Discovery of potent wall teichoic acid early stage inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.06.090

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with β-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure–activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant Staphylococci.

Full text of DOI:10.1016/j.bmcl.2016.06.090

Accepted Manuscript
Discovery of Potent Wall Teichoic Acid Early Stage Inhibitors
Marc A. Labroli, John P. Caldwell, Christine Yang, Sang Ho Lee, Hao Wang,
Sandra Koseoglu, Paul Mann, Shu-Wei Yang, Jing Xiao, Charles G. Garlisi,
Christopher Tan, Terry Roemer, Jing Su
PII:
S0960-894X(16)30701-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.090
BMCL 24043
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 May 2016
28 June 2016
29 June 2016
Please cite this article as: Labroli, M.A., Caldwell, J.P., Yang, C., Lee, S.H., Wang, H., Koseoglu, S., Mann, P.,
Yang, S-W., Xiao, J., Garlisi, C.G., Tan, C., Roemer, T., Su, J., Discovery of Potent Wall Teichoic Acid Early Stage
Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.090
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Discovery of Potent Wall Teichoic Acid
Early Stage Inhibitors
Leave this area blank for abstract info.
Marc A. Labroli*, John P. Caldwell*, Christine Yang, Sang Ho Lee, Hao Wang, Sandra Koseoglu, Todd
Mayhood, Charles Gill, Paul Mann, Sookhee Ha, Shu-Wei Yang, Lianzhu Liang, Paul Tawa, Yan Hou, Reshma
Kuvelkar, Kristine DeVito, Xiujuan Wen, Jing Xiao, Carl J. Balibar, Jenny Liu, Jianying Xiao, Nicholas
Murgolo, Charles G. Garlisi, Payal Sheth, Amy Flattery, Christopher Tan, Terry Roemer, and Jing Su
Herein we descirbe the optimization of a series of inhibitors termed tarocins which demonstrate by genetic and biochemical means inhibition of
TarO, the first step in wall teichoic acid (WTA) biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad
spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci.
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com

Bioorganic & Medicinal Chemistry Letters
Discovery of Potent Wall Teichoic Acid Early Stage Inhibitors
Marc A. Labroli*, John P. Caldwell*, Christine Yang, Sang Ho Lee, Hao Wang, Sandra Koseoglu, Paul
Mann, Shu-Wei Yang, Jing Xiao, Charles G. Garlisi, Christopher Tan, Terry Roemer, and Jing Su
Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has
dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for
novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified
small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to
be chemically synergistic with β-lactams. These previously disclosed inhibitors, termed
tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in
WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad
spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant
Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will
be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop
Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant
Staphylococci.
Keywords:
Keywords
Antibacterial
Wall teichoic acid
Antibiotic resistance
MRSA
2016 Elsevier Ltd. All rights reserved.
Methicillin-resistant Staphylococcus aureus (MRSA)
and the conditionally essential WTA late stage genes which are
responsible for WTA synthesis and eventual transport to the
and methicillin-resistant Staphylococcus epidermidis (MRSE) are
a major cause of bloodstream infections in the hospital and in the
community1-4 with MRSA being reported as the second leading
cause of mortality by drug-resistant bacterial pathogens in the
United States. Alarmingly, the rise in MRSA and MRSE
infections has conicided with an decline in the efficacy of β-
lactam antibiotics1-4. Numerous reports, including our previous
disclosures, detail a β-lactam combination agent strategy applied
to methicillin resistant Staphylococci which was contingent upon
the identification of new targets and pathways that normally
buffer the drug resistant pathogen from the effects of β-lactams
(either directly or indirectly) and that if inactivated by genetic or
small molecule inhibition, result in restored sensitization to this
important class of antibiotics5-10. A series of reports has shown
that inactivation or alteration of wall teichoic acid (WTA)
production causes MRSA to become highly susceptible to β-
lactam antibiotics both in vitro as well as in a murine infection
and offers an opportunity to test our β-lactam combination agent
extracellular environment17-24
.
