Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB)inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Article abstract of DOI:10.1016/j.bmcl.2019.04.034

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10)chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB)virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.

Full text of DOI:10.1016/j.bmcl.2019.04.034

Accepted Manuscript
Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB)
inhibitors: A polypharmacology strategy for treating Mycobacterium tubercu-
losis infections
Alex Washburn, Sanofar Abdeen, Yulia Ovechkina, Anne-Marie Ray, Mckayla
Stevens, Siddhi Chitre, Jared Sivinski, Yangshin Park, James Johnson, Quyen
Q. Hoang, Eli Chapman, Tanya Parish, Steven M. Johnson
PII:
S0960-894X(19)30254-9
https://doi.org/10.1016/j.bmcl.2019.04.034
BMCL 26401
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 March 2019
20 April 2019
23 April 2019
Please cite this article as: Washburn, A., Abdeen, S., Ovechkina, Y., Ray, A-M., Stevens, M., Chitre, S., Sivinski,
J., Park, Y., Johnson, J., Hoang, Q.Q., Chapman, E., Parish, T., Johnson, S.M., Dual-targeting GroEL/ES chaperonin
and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium
tuberculosis infections, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.04.034
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dual-targeting GroEL/ES chaperonin and protein
tyrosine phosphatase B (PtpB) inhibitors: A
polypharmacology strategy for treating Mycobacterium
tuberculosis infections
AUTHOR NAMES
Alex Washburn,a Sanofar Abdeen,a Yulia Ovechkina,b Anne-Marie Ray,a Mckayla Stevens,a Siddhi
Chitre,a Jared Sivinski,c Yangshin Park,a,d,e James Johnson,b Quyen Q. Hoang,a,d,e Eli Chapman,c Tanya
Parish,b and Steven M. Johnsona*
AUTHOR ADDRESSES
a Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, 635
Barnhill Dr., Indianapolis, IN 46202
b Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102
c The University of Arizona, College of Pharmacy, Department of Pharmacology and Toxicology, 1703
E. Mabel St., PO Box 210207, Tucson, AZ 85721
d Stark Neurosciences Research Institute, Indiana University School of Medicine. 320 W. 15th Street,
Suite 414, Indianapolis, IN 46202
e Department of Neurology, Indiana University School of Medicine. 635 Barnhill Drive, Indianapolis, IN
46202
*Correspondence: johnstm@iu.edu, Tel: 317-274-2458, Fax: 317-274-4686
KEYWORDS. GroEL, GroES, HSP60, HSP10, molecular chaperone, chaperonin, proteostasis,
phosphatases, small molecule inhibitors, Mycobacterium tuberculosis, antibiotics, polypharmacology.
1

Abstract
Current treatments for Mycobacterium tuberculosis infections require long and complicated
regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains,
and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve
tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting
molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two
GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma
formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping
chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB)
virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the
present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in
liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting
GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating
bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease –
which would be a significant advance to augment current therapeutics that primarily target actively
replicating bacteria.
2

Mycobacterium tuberculosis, the causative agent of tuberculosis, infects about 1 in 4 people
worldwide. In 2017, over 10 million active cases of tuberculosis infection were reported, with 1.6
million deaths attributed to this deadly disease.1 M. tuberculosis is a facultative intracellular bacterium
that is transmitted as an airborne particulate generated when people with active tuberculosis cough or
sneeze. When inhaled, the bacteria traverse the respiratory tract to alveoli where they infect phagocytic
cells, namely macrophages.2-4 The bacteria replicate within the macrophage and induce cytokines that
initiate an inflammatory response in the lungs. Macrophages and lymphocytes migrate to the site of
infection and form granulomas, which lead to asymptomatic latent disease where pools of bacteria can
reside in dormant states for years. However, a weakened immune system can lead to re-activation of
dormant M. tuberculosis from the granulomas, allowing bacteria to once again actively replicate, which
can be fatal if left untreated.
First-line antibiotics to treat tuberculosis include isoniazid, rifampicin, ethambutol, and
pyrazinamide.1, 3, 5 Unfortunately, these antibiotics are most effective against actively-replicating
bacteria, which is problematic since M. tuberculosis replicates slowly (18-54 h doubling times) and can
persist in granulomas in a more metabolically inactive state.6 To increase treatment efficacy,
combination therapy is typically administered for at least six-to-nine months. This extensive regimen
often leads to patient non-compliance, which contributes to the development of antibiotic resistant
strains. To help combat drug resistance, two new drugs have recently been approved – bedaquiline and
delamanid.7, 8 Unfortunately, resistance is also arising to these two drugs.9-11 Thus, new drug candidates
are needed that act on previously unexploited targets and pathways not predisposed to resistance, and
that are additionally effective against the asymptomatic latent phase of infections.
A new paradigm in antibiotic research is to exploit protein homeostasis pathways, such as
targeting molecular chaperones.12-24 A network of molecular chaperones and proteases collectively
functions to maintain protein homeostasis by assisting proteins to fold to their native, functional states,
or ensuring their proper degradation.25-31 In particular, all organisms contain at least one homolog of the
3

