and
substructures of this scaffold.
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MRSA cannot readily generate acute resistance to lead analogs.
After identifying which compounds selectively inhibited the GroEL chaperonin system
and killed bacteria, we next evaluated whether bacteria could quickly develop resistance to lead
candidate inhibitors. This was a concern we encountered with another series of GroEL/ES
inhibitors we have been studying, represented by the bis-sulfonamido compound “28R” shown
in Figure 5.38 For this experiment, we adapted a liquid culture resistance assay from the
previously established procedures of Kim et al., and used our MRSA strain as the test bacteria (a
detailed procedure is presented in the Supporting Information).38, 43 Briefly, test compounds
were incubated in dilution series with MRSA for 24 h and EC50 values were determined. The
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first well exhibiting OD600 readings >0.2 were then sub-cultured for another 24 h with test
compound again in dilution series. Serial passage in this manner was conducted for a total of 12
days, and each day EC50 values for the test compounds were determined: inhibitors to which
MRSA could rapidly generate resistance would exhibit increasing EC50 results over each
successive passage. Rapid resistance was observed for the previously reported bis-sulfonamide
compound, 28R, but was found to be reversible and likely owing to increased inhibitor efflux.38
We evaluated two of our lead GroEL inhibitors, 1 and 11, along with vancomycin, and found
that all three exhibited exemplary antibiotic efficacy to which this MRSA strain was not able to
easily generate resistance.
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Compound 1 is bactericidal to S. aureus within established biofilms
While we found that S. aureus is not able to easily generate resistance to compounds 1
and 11, what remained to be seen was whether this series of inhibitors would be effective at
preventing bacteria from establishing biofilms and killing bacteria within already established
biofilms. Establishing biofilms is another effective mechanism by which S. aureus can evade the
effects of many current antibiotics, including vancomycin. To gauge the efficacy of lead
inhibitors at preventing S. aureus from forming biofilms, we employed an assay similar to the
liquid culture assay we used to determine inhibition of bacterial proliferation, with a few
modifications (detailed procedures presented in the Experimental section). Briefly, test
compounds (1, 2, 5, 8, 11, and vancomycin) were incubated with S. aureus bacteria in media
supplemented with 0.5% glucose (to support biofilm formation) for 24 h at 37°C. After 24 h, the
supernatant was removed, the wells were gently washed, and the biofilm that had formed on the
well surfaces were stained with crystal-violet and quantified by UV-Vis spectroscopy. We found
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that all of the compound 1 analogs tested, and vancomycin, were able to prevent S. aureus from
forming biofilms with EC50 values nearly equipotent to antibacterial EC50s we determined
against planktonic bacterial growth. Representative dose-response curves for compound 1 and
vancomycin tested in these assays are presented in Figure 6, with a tabulation of test compound
EC50 results presented in Table 4. The high correlation of these results was not entirely
surprising as compound 1 and vancomycin are bactericidal against S. aureus, and thus dead
bacteria would not be able to form biofilms.31
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Next, we evaluated whether or not compounds would be bactericidal to S. aureus that
were within already established biofilms. In this assay, we first grew S. aureus bacteria for 24 h
in the absence of test compounds so that they could establish biofilms in the wells. After 24 h,
the cultures were removed, the wells were washed gently, and fresh media was added along with
test compounds or vancomycin. The cultures were incubated in the presence of test compounds
for another 24 h, then the wells were gently washed again to remove compounds and any
planktonic bacteria that had emerged. Fresh media was then added and the cultures were
incubated for another 24 h to allow any viable bacteria remaining in the biofilms to emerge and
grow planktonically again. While there were 5-15-fold shifts in EC50 values for the compound 1
analogs killing planktonic vs. biofilm bacteria (Figure 6 and Table 4), these are still exciting
results considering vancomycin was completely ineffective against biofilm bacteria (EC50 >100
M), and especially since this class of GroEL/ES inhibitors has yet to be fully optimized. Thus,
this scaffold shows considerable promise to take forward for further development as an
antibacterial candidate.
CONCLUSIONS
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In the present study, we developed a series of analogs of the previously identified hit
GroEL inhibitor, compound 1, and employed a molecular deconstruction strategy to
systematically evaluate the contributions that the R1 to R5 substituents and substructures make
towards selectively inhibiting the GroEL/ES chaperonin system and killing bacteria. We found
that the benzothiazole R1 group and hydroxyl R2 substituent were integral to inhibiting
GroEL/ES in vitro, but that the hydroxyl was the key determinant for being able to potently
inhibit S. aureus proliferation. While trends are noted between IC50 values from the GroEL/ES-
dMDH refolding assay and the E. faecium and S. aureus proliferation assays, further experiments
are warranted to conclusively determine whether or not inhibitors are on-target in bacteria.