Although Staphylococci without
WTA are viable, inactivation of late-stage WTA biosynthetic
steps results in a growth inhibitory bacteriostatic effect14,17,21,23
.
The essentiality of the late-stage genes can be suppressed by
concurrent inactivation of one of the non-essential WTA early
stage genes (Figure 1). This finding is an example of the
essential gene paradox20,21,23,24,27
.
Figure 1. Effect of TarO and TarG inhibition on wall teichoic acid
biosynthesis and S.aureus growth
We used the essential gene paradox to identify late-
stage WTA inhibitors whose antibacterial activity reproduces the
conditional essentiality of late-stage WTA genes in a phenotypic
screen17,26. These compounds should inhibit wild-type S. aureus
growth yet their activity is suppressed if an early non-essential
step in WTA biosynthesis is also inactivated. In this way,
multiple classes of late stage WTA inhibitors were identified,
including targocil and L-638, which both inhibit polymer
synthesis by targeting the WTA integral membrane transporter
strategy5, 11-15
.
WTA is
a
Gram-positive specific anionic
glycophosphate polymer comprising nearly 50% of the dry
weight of the S. aureus cell wall that is cross-linked to the
peptidoglycan and has critical roles in cell growth, division,
morphology and in vivo virulence12,17,18. The WTA biosynthetic
pathway is categorized into two phenotypically distinct groups;
the non-essential WTA early-stage genes tarO, tarA and mnaA,
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com

subunit, TarG 17,26,29,30 (Figure 1). Recently, we have disclosed a
phenotypic screening strategy which takes advantage of the
essential gene paradox in order to identify early-stage inhibitors
of wall teichoic acid production29. Furthermore, inhibition of
TarO should sensitize MRSA to β-lactam antibiotics29.
Table 1. WTA profile of amide analogs
Compounds from this phenotypic screen were
prioritized for follow up studies provided that they: i) were
synergistic in combination with imipenem below its clinical
breakpoint (4 µg/mL); ii) conferred resistance to cell lysis
mediated by the Staphylococcal-specific lytic bacteriophage ØK
(“PhageK”) that utilizes WTA as its receptor for bacteriophage
entry into the cell27; and iii) demonstrated a dose-dependent
suppression of the late-stage inhibitor L-63828. From our
compound collection of several million compounds we found two
compounds, an oxazolidinone (named “tarocin A”) and a
benzimidazole (named “tarocin B”, 1a)29, that met all pathway-
specific criteria. The SAR investigation of the “tarocin A” series
will be disclosed in due course. We investigated the SAR around
1a in an effort to improve potency, physicochemcial properties
and potentiation with a β-lactam.