60 kDa class of molecular chaperone (HSP60) that is essential under all conditions. Thus, targeting
HSP60 chaperonin systems with small molecule inhibitors should be an effective antibacterial strategy.
Escherichia coli GroEL is the prototypical member of the HSP60 chaperonin family, which has been
studied extensively. GroEL is a homo-tetradecameric protein that forms two, seven-subunit rings that
stack back-to-back with one another. Through a series of events driven by ATP binding and hydrolysis,
unfolded substrate polypeptides are bound within the central cavity of a GroEL ring and are
encapsulated by the GroES co-chaperonin lid, allowing protein folding within the sequestered chamber.
We refer readers to these other studies for a more detailed understanding of the structure and function of
GroEL/ES chaperonin systems.32, 33
M. tuberculosis has two GroEL homologs, GroEL1 and GroEL2, which interestingly diverge in
their sequences and share 61% amino acid identity with each other.34-40 GroEL2 is the putative
housekeeping molecular chaperone for folding proteins as it is essential for M. tuberculosis survival and
contains the GGM repeat motif found in the canonical chaperonins of other organisms.35 Thus, targeting
GroEL2 with small molecule inhibitors should be an effective strategy to kill actively replicating M.
tuberculosis. While GroEL1 is not essential, it is important in regulating cytokine-dependent granuloma
formation.35, 38 When infected with a strain of M. tuberculosis deficient in GroEL1 (∆cpn60.1), both
mice and guinea pigs produced equal numbers of bacteria as the WT-GroEL1 strain, but the mutant
strain failed to produce granulomatous inflammation.35 Thus, targeting GroEL1 with small molecule
inhibitors might be effective at preventing the formation of granulomas that contribute to latent disease,
or targeting bacteria that are already present in established granulomas. These enticing findings suggest
that a small molecule that could concurrently inhibit both GroEL1 and GroEL2 in M. tuberculosis might
be effective at treating active and latent stages of tuberculosis, which would be superior to current
tuberculosis therapeutics.
Towards our goal of exploiting HSP60/10 and GroEL/ES chaperonin systems as an antibiotic
strategy, we previously reported a high-throughput screen for small molecule inhibitors of the E. coli
4

GroEL/ES chaperonin system.41 This initial study identified compound 1 as one of our most potent
GroEL/ES inhibitors (Figure 1A). Our subsequent study found 1 lacked any appreciable antibacterial
efficacy against a panel of Gram-positive and Gram-negative bacteria known as the ESKAPE pathogens
– an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species.42 However, our third
study identified isostructural benzoxazole analogs (2-14, Figure 1B) that exhibit antibiotic effects
against Trypanosoma brucei parasites, the causative agents of African sleeping sickness.43 We refer to
these as “pseudosymmetric full-molecules” as they have two sulfonamide end-capping groups with R1
substructures being identical on both the right and left-hand sides of the molecule. Furthermore, our
fourth study identified 2-chlorothiophene-based analogs (15, 16R-34R, and 16L-34L, Figure 1C) that
exhibited antibiotic effects against Gram-positive bacteria, in particular Staphylococcus aureus and
methicillin-resistant S. aureus (MRSA).44 We refer to these as “asymmetric full-molecules” as they
have two sulfonamide end-capping groups, but the substructures differ between the Right- and Left-
hand sides of the molecule (i.e. R- and L-series full-molecules). Since the M. tuberculosis GroEL1 and
GroEL2 homologs share 52% and 59% sequence identity with E. coli GroEL, respectively, we reasoned
that they may also exhibit antibiotic effects against M. tuberculosis. Thus, in the present study, we
evaluated the previously developed compound 1 analogs for their ability to inhibit M. tuberculosis
growth in liquid media (see discussion below). Because we were unsure whether or not these
compounds required both of the sulfonamide end-capping groups to maintain potent inhibition, we
developed a new series that we term “half-molecules”, as they contain only one sulfonamide end-
capping group on either the Right- or Left-hand sides of the molecule (R- and L-series half-molecules,
Figure 1D). We also wanted to determine whether simplifying inhibitors in this manner might reduce
the cytotoxicity of this series to human cells, while still maintaining antibiotic efficacy.
5

Figure 1. Progression of compound 1 analog development and evaluation through our recent studies.41-
In each panel, the R1-R3 groups represent a variety of alkyl, aryl, and ortho-, meta-, and para-
44
substituted phenyl substructures (refer to the Tables S1-S6 in the Supporting Information for specific R-
substructures). Each successive study (panels A-C) included testing of compounds developed from the
previous studies in order to more thoroughly characterize SAR. Panel D presents compounds newly
developed in the current study, which we term “half-molecules” as they contain only one sulfonamide
end-capping group on either the Right- or Left-hand sides of the molecules (R- and L-series half-
6

molecules). We have evaluated these, as well as all the previously-developed “full-molecules” (i.e. two
sulfonamide end-capping groups as shown in panels A-C), for their ability to inhibit the proliferation of
M. tuberculosis, as well as the protein tyrosine phosphatase B (PtpB) virulence factor that they secrete
into macrophages to evade host cell immune responses.
Another possible strategy to treat tuberculosis infections is by targeting a virulence factor that M.
tuberculosis secretes into the cytoplasm of host macrophages, protein tyrosine phosphatase B (PtpB).
By secreting PtpB into the cytosol of host macrophages, M. tuberculosis disrupts host cell immune
responses by blocking ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by
interfering with Akt signaling.45 Deletion of PtpB has been shown to block intracellular survival of M.
tuberculosis in IFN-γ activated macrophages and reduce the bacterial load in a guinea pig model.45, 46
Several studies have shown the possibility of selectively inhibiting M. tuberculosis PtpB over human
phosphatases, which can result from structural differences such as PtpB containing alpha-helices that
can occlude the active site, yet are mobile enough to allow substrate and competitive inhibitors to
bind.45, 47-54 Furthermore, previous studies have demonstrated the feasibility of inhibiting M.
tuberculosis PtpB with small molecules and reducing bacterial loads in macrophages.45, 47, 48
In their 2007 study, Grundner et al. reported a crystal structure of PtpB in complex with the
selective inhibitor (oxalylamino-methylene)-thiophene sulfonamide (OMTS – Figure 2A).50 In that
structure, two OMTS molecules were found bound in the crystal structure, with the sulfonamides of
each interacting with a number of ordered water molecules and binding site residues (e.g. Arg59, Arg63,
His94, Glu129, and Arg136). Examining the crystal structure, we were intrigued that the sulfonamides
of each OMTS molecule reside ~11-12 Å apart from one another (Figure 2B). Since the two
sulfonamides of our compound 1 analogs are also ~11-12 Å apart (albeit in a head-to-head
configuration, whereas the OMTS sulfonamides are head-to-tail), we envisioned that our analog series
7