While some inhibitors were equipotent in the human HSP60/10-dMDH refolding assay, several
exhibited only moderate to low cytotoxicity to liver and kidney cells. Importantly, compounds 1
and 11 did not encounter acute resistance through a 12-day serial passage in MRSA, generally
maintaining efficacy <1 μM. Furthermore, compound 1 analogs were effective at killing S.
aureus bacteria within established biofilms, whereas vancomycin was ineffective. These
exemplary results support future med-chem derivatization efforts to optimize the in vitro and in
vivo pharmacological properties of this class of GroEL/ES inhibitors for antibacterial
development.
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EXPERIMENTAL
General Synthetic Methods.
Unless otherwise stated, all chemicals were purchased from commercial suppliers and
used without further purification. Reaction progress was monitored by thin-layer
chromatography on silica gel 60 F254 coated glass plates (EM Sciences). Flash chromatography
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was performed using a Biotage Isolera One flash chromatography system with elution through
Biotage KP-Sil Zip or Snap silica gel columns for normal-phase separations (hexanes:EtOAc
gradients) or Snap KP-C18-HS columns for reverse-phase separations (H2O:MeOH gradients).
Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using a
Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707
autosampler. For preparatory HPLC purification, samples were chromatographically separated
using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 Å pore size,
5 m particle size, 19x150 mm), eluting with a H2O:CH3CN gradient solvent system. Linear
gradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B (A = 95:5 H2O:CH3CN,
0.05% TFA; B = 5:95 H2O:CH3CN, 0.05% TFA. Products from normal-phase separations were
concentrated directly, and reverse-phase separations were concentrated, diluted with H2O,
frozen, and lyophilized. For primary compound purity analyses (HPLC-1), samples were
chromatographically separated using a Waters XSelect CSH C18 column (part number
186005282, 130 Å pore size, 5 m particle size, 3.0x150 mm), eluting with the above
H2O:CH3CN gradient solvent systems. For secondary purity analyses (HPLC-2) of final test
compounds, samples were chromatographically separated using a Waters XBridge C18 column
(either part number 186003027, 130 Å pore size, 3.5 m particle size, 3.0x100 mm, or part
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number 186003132, 130 Å pore size, 5.0 m particle size, 3.0x100 mm), eluting with a
H2O:MeOH gradient solvent system. Linear gradients were run from either 100:0, 80:20, 60:40,
or 20:80 A:B to 0:100 A:B (A = 95:5 H2O:MeOH, 0.05% TFA; B = 5:95 H2O:MeOH, 0.05%
TFA). Test compounds were found to be >95% pure from both RP-HPLC analyses. Mass
spectrometry data were collected using either an Agilent analytical LC-MS at the IU Chemical
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were recorded on a Bruker 300 MHz spectrometer at the CGCF. Chemical shifts are reported in
parts per million and calibrated to the d6-DMSO solvent peaks at 2.50 ppm. Synthesis and
characterization of intermediates 45-49 are presented below. General amide coupling and
methoxy deprotection steps are presented as follows using compounds 29 and 1 as representative
examples. Specific synthetic procedures and compound characterizations are presented in the
Supporting Information for the remaining analogs.
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45: 2-((2-Chloro-4-nitrophenyl)thio)benzo[d]thiazole. 2-Mercaptobenzothiazole (11.9 g, 71.1
mmol), 3,4-dichloronitrobenzene (11.8 g, 61.5 mmol), and potassium carbonate (11.7 g, 84.7
mmol) were stirred together in DMF (60 mL) at R.T. overnight, then at 80°C for 4 h. The
reaction was then diluted with water and the precipitate was filtered, rinsed with water, and dried
1
to afford 45 as a yellow powder (19.5 g, 98% yield). H-NMR (300 MHz, d6-DMSO) δ 8.51 (d,
J = 2.4 Hz, 1H), 8.23 (dd, J = 8.7, 2.5 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.07-8.13 (m, 1H), 7.91
(d, J = 8.7 Hz, 1H), 7.44-7.59 (m, 2H); MS (ESI) C13H8ClN2O2S2 [MH]+ m/z expected = 323.0,
observed = 323.1; HPLC-1 = 98%.