COL
EC50
Targocil
Reversal
(M)
MITC95
+IPM
(M)
EC50
PhageK
M)
#
R1
R2
cLogP
(R)-(-)-
1.6
7.9
2.4
6.9
6.9
1a
1b
-H
-H
isonorbornyl
(R)-(+)-
bornyl
16.7
178.5
57.7
50
4.7
1.9
12.5
6.2
4.7
25
200
200
10.1
200
200
25.0
200
200
200
6.2
29.7
7.8
11.4
138.3
5.7
6.6
6.0
5.5
6.6
5.5
5.1
2
3
4
5
6
7
8
- Ph
-Et
-Et
-Et
-Et
-nPr
-CH3
-H
-c-heptyl
-c-hexyl
-c-pentyl
-c-hexyl
-c-hexyl
-c-hexyl
In the cellular assays, the initial lead 1a when combined
with 4 µg/mL of imipenem (IPM) exhibited synergistic effects
with an MITC95 = 1.56 M31, conferred resistance to PhageK-
mediated cell lysis (EC50 = 7.9 M), and suppressed the
inhibition of targocil (EC50 = 2.4 M). When tested without
imipenem, 1a was inactive in these cellular assays; therefore,
potentiating the effect of imipenem as expected from a WTA
early-stage inhibitor. Initially we investigated the SAR of the
amide region to explore the structural requirements to retain the
potency of 1a. Analogs were prepared in a parallel fashion using
standard amidation conditions as shown in Scheme 1 from the
carboxylic acid precursor. Select analogs are highlighted in
Table 1. There was a stereochemical preference for one of the
bornyl analogs over the other ((-)-1a vs. (+)-1b) (Table 1). In
general, aromatic substitution was not well tolerated as illustrated
by 2 with decreased potency with a MITC95 = 50 M and
inactivity (EC50 ≥ 200) in the PhageK lysis and targocil reversal
assays. However, several cycloalkyl tertiary amides (3–7) were
well tolerated in terms of imipenem potentiation. We were
gratified to find that compound 4 approached the potency of 1a
(MITC95 = 1.95 M) but also showed good activity in the
PhageK lysis and targocil reversal assays. Other than 1a, most
secondary amides explored were not well tolerated in terms of
potency with the exception of compound 8, possessing a
cyclohexyl ring, which had a modest MITC95 of 25 M.
However, 8 was inactive in the PhageK lysis and targocil reversal
assays. In general, the incorporation of polar, hydrophilic
moieties was not tolerated nor were aromatic and heteroaromatic
groups (data not shown). Indeed, even subtle modifications of
this tertiary amide led to notable drops in activity, especially in
the PhageK lysis and targocil reversal assays.
Compounds 9–19 were prepared as shown in Scheme 2.
Initial studies were focused on the isonorbornyl amide series
(Table 2). Unfortunately, it appeared that hydrophobic groups
were necessary to retain potency since the introduction of
polarity via the piperidine analogs 9–13 typically led to a
significant loss of activity. Turning our attention to the N'-
cyclohexyl, N'-ethyl amide subseries (Table 3), we found that the
introduction of 4′-alkylcyclohexyl substituents led to the most
potent compounds within this subseries (14, 15) in terms of
MITC95 potencies, PhageK lysis and targocil reversal activities.
However, this increased potency came at the expense of an
increasing hydrophobicity (15, cLogP 7.9).
Based on the
necessity of a hydrophobic portion in the amide region, we
sought to improve the overall physicochemical properties
through the introduction of polarity at the 2-position of the
benzimidazole. Gratifyingly, we were able to retain some of the
nanomolar potency from compound 15 while introducing polarity
through amination of the benzimidizole bromide thereby
reducing the cLogP by a log unit resulting in 16 (cLogP 6.8). An
attempt to remove the 4′-alkyl substituent altogether (17, cLogP
5.0) resulted in a drastic loss of activity in all three assays.
Activity was reclaimed across all three assays by installation of a
4′-ethyl substituent, 18 (cLogP 6.1). Truncation of the 4′-ethyl to
the 4′-methyl (19, cLogP 5.5) resulted in a modest loss in
activity. Here again, activity in the assays improved at the
expense of increased hydrophobicity.
Scheme 1. Reagents and conditions: HATU, DIEA, amine, DMF, room
temperature.
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com

Table 3. WTA profile of C2-substituted analogs
COL
MITC95
+IPM
(M)
EC50
Phage
K
EC50
Targocil
Reversal
(M)
#
R
M)
cLogP
0.03
0.015
0.016
4.7
0.22
0.25
0.25
>200
0.39
3.8
0.24
0.05
0.13
7.0
7.1
14
7.9
6.8
5.0
6.1
5.5
15
16
17
18
19
0.10
1.3
0.22
0.91
After the initial SAR exploration on two regions of the
compound, it appeared that activity was dependent on increasing
hydrophobicity. This finding is consistent with our finding that
our TarO inhibitors are likely binding by hydrophobic
interactions with an extracellularly exposed hydrophobic pocket
of TarO determined through a TarO resistance map with a protein
model31. Finally, we turned our attention to the benzimidazole
core for additional opportunities to introduce polarity. Originally
we intended to use bromobenzimidazoles to serve as functional
handles for further derivatization through metal catalyzed
coupling reactions. However, most of the modifications only
improved potency at the expense of increased hydrophobicity
(data not shown). Upon testing, bromides 20–22 were active
across the cell assays (Table 4). The 4-methyl analog, 23,
although showing a modest decrease in cellular potency, did
show a profound improvement in coverage against both 25
MRSA clinical isolates and 24 MRSE clinical isolates. It is
noteworthy that compounds which display nanomolar potency
against the COL strain (cf. 16, 20, and 21), do not necessarily
possess broad coverage against MRSE strains.