might also be capable of inhibiting M. tuberculosis PtpB in a conformation that would bridge across the
two OMTS binding sites, making similar polar interactions as OMTS, while positioning the sulfonamide
end-capping groups upwards to what would be the phosphotyrosine active site catalytic residues and
adjacent pocket. This raises the enticing possibility that this class of molecules could inhibit the two M.
tuberculosis GroEL homologs to target actively replicating mycobacteria and dormant mycobacteria in
granulomas, as well as inhibit PtpB to target intracellular M. tuberculosis that are evading host cell
immune responses. Thus, this class of inhibitors could be effective against all stages of M. tuberculosis
infection. The present study was designed to explore such dual-targeting GroEL/ES and PtpB inhibitors
in vitro and in cell culture, with future studies envisioned to build from the established structure-activity
relationships (SAR) to further optimize lead candidates for evaluating in animal infection models.
Figure 2. A. Grundner et al. previously reported that the OMTS compound was a potent and selective
inhibitor of M. tuberculosis PtpB.50 B. Two OMTS molecules were found occupying the active site of
M. tuberculosis PtpB (PDB ID 2OZ5). On examination, the sulfonamides of each OMTS molecule
reside ~11-12 Å apart, similar to our compound 1 analogs. Thus, we envisioned compound 1 analogs
might be able to bind in a manner that bridges the distal and proximal parts of the active site. In such a
conformation, the sulfonamides could interact with the binding site similarly to those of OMTS, with the
R-substructures pointing upwards to fill the binding cavity and engaging the catalytic residues located in
the proximal part of the active site.
8

Extending from our previous studies that explored the bis-sulfonamide full-molecule series
(compounds 1-34 – see Tables S1-S4 in the Supporting Information), we synthesized a library of half-
molecule analogs that had the R3-substituted sulfonamide end caps on either the Right (R-series) or Left
(L-series) sides of the 2-phenylbenzoxazole core (Scheme 1).43, 44 Representative synthetic protocols
for the sulfonamide coupling and methoxy-to-hydroxy deprotection reactions are presented in the
Supporting Information along with complete compound characterizations (1H-NMR, MS, and RP-
HPLC). These syntheses resulted in the development of 58 new half-molecules – 29 each of the R- and
L-series analogs. As we previously found that aryl-sulfonamides were the most potent GroEL/ES
inhibitors, we developed analogs bearing substituted phenyl-sulfonamides. Furthermore, we biased the
analogs to contain a variety of halide substituents and substitution patterns as our previous antibacterial
study indicated that halide-bearing compounds were typically more effective at inhibiting bacterial
proliferation than compounds with other substituents.44
Scheme 1a
a Reagents and conditions: (a) R3-SO2Cl, pyridine, CH2Cl2, rt; (b) BBr3, CH2Cl2, rt.
We next employed a series of established biochemical assays to evaluate the inhibitory effects of
the new half-molecules against the GroEL/ES chaperonin system.41-44, 55 For these assays, we used E.
coli GroEL/ES as a surrogate, as obtaining functional GroEL oligomeric rings from M. tuberculosis has
9

so far proven difficult.12, 36, 37, 39 However, we anticipate a high probability that inhibitors would bind to
and inhibit the GroEL/ES chaperonin systems in M. tuberculosis since E. coli GroEL shares 52%
identity with the GroEL1 homolog, and 59% identity with GroEL2. We employed two chaperonin-
mediated folding assays using malate dehydrogenase (MDH) and rhodanese (Rho) as the unfolded
reporter enzymes. We have described these assays elsewhere, and have included detailed protocols of
each in the Supporting Information.41-44, 55 Briefly, these are coupled assays where we monitor the
enzymatic activity of two reporter enzymes (MDH and Rho), which are denatured and then get
efficiently refolded by GroEL/ES in the absence of inhibition. We further counter-screened compounds
for their ability to inhibit native MDH and Rho to identify false-positives that inhibit the enzymatic
reporter reactions of the refolding assays. IC50 results for the testing of compounds in these assays are
shown in Tables S5A-S6A in the Supporting Information. The pseudosymmetric and asymmetric full-
molecules had been evaluated in these assays in our previous studies, and thus we refer readers to those
publications for a detailed account of their results.43, 44
As visualized in the correlation plot in Figure 3A, compounds were nearly equipotent at
inhibiting in both of the GroEL/ES-mediated folding assays (Spearman correlation coefficient is 0.923,
p < 0.0001). As SAR for the full-molecule series has been more thoroughly discussed for these assays
in our previous studies,43, 44 we will primarily present comparisons between the full vs. half-molecule
scaffolds herein. In this context, the full-molecules were dramatically more potent than the half-
molecules, indicating that the presence of both aryl-sulfonamide substructures are required for potent
inhibition. While some compounds were found to inhibit in either the native MDH or Rho reporter
activity counter-screens, only one previously reported compound (28R) inhibited in both native assays,
and only weakly (Figure 3B).43, 44 As the IC50 values for all the compounds in the native MDH and Rho
reporter counter-screens were much higher than in the corresponding refolding assays, these results
support that compounds were on target for inhibiting the GroEL/ES-mediated folding cycle.
10