46: 4-(Benzo[d]thiazol-2-ylthio)-3-chloroaniline. Tin powder (5.64 g, 47.5 mmol) was added
slowly to a stirring mixture of 45 in a 1:10 mixture of HCl:AcOH (15 mL). The reaction was
allowed to stir at R.T. for 2 days, then diluted with EtOAc and H2O, neutralized with NaHCO3,
and filtered. The filtrate was extracted with EtOAc and the organics dried over Na2SO4, filtered,
and concentrated. The crude product was then chromatographed over silica (hexanes:EtOAc
gradient) and concentrated. The residue was diluted in a 4:1 mixture of hexanes:DCM and the
precipitate was filtered and dried to afford 46 as a yellow powder (3.73 g, 81% yield). 1H-NMR
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(300 MHz, d6-DMSO) δ 7.87-7.96 (m, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H),
7.43 (td, J = 7.7, 1.3 Hz, 1H), 7.26-7.34 (m, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 8.5, 2.4
Hz, 1H), 6.18 (s, 2H); MS (ESI) C13H10ClN2S2 [MH]+ m/z expected = 293.0, observed = 293.0;
HPLC-1 = 98%.
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47: 4-(Benzo[d]thiazol-2-ylthio)aniline. 2-Chlorobenzothiazole (2.00 g, 11.8 mmol), 4-
aminothiophenol (1.70 g, 13.6 mmol), and potassium carbonate (3.24 g, 23.4 mmol) were stirred
together in EtOH (15 mL) for 18 h. The reaction was then diluted with water and the precipitate
was filtered, rinsed with water, and collected. Flash chromatographic purification
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(hexanes:EtOAc gradient) afforded 47 as an off-white solid (2.71 g, 89% yield). H-NMR (300
MHz, d6-DMSO) δ 7.85-7.91 (m, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.37-7.45 (m, 3H), 7.25-7.34 (m,
1H), 6.66-6.73 (m, 2H), 5.84 (s, 1H); MS (ESI) C13H11N2S2 [MH]+ m/z expected = 259.0,
observed = 259.0; HPLC-1 = >99%.
48: 3,5-Dibromo-2-methoxybenzoic acid. Iodomethane (6.30 mL, 101 mmol), 3,5-
dibromosalicylic acid (10.0 g, 33.7 mmol), and K2CO3 (14.0 g, 101 mmol) were stirred at R.T.
overnight, then at 80°C for 4 h. The reaction was diluted into water and extracted into DCM.
The organics were dried over Na2SO4, filtered, and concentrated. The intermediate ester was
then stirred overnight with LiOH•H2O (5.70 g, 136 mmol) in a 3:1:1 mixture of
THF:MeOH:H2O (35 mL). The reaction was diluted with water and acidified with HCl. The
precipitate was filtered, washed with water, and dried to afford 48 as a white solid (9.85 g, 94%
1
yield). H-NMR (300 MHz, d6-DMSO) δ 13.56 (br s, 1H), 8.09 (d, J = 2.5 Hz, 1H), 7.83 (d, J =
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2.5 Hz, 1H), 3.81 (s, 3H); MS (ESI) C8H5Br2O3 [M-H]- m/z expected = 308.9, observed = 309.1;
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HPLC-1 = >99%.
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49: 5-Bromo-2-methoxybenzoic acid. 5-Bromosalicylic acid (10.0 g, 46.1 mmol), iodomethane
(8.60 mL, 138 mmol), and K2CO3 (19.0 g, 137 mmol) were stirred at R.T. overnight, then at
80°C for 4 h. The reaction was diluted into water and the precipitate was filtered, rinsed with
water, and dried. The intermediate ester was then stirred overnight with LiOH•H2O (7.70 g, 184
mmol) in a 3:1:1 mixture of THF:MeOH:H2O (45 mL). The reaction was diluted with water and
acidified with HCl. The precipitate was filtered, washed with water, and dried to afford 49 as a
1
white solid (8.70 g, 82% yield). H-NMR (300 MHz, d6-DMSO) δ 12.98 (br s, 1H), 7.72 (d, J =
2.6 Hz, 1H), 7.66 (dd, J = 8.8, 2.6 Hz, 1H), 7.10 (d, J = 8.9 Hz, 1H), 3.81 (s, 3H); MS (ESI)
C8H6BrO3 [M-H]- m/z expected = 229.0, observed = 229.0; HPLC-1 = 98%.