Scheme 2. Reagents and conditions: a) Boc2O, CH2Cl2; b) Michael acceptor,
K2CO3, DMF, 120 C; c) TFA, CH2Cl2; d) N-derivatization; e) PPA, 200 C;
f) amine, DMF, 140 C.
Table 2. WTA profile of C2-substituted analogs
Table 4. WTA profile of benzimidazole analogs
COL
MITC95
+IPM
(M)
EC50
EC50
Phage Targocil
K
Reversal
COL
EC50
% MRSA
MITC95
% MRSE
MITC95
#
9
R
M)
(M)
cLogP
MITC95
EC50
Targocil
9.4
162
58.7
>200
>200
6.5
+IPM PhageK Reversal
(M) M) (M)
0.031 0.24 0.33
≤ 25 M
for 25 isolates
≤ 25 M
for 24 isolates
#
cLogP
8.0
>200
>200
>200
>200
4.5
5.5
10
11
68%
80%
68%
68%
76%
42%
46%
83%
92%
29%
20
21
22
23
16
0.004
0.3
0.027
3.4
0.015
1.3
8.0
8.0
0.1
0.9
0.8
7.6
0.016
0.25
0.13
7.6
>200
>200
>200
>200
>200
>200
4.2
3.6
12
13
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com

During the course of this exploration, we were also able to
discover compounds which could tolerate polar modifications.
Finally, through elaboration of the benzimidazole region, we
discovered very active inhibitors of WTA and serendipitously
found compounds active among a broad spectrum of clinical
isolates of both MRSA and MRSE. Unfortunately, most of these
gains in potency were made at the expense of adding lipophilicity
to the compound. This has significant implications since it is
expected that in a clinical setting a WTA inhibitor would be
coadministered intravenously in combination with an existing
antibiotic. Further work detailing efforts to improve the
physicochemical properties of this tarocin series will be
presented in due course.
To summarize, through a high throughput phenotypical
cell-based screen we were able to identify several potential early
stage inhibitors of wall teichoic acid biosynthesis. We initiated an
SAR campaign and interrogated three major regions of the initial
pharmacophore 1a. We successfully found a suitable replacement
for the isonorbornyl amide and were able to produce compounds
which displayed nanomolar activity in the cell-based assays.
References and notes
1.
2.
Bancroft, E. A.; J. Am. Med. Assoc. 2007, 298, 1803.
Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D. ; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009,
48, 1.
3.
4.
Johnson, A. P.; J. Antimicrob. Chemother. 2011, 66(4), 43.
Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.;
Zell, E. R.; Fosheim, G. E.; McDougal, L. K.; Carey, R. B.; Fridkin, S. K. J. Am. Med. Assoc., 2007, 298, 1763.
Lee, S. H.; Jarantow, L. W.; Wang, H.; Sillaots, S.; Cheng, H.; Meredith, T. C.; Thompson, J.; Roemer, T. Chem. Biol. 2011, 18, 1379.
Tan, C. M.; Therien, A. G.; Lu, J.; Lee, S. H.; Caron, A.; Gill, C. J.; Lebeau-Jacob, C.; Benton-Perdomo, L.; Monteiro, J. M.; Pereira, P. M.; Elsen, N.