Figure 3. Correlation plots of IC50 and EC50 values for compounds tested in the respective GroEL/ES-
dMDH and dRho refolding assays (A), native MDH and Rho reporter counter-screens (B), and M.
tuberculosis proliferation assay (C). Each data point represents results for an individual compound
(plotted from results presented in Tables S1-S6 in the Supporting Information). Full molecules (i.e.
compounds 1-34) are represented by blue circles, and half-molecules (i.e. compounds 35-51) are
represented by yellow squares. A & B. Compounds inhibit nearly equipotently in the GroEL/ES-dMDH
and -dRho refolding assays (Spearman correlation coefficient is 0.923, p < 0.0001), but are weak or
inactive in the native MDH or Rho enzymatic reporter counter-screens, supporting on-target effects for
inhibiting chaperonin-mediated substrate refolding. As indicated in panel A, we consider compounds
with IC50 values plotted in the grey zones to be inactive (i.e. greater than the maximum concentrations
tested), >30 M to be weak inhibitors, 10-30 M moderate inhibitors, 1-10 M potent inhibitors, and <1
M very potent and acting near stoichiometrically since the concentration of GroEL subunits is 700 nM.
C. Some GroEL/ES inhibitors exhibit weak-to-moderate inhibition of M. tuberculosis proliferation, but
the correlation with inhibiting GroEL/ES refolding functions in vitro is weak (Spearman correlation
coefficient is 0.439, p = 0.0011). Compounds showing <50% inhibition of M. tuberculosis proliferation
when tested at a single 200 μM concentration were excluded from this analysis.
11

We next evaluated how effective our GroEL/ES inhibitors would be at inhibiting the growth of
M. tuberculosis in liquid culture, as inhibiting the essential GroEL2 homolog should be bactericidal. We
tested the ability of each compound to inhibit growth at a fixed concentration of 200 µM after 5 days
(see the Supporting Information for the assay protocol).56 Compounds exhibiting >50% inhibition were
then tested as serial dilutions in order to determine EC50 values, defined as the concentration at which
growth was inhibited by 50%. Percent inhibition and EC50 results are presented in Tables S1-S6 in the
Supporting Information. Intriguingly, while the half-molecules were twice as likely to exhibit >50%
inhibition at the single 200 µM test concentration (28/58, or 48%) compared to the full-molecules
(22/89, or 25%), the only analogs with EC50s <100 µM were the full molecules. This latter finding was
not entirely surprising since the full-molecules were much more potent GroEL/ES inhibitors than the
half-molecules. While this coarse SAR helps support that inhibitors are on target for GroEL/ES in
mycobacteria, we note only a weak correlation between M. tuberculosis EC50 results compared to IC50
results for the GroEL/ES-dMDH refolding assay (Figure 3C – Spearman correlation coefficient is
0.439, p = 0.0011). More thorough experiments need to be conducted in the future to conclusively
determine the mechanism of action in cells. While the most potent compounds exhibited only moderate
EC50s in the 20-30 µM range, these were encouraging results since this was the first exploratory study
we have conducted to see whether or not GroEL/ES inhibitors would have antibiotic efficacy against M.
tuberculosis. Thus, the current results have provided valuable insights to guide future pharmacological
optimization studies.
A potential caveat to targeting GroEL/ES chaperonin systems as an antibiotic strategy is that
human cells contain homologous machinery in their mitochondria. Human mitochondrial HSP60 shares
48% sequence identity with E. coli GroEL (similar to the M. tuberculosis GroEL1 and GroEL2
homologs), which raises the possibility of potential HSP60-dependent cytotoxicity to human cells.
However, as we continue to study this and other chaperonin inhibitor scaffolds, we become less and less
concerned with potential cytotoxicities associated with targeting human HSP60/10. In many instances,
12

compounds that we have found to inhibit HSP60/10 in vitro exhibit low-to-no cytotoxicity when tested
in our liver and kidney cell viability assays.42, 43, 55 Furthermore, we have recently identified numerous
known drugs and natural products that are able to inhibit HSP60/10 in vitro.43, 55, 57 For example, while
we found that suramin can inhibit human HSP60/10 in vitro, it has been safely used for over 100 years
as a first-line therapeutic for treating African sleeping sickness, which is caused by Trypanosoma brucei
parasites. Nonetheless, we continue to counter-screen compounds in our standard HSP60/10-dMDH
refolding assay as it does prove useful for helping to understand inhibitor mechanisms of action at the
protein level. This assay is analogous to the GroEL/ES-dMDH assay so that IC50 results can be directly
compared between the two chaperonin systems. A detailed protocol is presented in the Supporting
Information, with results for the half-molecules presented in Tables S5A-S6A. For an account of results
for the pseudosymmetric and asymmetric full-molecules, we refer readers to our previous studies.43, 44
As we observed with GroEL/ES, the full-molecules were more potent at inhibiting HSP60/10
than the half-molecules (Figure 4A). While some compounds were nearly equipotent between the two
chaperonin systems, many exhibited selectivity towards E. coli GroEL/ES. We previously noted this
SAR for the pseudosymmetric (1-15) and R-series asymmetric full molecules (16R-34R), demonstrating
that it is possible to tune this scaffold to selectively target GroEL/ES.43, 44 However, as noted above, this
may be inconsequential for cytotoxicity purposes as the mitochondrial membrane is highly impermeable
to penetration by small molecules, and thus inhibitors may never reach HSP60/10 in the mitochondrial
matrix.
As a moderate-throughput first-pass indicator of cellular toxicity, we typically employ two
Alamar Blue-based cell viability assays, where compounds are incubated with human liver (THLE-3)
and kidney (HEK 293) cells over a 48 h time period. A detailed protocol for these assays is presented in
the Supporting Information, with cell viability results (cytotoxicity CC50 values) presented in Tables
S5B-S6B. We again refer readers to our previous studies for cell viability CC50 results for the
pseudosymmetric and asymmetric full-molecules (compounds 1-34).43, 44 Similar to results from the
13