General procedure for the amide coupling step using analog 29 as a representative
example: N-(4-(Benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-
methoxybenzamide. Compound 48 (225 mg, 0.726 mmol) was stirred in SOCl2 (2 mL) at 60°C
for 1 h, then was concentrated. Anhydrous DCM (5 mL), compound 46 (148 mg, 0.505 mmol),
and pyridine (62.0 L, 0.760 mmol) were added and the reaction was stirred at R.T. for 18 h
(under Ar). Flash chromatographic purification (hexanes:EtOAc gradient) afforded 29 as a
1
yellow solid (58.6 mg, 20% yield). H-NMR (300 MHz, d6-DMSO) δ 11.00 (s, 1H), 8.21 (d, J =
2.1 Hz, 1H), 8.10 (d, J = 2.3 Hz, 1H), 7.97 (t, J = 8.3 Hz, 2H), 7.82-7.87 (m, 2H), 7.78 (dd, J =
8.6, 2.2 Hz, 1H), 7.47 (td, J = 7.7, 1.3 Hz, 1H), 7.32-7.38 (m, 1H), 3.84 (s, 3H); MS (ESI)
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C21H12Br2ClN2O2S2 [M-H]- m/z expected = 580.8, observed = 580.7; HPLC-1 = >99%; HPLC-2
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= >99%.
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General procedure for the methoxy deprotection step to give hydroxylated compounds
using analog 1 as a representative example: N-(4-(Benzo[d]thiazol-2-ylthio)-3-
chlorophenyl)-3,5-dibromo-2-hydroxybenzamide. To a stirring mixture of 29 (51.0 mg,
0.0872 mmol) in anhydrous DCM (5 mL) was added BBr3 (0.26 mL of 1 M in DCM, 0.26
mmol). The reaction was allowed to stir at R.T. (under Ar) for 18 h and then quenched with
MeOH. Flash chromatographic purification (hexanes:EtOAc gradient) afforded 1 as an off-white
1
solid (34.4 mg, 69% yield). H-NMR (300 MHz, d6-DMSO) δ 11.14 (br s, 1H), 8.19 (dd, J =
6.2, 2.2 Hz, 2H), 7.94-8.05 (m, 3H), 7.82-7.89 (m, 2H), 7.47 (td, J = 7.7, 1.3 Hz, 1H), 7.33-7.40
(m, 1H); MS (ESI) C20H10Br2ClN2O2S2 [M-H]- m/z expected = 566.8, observed = 566.6; HPLC-1
= 99%; HPLC-2 = 98%.
Protein Expression and purification.
E. coli GroEL and GroES, and human HSP60 and HSP10, were expressed and purified as
previously reported.30, 31, 38, 40, 44 Detailed protocols for these protein purifications are presented
in the Supporting Information.
Control compounds for assays.
For all of the biochemical assays (GroEL/ES and HSP60/10-mediated dMDH and dRho
refolding assays, and native MDH and Rho enzymatic activity counter-screens), DMSO was
used as negative control, and a panel of our previously discovered and reported chaperonin
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inhibitors were used as positive controls: e.g. compound 1 herein; compounds 9 and 18 from
Johnson et. al 2014 and Abdeen et. al 201630, 31; suramin and compound 2h-p from Abdeen et. al
201640; and compounds 20R, 20L, and 28R from Abdeen et. al 2018.38 For the bacterial
proliferation assays, control compounds included the aforementioned panel of previously
reported chaperonin inhibitors as well as vancomycin, daptomycin, ampicillin, and rifampicin.
For the human cell viability assays, control compounds include the aforementioned compounds
as well as other protein homeostasis inhibitors, such as bortezomib (proteasome inhibitor); VER-
155008 (HSP70 inhibitor); and ganetespib and 17-DMAG (HSP90 inhibitors).
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Evaluation of compounds in GroEL/ES and HSP60/10-mediated dMDH and dRho
refolding assays.
All compounds were evaluated for inhibiting E. coli GroEL/ES and human HSP60/10-
mediated refolding of the denatured MDH and denatured Rho reporter enzymes as per previously
reported procedures.30, 31, 38, 40 Detailed protocols for these assays are presented in the
Supporting Information.
Counter-screening compounds for inhibition of native MDH and Rho enzymatic activity.
All compounds were counter-screened for inhibiting the enzymatic activity of the native
MDH and native Rho reporter enzymes as per previously reported procedures.30, 31, 38, 40 Detailed
protocols for the assays are presented in the Supporting Information.
Cell information for compound evaluation.