L.; Wu, J.; Deschamps, K.; Petcu, M.; Wong, S.; Daigneault, E.; Kramer, S.; Liang, L.; Maxwell, E.; Claveau, D.; Vaillancourt, J.; Skorey, K.; Tam,
J.; Wang, H.; Meredith, T. C.; Sillaots, S.; Wang-Jarantow, L.; Ramtohul, Y.; Langlois, E.; Landry, F.; Reid, J. C.; Parthasarathy, G.; Sharma, S.;
Baryshnikova, A.; Lumb, K. J.; Pinho, M. G.; Soisson, S. M.; Roemer, T. Sci. Trans. Med. 2012, 4, 126.
Huber, J.; Donald, R. G.; Lee, S. H.; Jarantow, L. W.; Salvatore, M. J.; Meng, X.; Painter, R.; Onishi, R. H.; Occi, J.; Dorso, K.; Young, K.; Park, Y.
W.; Skwish, S.; Szymonifka, M. J.; Waddell, T. S.; Miesel, L.; Phillips, J. W.; Roemer, T. Chem. Biol. 2009, 16, 837.
de Lencastre, H.; Wu, S. W.; Pinho, M. G.; Ludovice, A. M.; Filipe, S.; Gardete, S.; Sobral, R.; Gill, S.; Chung, M.; Tomasz, A. Microb. Drug Res.
1999, 5, 163.
5.
6.
7.
8.
9.
Berger-Bachi, B.; Rohrer, S. Arch. Microbiol. 2002, 178, 165.
10. Roemer, T.; Schneider, T.; Pinho, M. G. Curr. Opin. Microbiol. 2013, 16, 538.
11. Brown, S.; Xia, G.; Luhachack, L. G.; Campbell, J.; Meredith, T. C.; Chen, C.; Winstel, V.; Gekeler, C.; Irazoqui, J. E.; Peschel, A.; Walker, S. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 18909.
12. Campbell, J.; Singh, A. K.; Santa Maria, Jr. J. P.; Kim, Y.; Brown, S.; Swoboda, J. G.; Mylonakis, E.; Wilkinson, B. J.; Walker, S. ACS Chem. Biol.
2011, 6, 106.
13. Farha, M. A.; Leung, A.; Sewell, E. W.; D'Elia, M. A.; Allison, S. E.; Ejim, L.; Pereira, P. M.; Pinho, G.; Wright, G. D.; Brown, E. D. ACS Chem.
Biol. 2012, 8, 226.
14. Maki, H.; Yamaguchi, T.; Murakami, K. J. Bacteriol. 1994, 176, 4993.
15. Wang, H.; Gill, C. J.; Lee, S. H.; Mann, P.; Zuck, P.; Meredith, T. C.; Murgolo, N.; She, X.; Kales, S.; Liang, L.; Liu, J.; Wu, J.; Santa Maria, J.; Su,
J.; Pan, J.; Hailey, J.; McGuinness, D.; Tan, C. M.; Flattery, A.; Walker, S.; Black, T.; Roemer, T. Chem. Biol. 2013, 20, 272.
16. Weidenmaier, C.; Peschel, A. Nat. Rev. Microbiol. 2008, 6, 276.
17. Swoboda, J. G.; Campbell, J.; Meredith, T. C.; Walker, S. Chembiochem 2010, 11, 35.
18. D'Elia, M. A.; Henderson, J. A.; Beveridge, T. J.; Heinrichs, D. E.; Brown, E. D. J. Bacteriol. 2009, 191, 4039.
19. D'Elia, M. A.; Millar, K. E.; Beveridge, T. J.; Brown, E. D. J. Bacteriol. 2006, 188, 8313.
20. D'Elia, M. A.; Millar, K. E.; Bhavsar, A. P.; Tomljenovic, A. M.; Hutter, B.; Schaab, C.; Moreno-Hagelsieb, G.; Brown, E. D. Chem. Biol. 2009, 16,
548.