biochemical assays, the full-molecules were more potent inhibitors (i.e. more cytotoxic) than the half-
molecules were in the two cell viability assays (Figure 4B). However, we note that there is a poor
correlation when comparing IC50 and CC50 results from the HSP60/10-dMDH and cell viability assays
(Figure 4C – Spearman correlation coefficient is 0.244, p = 0.0027 when comparing with liver cells),
which could indicate off-target effects not related to inhibiting the HSP60/10 chaperonin system, and/or
potential cell or mitochondrial permeability differences between compounds. These possibilities
highlight that results from the cell viability assays are the critical indicators of potential cytotoxic effects
that may be encountered in vivo.
Figure 4. Correlation plots of IC50 and CC50 values for compounds tested in the respective GroEL/ES-
dMDH and HSP60/10-dMDH refolding assays (A), and liver/kidney cell viability assays (B & C). A.
While many compounds inhibit human HSP60/10 nearly as well as E. coli GroEL/ES (Spearman
correlation coefficient for all molecules is 0.740, p < 0.0001), we previously reported being able to tune
for selectivity with the pseudosymmetric and asymmetric R-series full molecules.43, 44 B. Cytotoxicities
of compounds are comparable between human THLE-3 liver and HEK 293 kidney cells (Spearman
correlation coefficient is 0.857, p < 0.0001). C. HSP60/10 inhibitors exhibit weak-to-moderate
cytotoxicity to human THLE-3 liver cells, with a low correlation evident between the two assays
(Spearman correlation coefficient is 0.244, p = 0.0027).
14

While the above results support the feasibility of identifying GroEL/ES inhibitors that can kill
actively replicating M. tuberculosis, we were particularly intrigued by the possibility of this series also
being able to target the protein tyrosine phosphatase B (PtpB) virulence factor that intracellular M.
tuberculosis secrete into macrophages to evade host cell immune responses. To investigate this
possibility, we obtained a His-tagged version of M. tuberculosis PtpB, which was previously developed
by Grundner et al., to generate recombinantly-expressed and purified enzyme.49, 50 Following
previously reported procedures by Zhou et al., which monitored for phosphatase activity using para-
nitrophenyl phosphate (pNPP), we evaluated all compounds in dose-response format to obtain IC50
values.45, 47, 49, 50 As a preliminary indication of selectively targeting M. tuberculosis PtpB, we counter-
screened against three human phosphatases, PTPN1 (PTP1B), PTPN2 (TCPTP), and PTPN5 (STEP),
using analogous procedures. Detailed protocols are presented in the Supporting Information for these
assays, with IC50 results for all compounds presented in Tables S1-S6.
While both the full- and half-molecules were able to inhibit M. tuberculosis PtpB phosphatase
activity, as noted above for the other assays, the full-molecules were generally more potent than the half-
molecules (Figure 5A). An interesting discovery was that, while the full-molecules all inhibit M.
tuberculosis PtpB activity with increasing compound concentrations, most half-molecules exhibit a bi-
phasic response first activating, then inhibiting phosphatase activity (Figure 5B). This could be the
result of two half-molecules binding to PtpB in a manner like that previously observed for OMTS.50
Thus, binding of the first molecule at the distal part of the active site may open the alpha-helical lid,
allowing pNPP to more effectively occupy the proximal part next to the catalytic residues. Then at
higher concentrations, the second half-molecule could competitively displace the pNPP, thereby
showing inhibition. It should be noted, though, that this may be an artifact of this particular assay
protocol using pNPP, as binding to either site would theoretically compete with a phosphorylated
15

peptide. Thus, IC50 values could be more potent in a physiological context than what we have reported
(presuming binding affinities were high enough), although future studies would need to confirm this.
Figure 5. A. Correlation plot of IC50 values for compounds tested in the GroEL/ES-dMDH refolding
and M. tuberculosis PtpB phosphatase assays (Spearman correlation coefficient is 0.756, p < 0.0001).
16

B. Dose-response curves for analogs 2h-m, R51-m, and L51-m evaluated in the M. tuberculosis PtpB
phosphatase assay. While the full-molecules all inhibit M. tuberculosis PtpB phosphatase activity with
increasing compounds concentrations, most half-molecules exhibit a bi-phasic response where
compounds activate then inhibit phosphatase activity, to varying extents, as shown for analogs R51-m
and L51-m. Data point errors represent standard deviations from 6-8 replicates.
While we were excited to see so many GroEL/ES inhibitors were also able to potently inhibit
PtpB, we were intrigued that such a high correlation was observed between the two data sets (Spearman
correlation coefficient is 0.756, p < 0.0001) as there is no a priori reason why the binding sites of GroEL
and PtpB would be similar. However, we note that the SAR is relatively flat for the full-molecules for
inhibiting PtpB (i.e. most are within the 1-10 μM range), which could be consistent with the bis-
sulfonamide core predominantly contributing to binding affinity and the end-capping groups not fully
occupying the remainder of the active site. This is important since, on the whole, this scaffold class
exhibits only slight selectivity for M. tuberculosis PtpB over the three human phosphatases (Figure 6).
Thus, with extra space that compounds could more fully occupy in the active site, there should be room
to optimize inhibitor potency and selectivity profiles against M. tuberculosis PtpB. This also boosts
confidence that there should be ample chemical space to simultaneously optimize inhibition of
GroEL/ES and PtpB function to target M. tuberculosis in both active and latent stages of infection. Such
optimization studies will be the focus of future investigations.
17

Figure 6. Correlation plots of IC50 values for compounds tested against the M. tuberculosis PtpB
phosphatase compared to the human PTPN1 (PTP1B, panel A), PTPN2 (TCPTP, panel B), and PTPN5
(STEP, panel C). While some individual compounds are able to selectively inhibit the M. tuberculosis
PtpB over the human phosphatases, on the whole, this scaffold class only exhibits a slight selectivity for
the bacterial phosphatase – Spearman correlation coefficients are 0.828 (p < 0.0001) for PTPN1, 0.755
(p < 0.0001) for PTPN2, and 0.786 (p < 0.0001) for PTPN5.
In summary, in the present study, we evaluated whether or not a series of GroEL/ES inhibitors,
based on the compound 1 scaffold, would be effective at inhibiting the growth of M. tuberculosis.
Furthermore, we envisioned that these could be dual-targeting molecules capable of inhibiting the M.
tuberculosis GroEL/ES chaperonin systems as well as the PtpB virulence factor that they secrete into
macrophages. This study identified 14 GroEL/ES inhibitors that exhibited EC50 values <100 μM against
M. tuberculosis growth in liquid culture (Table 1). We have categorized these into three groups,
depending on their selectivity profiles for inhibiting GroEL/ES over human HSP60/10, as well as for
inhibiting M. tuberculosis PtpB over the panel of three human phosphatases. Compounds within the
blue category exhibit selectivity for both GroEL/ES and PtpB; compounds within the green category
exhibit selectivity for either GroEL/ES or PtpB, but not both; and compounds in the yellow category are
18