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The ESKAPE bacteria were purchased from the American Type Culture Collection
(ATCC): E. faecium (Orla-Jensen) Schleifer and Kilpper-Balz strain NCTC 7171 (ATCC
19434); S. aureus subsp. aureus Rosenbach strain Seattle 1945 (ATCC 25923); Multi-drug
resistant S. aureus (MRSA) subsp. aureus Rosenbach strain HPV107 (ATCC BAA-44); K.
pneumonia, subsp. pneumoniae (Schroeter) Trevisan strain NCTC 9633 (ATCC 13883); A.
baumannii Bouvet and Grimont strain 2208 (ATCC 19606); P. aeruginosa (Schroeter) Migula
strain NCTC 10332 (ATCC 10145); E. cloacae, subsp. cloacae (Jordan) Hormaeche and
Edwards strain CDC 442-68 (ATCC 13047). Human THLE-3 liver cells (ATCC CRL-11233)
and HEK 293 kidney cells (ATCC CRL-1573) were used for the cell viability assays.
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Evaluation of compounds for inhibition of bacterial cell proliferation.
All compounds were evaluated for inhibiting the proliferation of each of the ESKAPE
bacteria as per previously reported procedures.38 Detailed protocols for bacterial growth assays
are presented in the Supporting Information.
Evaluation of compound effects on HEK 293 and THLE-3 cell viability.
All compounds were evaluated for cytotoxicities to THLE-3 liver and HEK 293 kidney
cells using Alamar Blue-based viability assays as per previously reported procedures.31, 38, 40
Detailed protocols for these assays are presented in the Supporting Information.
Evaluation of MRSA resistance generation against lead inhibitors.
To identify potential resistance toward compounds 1 and 11, a liquid culture, 12-day
serial passage assay was employed as per previously reported procedures, and using the ATCC
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BAA-44 MRSA strain.38, 43, 45 A detailed protocol for this assay is presented in the Supporting
Information.
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S. aureus Biofilm Prevention Assay.
The biofilm prevention assay was carried out with S. aureus Rosenbach (ATCC 25923)
using a quantitative crystal violet-based adherence assay on 96-well plates as described
previously by Kwasny et al.46 S. aureus (ATCC 25923) bacteria were streaked onto a Tryptic
Soy Broth (TSB) agar plate and grown overnight at 37°C. A fresh aliquot of TSB media was
inoculated with a single bacterial colony and the cultures were grown overnight at 37°C with
shaking (250 rpm). The overnight culture was then sub-cultured (1:5 dilution) into a fresh
aliquot of TSB media supplemented to a final concentration of 0.5% glucose and grown at 37°C
for 1 h with shaking, then diluted into fresh TSB media supplemented with 0.5% glucose to
achieve a final OD600 reading of 0.01. Aliquots of the diluted culture (100 µL) were dispensed to
96 well polystyrene plates along with addition of 1 µL test compounds in DMSO. The inhibitor
concentration range during the assay was 100 µM to 46 nM (3-fold dilution series). A second set
of baseline control plates were prepared analogously, but without any bacteria added, to correct
for possible compound absorbance and/or precipitation. Plates were sealed with "Breathe Easy"
oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking
(stagnant assay) until the biofilm was formed. After 24 h, the planktonic cultures were removed
and the plates were washed gently 2-3 times with 200 μl of water. Next, the plates were air dried
and the adherent biofilms were stained with 150 μL of crystal violet solution (2.3% crystal violet
in 20% Ethanol, Sigma Aldrich #HT90132) for 15 minutes at R.T. The unbound crystal violet
stain was removed, then plates were gently washed again with running water and air dried for 10
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min. Quantitative assessment of biofilm formation was obtained by adding 100 μL of developer
solution (4:1:5 mixture of MeOH:AcOH:H2O) per well. Well absorbance was then read at 595
nm using a Molecular Devices SpectraMax Plus384 microplate reader. EC50 values for the test
compounds were obtained by plotting the A595 nm results in GraphPad Prism 6 and analyzing by
non-linear regression using the log(inhibitor) vs. response (variable slope) equation. Results
presented represent the averages of EC50 values obtained from at least triplicate experiments.
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S. aureus Biofilm Penetration and Bactericidal Activity Assay.