21. D'Elia, M. A.; Pereira, M. P.; Chung, Y. S.; Zhao, W.; Chau, A.; Kenney, T. J.; Sulavik, M. C.; Black, T. A.; Brown, E. D. J. Bacteriol. 2006, 188,
4183.
22. Sewell, E. W.; Brown, E. D. J. Antibiot. 2014, 67, 43.
23. Wolff, K.; Mann, P.; Fischmann, T.; Müller, A.; Wang, H.; Hou, Y.; Xiao, J.; Murgolo, N.; Sher, X.; Reed, P.; Sheth, P.; Mirza, A.; Labroli, M.;
McCoy, M.; Pinho, M. G.; Gill, C. J.; Schneider, T.; Roemer, T.
24. Swoboda, J. G.; Meredith, T. C.; Campbell, J.; Brown, S.; Suzuki, T.; Bollenbach, T.; Malhowski, A. J.; Kishony, R.; Gilmore, M. S.; Walker, S. ACS
Chem. Biol. 2009, 4, 875.
25. D'Elia, M. A.; Pereira, M. P.; Brown, E. D. Trends Microbiol. 2009, 17, 433.
26. Schirner, K.; Stone, L. K.; Walker, S. ACS Chem. Biol. 2011, 6, 407.
27. A. N. Chatterjee, Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus. J. Bacteriol.
1969, 98, 519. For the bacteriophage ØK (ATCC 19685-B1) resistance assay, 5x105 cfu/mL of MRSA COL was infected with bacteriophage ØK
(final MOI=0.4) and mixed with increasing concentrations of test compounds, and EC50 was calculated compared to DMSO control.
28. Pasquina, L. W.; Santa Maria, J. P.; Walker, S. Curr. Opin. Microbiol. 2013, 16, 531. For TarG inhibitor suppression assay, 5x105 cfu/mL of MRSA
COL was mixed with increasing concentrations of test compound in combination with L-638 (27) 4 µg/mL (2x MIC), after 20 hrs of incubation at
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com

37°C, OD600 was read and percent suppression is calculated compared to DMSO control.
29. Lee, S. H.; Wang, H.; Labroli, M.; Koseoglu, S.; Zuck, P.; Mayhood, T.; Gill, C.; Mann, P.; Sher, X.; Ha, S.; Yang, S.-W.; Mandal, M.; Yang, C.;
Liang, L.; Tan, Z.; Tawa, P.; Hou, Y.; Kuvelkar, R.; DeVito, K.; Wen, X.; Xiao, J.; Batchlett, M.; Balibar, C. J.; Liu, J.; Xiao, J.; Murgolo, N.; Garlisi,
C. G.; Sheth, P. R.; Flattery, A.; Su, J.; Tan, C.; Roemer, T. Sci. Transl. Med. 8, 329 (8), 329ra32.
30. Bacterial sources. S. aureus COL was obtained from Brian Wilkinson (Illinois State University), who has maintained the culture as a frozen stock
since 1976. The COL strain was reportedly isolated as a penicillinase-negative strain in the early 1960s from the operating theatre in a hospital in
Colindale, England. COL was one of the first MRSA isolates to be identified and has been used extensively in biochemical investigations of
methicillin and vancomycin resistance.
31. Compound 1a effectively inhibits TarO enzymatic activity, demonstrating a 50% inhibitory concentration at IC50= 0.4 µM. Compound 1a does not
inhibit either TarA or MnaA in vitro at the highest (200 µM) concentration tested demonstrating highly selective inhibitory activity towards TarO
within the WTA biosynthetic pathway. MITC95 is the concentration of compound that potentiated the action of antibiotic (imipenem) affecting 95%
inhibition of bacterial growth.
 Corresponding authors MAL e-mail: marc.labroli@merck.com; JPC e-mail: john.caldwell@merck.com