not inherently selective. From our continued studies, though, we note that even if compounds can
inhibit human HSP60/10 in vitro, many exhibit only low or no cytotoxicity to human cells.
Furthermore, as PtpB is highly adaptable to compounds that bind within the active site, there should still
be ample chemical space to optimize inhibitors for binding affinity, efficacy, and selectivity.
Two of the lead compounds we identified were 20R and 20L, which had primary amines either
on the right or left-hand sides of the scaffold. These were unique compounds within this study as they
were the only analogs to contain primary amines that would be charged under physiological conditions.
Thus, future studies will explore new analogs that contain amines on either the right or left-hand
sulfonamides, while varying groups on the other side. The 2h-o/m/p analogs were also unique lead
molecules – the only hydroxylated full-molecule analogs – to which a similar SAR strategy will be
applied. In either case, additional areas of exploration to optimize inhibitors are varying the
sulfonamide linkers and employing scaffolds other than the 2-phenylbenzoxazole core. By doing so, we
are optimistic that we will be able to enhance the potency and selectivity of lead inhibitors for M.
tuberculosis GroEL/ES chaperonin systems and PtpB, and increase their selectivity indices for inhibiting
M. tuberculosis proliferation over cytotoxicity to human cells. Once we have developed more viable
lead candidates, we will pursue additional studies looking at the effects of inhibiting M. tuberculosis
PtpB in macrophage models, as well as antibiotic efficacy in an in vivo infection model. With an
antibiotic strategy that concomitantly inhibits the two M. tuberculosis GroEL homologs and PtpB, these
polypharmacologic agents are predicted to target actively replicating bacteria, intracellular bacteria that
are evading host cell immune responses, and dormant bacteria that have formed granulomas. Thus, we
are hopeful that this could be an effective strategy for treating all stages of tuberculosis.
Table 1. Biochemical and cell-based assay results for lead analogs exhibiting M. tuberculosis
proliferation EC50 values <100 μM. Fold-selectivities for inhibiting GroEL/ES over human HSP60/10,
and M. tuberculosis PtpB over the three human phosphatases, are shown in brackets in the shaded
19

columns. Compounds colored blue exhibit >5x selectivities for both GroEL and PtpB; compounds
colored green exhibit >5x selectivities for either GroEL or PtpB (but not both); and compounds colored
yellow exhibit between 1-5x selectivities for GroEL or PtpB.
Cell Assay EC50 and CC50 (M)
Biochemical Assay IC50 (M)
Mtb
Liver
Kidney
GroEL/ES- HSP60/10-
dMDH Refolding
Mtb
Human
Compound Structure & Number
Proliferation
Cell Viability
PtpB
PTPN1 PTPN2 PTPN5
(37x)
(8.0x)
(18x)
(20x)
(7.3x)
(20x)
(61x)
(2.3x)
(2.3x)
(1.6x)
(1.4x)
(2.9x)
(1.4x)
(3.1x)
20R
20L
2h-m
2a
26
59
84
28
34
66
94
63
36
40
43
45
58
66
26
28
40
22
39
22
46
19
43
31
75
40
69
25
23
29
37
19
31
29
57
19
39
34
55
31
58
24
1.3
4.6
0.97
4.0
5.5
3.3
0.23
7.7
4.0
5.6
35
48
37
17
80
40
67
14
18
9.1
9.0
49
57
13
43
9.1
(6.2x)
(9.5x)
(25x)
56
42
38
24
31
27
27
23
13
14
37
36
15
52
91
61
84
29
41
28
75
39
31
28
35
31
28
95
75
62
47
23
33
23
35
25
23
24
27
26
24
52
4.4
1.5
18
(1.3x)
(2.6x)
(1.3x)
(2.3x)
(10x)
6
12
2b-p
2h-p
2h-o
17L
18L
2k-p
2b-o
33R
16R
18
12
2.3
5.1
8.8
12
(2.5x)
(1.6x)
(2.3x)
(1.6x)
(2.5x)
(1.4x)
20
16
9.3
14
5.9
37
20

Supporting Information: Supporting information associated with this article can be found in the online
version, which includes tabulation of all biochemical IC50, bacterial proliferation EC50, and human cell
viability CC50 results; log(IC50), log(EC50), and log(CC50) results with standard deviations; synthetic
protocols and characterization data for test compounds (HPLC purity, MS, and 1H-NMR); experimental
protocols for protein synthesis and purification, and all biochemical and cell-based assays.
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 by startup funds from the IU
School of Medicine (SMJ) and the University of Arizona (EC). Technical assistance from Megha
Gupta, Junitta Guzman, and Aaron Korkegian (IDRI, Seattle, WA) is also greatly appreciated. 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. We thank Dr. Zhong-Yin Zhang (Purdue University, West
Lafayette, IN, USA) for providing the M. tuberculosis PtpB expression plasmid, with permission from
Dr. Christoph Grundner (Center for Infectious Disease Research, Seattle, WA, USA), who originally
developed the plasmid.
21