The biofilm penetration and bactericidal activity assay was carried out with S. aureus
Rosenbach (ATCC 25923) as described previously by Kwasny et al.46 S. aureus bacteria were
streaked onto a Tryptic Soy Broth (TSB) agar plate and grown overnight at 37°C. A fresh
aliquot of TSB media was inoculated with a single bacterial colony and the cultures were grown
overnight at 37°C with shaking (250 rpm). The overnight culture was then sub-cultured (1:5
dilution) into a fresh aliquot of TSB media supplemented with 0.5% glucose and grown at 37°C
for 1 h with shaking, then diluted into fresh TSB media supplemented with 0.5% glucose to
achieve a final OD600 reading of 0.01. Aliquots of the diluted culture (100 µL) were dispensed to
96 well polystyrene plates without any compounds added. A second set of control plates were
prepared analogously, but without any bacteria added. Plates were sealed with "Breathe Easy"
oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking
(stagnant assay) until biofilm was formed. After 24 h, the planktonic cultures (or media blanks
in the control plates) were removed and the plates were washed gently 3 times with 200 μL of
sterile phosphate buffered saline (PBS). Then, 100 µL aliquots of fresh TSB media were
dispensed to the plates along with addition of 1 µL of test compounds in DMSO. The inhibitor
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concentration range during the assay was 100 µM to 46 nM (3-fold dilution series). The plates
were sealed with "Breathe Easy” membranes and incubated at 37°C without shaking to allow
compounds to penetrate and kill bacteria in the biofilms. After 24 h, the cultures were removed
and plates were washed again gently 3 times with 200 μL of sterile PBS. The remaining bacteria
in the biofilms were allowed to recover by adding 100 μL of fresh TSB media per well and
incubating for 24 h at 37°C. At the end of this final incubation, bacterial growth was monitored
by measuring the OD600 nm using a Molecular Devices SpectraMax Plus384 microplate reader.
EC50 values for the test compounds were obtained by plotting the OD600 nm results in GraphPad
Prism 6 and analyzing by non-linear regression using the log(inhibitor) vs. response (variable
slope) equation. Results presented represent the averages of EC50 values obtained from at least
triplicate experiments.
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Calculation of IC50 / EC50 / CC50 values and statistical considerations.
All IC50 / EC50 / CC50 results reported are averages of values determined from individual
dose-response curves in assay replicates as follows: 1) Individual I/E/CC50 values from assay
replicates were first log-transformed and the average log(I/E/CC50) values and standard
deviations (SD) calculated; 2) Replicate log(I/E/CC50) values were evaluated for outliers using
the ROUT method in GraphPad Prism 6 (Q of 10%); and 3) Average I/E/CC50 values were then
back-calculated from the average log(I/E/CC50) values. To compare log(I/E/CC50) values
between different assays, two-tailed Spearman correlation analyses were performed using
GraphPad Prism 6 (95% confidence level). For compounds where log(I/E/CC50) values were
greater than the maximum compound concentrations tested (i.e. >2.0, or >100 M), results were
represented as 0.1 log units higher than the maximum concentrations tested (i.e. 2.1, or 126 M),
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so as not to overly bias comparisons because of the unavailability of definitive values for these
inactive compounds.
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Corresponding Author
* E-mail: johnstm@iu.edu, Phone: 317-274-2458, Fax: 317-274-4686.
ACKNOWLEDGMENTS
Research reported in this publication was supported by the National Institute of General Medical
Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number
R01GM120350. QQH and YP additionally acknowledge support by NIH grants
5R01GM111639 and 5R01GM115844. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the NIH. This work was also supported
in part by startup funds from the IU School of Medicine (SMJ) and the University of Arizona
(EC). The human HSP60 expression plasmid (lacking the 26 amino acid N-terminal
mitochondrial signal peptide) was generously donated by Dr. Abdussalam Azem from Tel Aviv
University, Faculty of Life Sciences, Department of Biochemistry, Israel.
ABBREVIATIONS
MDH, malate dehydrogenase; Rho, rhodanese; IC50 - Inhibitory concentration for half-maximal
signal in biochemical assay; EC50, effective concentration for half-maximal signal in bacterial
proliferation assays; CC50, cytotoxicity concentration for half-maximal signal in human cell
viability assays.
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SUPPORTING INFORMATION
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Supporting Information associated with this article can be found in the online version,
which includes tabulation of all log(IC50), log(EC50), and log(CC50) results with standard
deviations; synthetic protocols and characterization data for test compounds (MS, 1H-NMR, and
HPLC purity); experimental protocols for protein synthesis and purification, and biochemical,
bacterial proliferation, and human cell viability assays; and SMILES strings of compound
structures.
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