References
1. World Health Organization. Global Tuberculosis Report 2018; Geneva, Switzerland: World Health
Organization, 2018.
2. Flynn, J. L.; Chan, J. Infect. Immun. 2001, 69, 4195.
3. Pai, M.; Behr, M. A.; Dowdy, D.; Dheda, K.; Divangahi, M.; Boehme, C. C.; Ginsberg, A.;
Swaminathan, S.; Spigelman, M.; Getahun, H.; Menzies, D.; Raviglione, M. Nat. Rev. Dis. Primers
2016, 2, 16076.
4. Smith, I. Clin. Microbiol. Rev. 2003, 16, 463.
5. World Health Organization. Treatment of Tuberculosis: Guidelines.; Geneva, Switzerland: World
Health Organization, 2010.
6. Gill, W. P.; Harik, N. S.; Whiddon, M. R.; Liao, R. P.; Mittler, J. E.; Sherman, D. R. Nat. Med.
2009, 15, 211.
7. D'Ambrosio, L.; Centis, R.; Tiberi, S.; Tadolini, M.; Dalcolmo, M.; Rendon, A.; Esposito, S.;
Migliori, G. B. J. Thorac. Dis. 2017, 9, 2093.
8. Migliori, G. B.; Pontali, E.; Sotgiu, G.; Centis, R.; D'Ambrosio, L.; Tiberi, S.; Tadolini, M.;
Esposito, S. Int. J. Mol. Sci. 2017, 18.
9. Nguyen, T. V. A.; Anthony, R. M.; Banuls, A. L.; Nguyen, T. V. A.; Vu, D. H.; Alffenaar, J. C.
Clin. Infect. Dis. 2018, 66, 1625.
10. Fujiwara, M.; Kawasaki, M.; Hariguchi, N.; Liu, Y.; Matsumoto, M. Tuberculosis (Edinb) 2018,
108, 186.
11. Stinson, K.; Kurepina, N.; Venter, A.; Fujiwara, M.; Kawasaki, M.; Timm, J.; Shashkina, E.;
Kreiswirth, B. N.; Liu, Y.; Matsumoto, M.; Geiter, L. Antimicrob. Agents Chemother. 2016, 60,
3316.
12. Lupoli, T. J.; Vaubourgeix, J.; Burns-Huang, K.; Gold, B. ACS Infect. Dis. 2018, 4, 478.
13. Kumar, A.; Balbach, J. Sci. Rep. 2017, 7, 42141.
22

14. Chiappori, F.; Fumian, M.; Milanesi, L.; Merelli, I. Plos One 2015, 10, e0124563.
15. Arita-Morioka, K.; Yamanaka, K.; Mizunoe, Y.; Ogura, T.; Sugimoto, S. Antimicrob. Agents
Chemother. 2015, 59, 633.
16. Sass, P.; Josten, M.; Famulla, K.; Schiffer, G.; Sahl, H. G.; Hamoen, L.; Brotz-Oesterhelt, H. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 17474.
17. Evans, C. G.; Chang, L.; Gestwicki, J. E. J. Med. Chem. 2010, 53, 4585.
18. Piper, P. W.; Millson, S. H. Open Biol. 2012, 2, 120138.
19. Liebscher, M.; Jahreis, G.; Lucke, C.; Grabley, S.; Raina, S.; Schiene-Fischer, C. J. Biol. Chem.
2007, 282, 4437.
20. Otvos, L., Jr.; O, I.; Rogers, M. E.; Consolvo, P. J.; Condie, B. A.; Lovas, S.; Bulet, P.; Blaszczyk-
Thurin, M. Biochemistry 2000, 39, 14150.
21. Otvos, L., Jr.; Wade, J. D.; Lin, F.; Condie, B. A.; Hanrieder, J.; Hoffmann, R. J. Med. Chem. 2005,
48, 5349.
22. Kragol, G.; Hoffmann, R.; Chattergoon, M. A.; Lovas, S.; Cudic, M.; Bulet, P.; Condie, B. A.;
Rosengren, K. J.; Montaner, L. J.; Otvos, L., Jr. Eur. J. Biochem. 2002, 269, 4226.
23. Kragol, G.; Lovas, S.; Varadi, G.; Condie, B. A.; Hoffmann, R.; Otvos, L., Jr. Biochemistry 2001,
40, 3016.
24. Mohammadi-Ostad-Kalayeh, S.; Hrupins, V.; Helmsen, S.; Ahlbrecht, C.; Stahl, F.; Scheper, T.;
Preller, M.; Surup, F.; Stadler, M.; Kirschning, A.; Zeilinger, C. Bioorg. Med. Chem. 2017, 25,
6345.
25. Chapman, E.; Farr, G. W.; Usaite, R.; Furtak, K.; Fenton, W. A.; Chaudhuri, T. K.; Hondorp, E. R.;
Matthews, R. G.; Wolf, S. G.; Yates, J. R.; Pypaert, M.; Horwich, A. L. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 15800.
26. Hartl, F. U.; Bracher, A.; Hayer-Hartl, M. Nature 2011, 475, 324.
27. Hartl, F. U.; Hayer-Hartl, M. Science 2002, 295, 1852.
23

28. Stefani, M.; Dobson, C. M. J. Mol. Med. (Berl.) 2003, 81, 678.
29. Maisonneuve, E.; Ezraty, B.; Dukan, S. J. Bacter. 2008, 190, 6070.
30. Carmichael, J.; Chatellier, J.; Woolfson, A.; Milstein, C.; Fersht, A. R.; Rubinsztein, D. C. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 9701.
31. Bao, Y. P.; Cook, L. J.; O'Donovan, D.; Uyama, E.; Rubinsztein, D. C. J. Biol. Chem. 2002, 277,
12263.
32. Horwich, A. L.; Fenton, W. A.; Chapman, E.; Farr, G. W. Annu. Rev. Cell. Dev. Biol. 2007, 23,
115.
33. Horwich, A. L.; Farr, G. W.; Fenton, W. A. Chem. Rev. 2006, 106, 1917.
34. Kong, T. H.; Coates, A. R. M.; Butcher, P. D.; Hickman, C. J.; Shinnick, T. M. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 2608.
35. Hu, Y. M.; Henderson, B.; Lund, P. A.; Tormay, P.; Ahmed, M. T.; Gurcha, S. S.; Besra, G. S.;
Coates, A. R. M. Infect. Immun. 2008, 76, 1535.
36. Qamra, R.; Mande, S. C.; Coates, A. R.; Henderson, B. Tuberculosis (Edinb) 2005, 85, 385.
37. Qamra, R.; Srinivas, V.; Mande, S. C. J. Mol. Biol. 2004, 342, 605.
38. Hu, Y. M.; Coates, A. R. M.; Liu, A.; Lund, P. A.; Henderson, B. Tuberculosis 2013, 93, 442.
39. Fan, M.; Rao, T.; Zacco, E.; Ahmed, M. T.; Shukla, A.; Ojha, A.; Freeke, J.; Robinson, C. V.;
Benesch, J. L.; Lund, P. A. Mol. Microbiol. 2012, 85, 934.
40. Henderson, B.; Lund, P. A.; Coates, A. R. Tuberculosis (Edinb) 2010, 90, 119.
41. Johnson, S. M.; Sharif, O.; Mak, P. A.; Wang, H. T.; Engels, I. H.; Brinker, A.; Schultz, P. G.;
Horwich, A. L.; Chapman, E. Bioorg. Med. Chem. Lett. 2014, 24, 786.
42. Abdeen, S.; Salim, N.; Mammadova, N.; Summers, C. M.; Frankson, R.; Ambrose, A. J.; Anderson,
G. G.; Schultz, P. G.; Horwich, A. L.; Chapman, E.; Johnson, S. M. Bioorg. Med. Chem. Lett. 2016,
26, 3127.
24

43. Abdeen, S.; Salim, N.; Mammadova, N.; Summers, C. M.; Goldsmith-Pestana, K.; McMahon-Pratt,
D.; Schultz, P. G.; Horwich, A. L.; Chapman, E.; Johnson, S. M. Bioorg. Med. Chem. Lett. 2016,
26, 5247.
44. Abdeen, S.; Kunkle, T.; Salim, N.; Ray, A. M.; Mammadova, N.; Summers, C.; Stevens, M.;
Ambrose, A. J.; Park, Y.; Schultz, P. G.; Horwich, A. L.; Hoang, Q. Q.; Chapman, E.; Johnson, S.
M. J. Med. Chem. 2018, 61, 7345.
45. Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.; Wang, Y.; Franzblau, S. G.; Yang, Z.; Chan, R. J.;
Liu, Y.; Zheng, J.; Zhang, Z. Y. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4573.
46. Singh, R.; Rao, V.; Shakila, H.; Gupta, R.; Khera, A.; Dhar, N.; Singh, A.; Koul, A.; Singh, Y.;
Naseema, M.; Narayanan, P. R.; Paramasivan, C. N.; Ramanathan, V. D.; Tyagi, A. K. Mol.
Microbiol. 2003, 50, 751.
47. Chen, L.; Zhou, B.; Zhang, S.; Wu, L.; Wang, Y.; Franzblau, S. G.; Zhang, Z. Y. ACS Med. Chem.
Lett. 2010, 1, 355.
48. Beresford, N. J.; Mulhearn, D.; Szczepankiewicz, B.; Liu, G.; Johnson, M. E.; Fordham-Skelton,
A.; Abad-Zapatero, C.; Cavet, J. S.; Tabernero, L. J. Antimicrob. Chemother. 2009, 63, 928.
49. Grundner, C.; Ng, H. L.; Alber, T. Structure 2005, 13, 1625.
50. Grundner, C.; Perrin, D.; Hooft van Huijsduijnen, R.; Swinnen, D.; Gonzalez, J.; Gee, C. L.; Wells,
T. N.; Alber, T. Structure 2007, 15, 499.
51. Chiaradia, L. D.; Martins, P. G.; Cordeiro, M. N.; Guido, R. V.; Ecco, G.; Andricopulo, A. D.;
Yunes, R. A.; Vernal, J.; Nunes, R. J.; Terenzi, H. J. Med. Chem. 2012, 55, 390.
52. Mascarello, A.; Mori, M.; Chiaradia-Delatorre, L. D.; Menegatti, A. C.; Delle Monache, F.; Ferrari,
F.; Yunes, R. A.; Nunes, R. J.; Terenzi, H.; Botta, B.; Botta, M. Plos One 2013, 8, e77081.
53. Mascarello, A.; Chiaradia-Delatorre, L. D.; Mori, M.; Terenzi, H.; Botta, B. Curr. Pharm. Design
2016, 22, 1561.
25

54. Mascarello, A.; Orbem Menegatti, A. C.; Calcaterra, A.; Martins, P. G. A.; Chiaradia-Delatorre, L.
D.; D'Acquarica, I.; Ferrari, F.; Pau, V.; Sanna, A.; De Logu, A.; Botta, M.; Botta, B.; Terenzi, H.;
Mori, M. Eur. J. Med. Chem. 2018, 144, 277.
55. Kunkle, T.; Abdeen, S.; Salim, N.; Ray, A. M.; Stevens, M.; Ambrose, A. J.; Victorino, J.; Park, Y.;
Hoang, Q. Q.; Chapman, E.; Johnson, S. M. J. Med. Chem. 2018, 61, 10651.
56. Ollinger, J.; Bailey, M. A.; Moraski, G. C.; Casey, A.; Florio, S.; Alling, T.; Miller, M. J.; Parish, T.
Plos One 2013, 8, e60531.
57. Stevens, M.; Abdeen, S.; Salim, N.; Ray, A. M.; Washburn, A.; Chitre, S.; Sivinski, J.; Park, Y.;
Hoang, Q. Q.; Chapman, E.; Johnson, S. M. Bioorganic & medicinal chemistry letters 2019, 29,
1106.
26

Table of Contents Graphic
27