Potent Small-Molecule Suppression of Oxacillin Resistance in Methicillin-Resistant Staphylococcus aureus

Article abstract of DOI:10.1002/anie.201206911

Shields down. Adjuvant molecules that have the ability to restore the susceptibility of multi-drug-resistant bacteria, such as MRSA, to clinically available antibiotics are a promising alternative to the development of novel antimicrobials. Pictured is a

Full text of DOI:10.1002/anie.201206911

.
Angewandte
Communications
DOI: 10.1002/anie.201206911
Disarming Bacterial Resistance
Potent Small-Molecule Suppression of Oxacillin Resistance in
Methicillin-Resistant Staphylococcus aureus**
Tyler L. Harris, Roberta J. Worthington, and Christian Melander*
The emergence of resistance to multiple antimicrobial agents
in pathogenic bacteria is a significant global public health
threat and causes considerable patient mortality and morbid-
ity. In the United States, methicillin-resistant Staphylococcus
aureus (MRSA) accounts for 80% of all hospital-acquired S.
aureus infections.[1] In 2005, almost 95000 people acquired
MRSA infections in the United States, of which nearly 19000
people died — more than die annually from HIV/AIDS,
emphysema, Parkinsonꢀs disease, and homicide combined.[2]
Furthermore, MRSA infections, which are traditionally only
observed among hospitalized patients, have now become
prevalent outside of the hospital setting, with the emergence
of community-associated MRSA (CA-MRSA).[3] In the USA,
the USA300 clone is the most prevalent CA-MRSA clone.[3]
b-Lactam antibiotics have typically been the most effective
drugs for the treatment of infections caused by staphylococci;
however, increasing occurrence of resistance means that they
are often no longer efficacious.
biofilms of several pathogenic bacteria.[11,12] Some members
of this class of compounds also possess the ability to suppress
the resistance of planktonic bacteria to b-lactam antibiotics.[7]
We recently reported 2-AIT 1 (Scheme 1), which lowers the
minimum inhibitory concentration (MIC) of oxacillin against
Although the design of new antibiotics could address the
threat of multi-drug resistance, the sobering fact is that there
have been very few novel classes of antibiotics marketed in
the last four decades.[4] Furthermore, bacteria inevitably
develop resistance to all microbicidal agents that are intro-
duced into the clinic.[5] An orthogonal approach to the
development of new antibiotic entities is the use of small-
molecule adjuvants.[6] Recently, our group and others, have
been exploring the use of small molecules that are able to
render multi-drug resistant bacteria sensitive to the effects of
conventional antibiotics.[7–10] One of the key features of this
approach is the targeting of pathways within the bacteria that,
by themselves, are not essential for bacterial growth, by which
the rate of resistance acquisition may be significantly reduced.
We have developed a class of 2-aminoimidazole/triazole
conjugates (2-AIT) that are able to inhibit and disperse
Scheme 1. Synthesis of N1-substituted 2-AITs. Reagents and condi-
tions: a) tBuOK, THF, À788C–RT 72 h; b) N-(2-azidoethyl)-4-pentyl-
benzamide, CuSO4, sodium ascorbate, H2O/EtOH/CH2Cl2, RT, 3 h;
c) 2m HCl/Et2O, RT, 2 h; d) 1) RCHO, LiOH·H2O, MeOH, RT, 2 h,
2) NaBH4, RT, 1 h; e) Boc2O, Et3N, 1:1 dioxane/water, RT, 16 h; f) HN-
(OMe)Me·HCl, iPrMgCl, THF, À208C–RT, 18 h; g) DIBAL-H, THF,
À788C, 2 h; h) 9:1 CH2Cl2/TFA, RT, 15 min; i) H2O/EtOH, pH 4.3,
H2NCN, 958C, 3 h. Boc=tert-butoxycarbonyl, DIBAL-H=diisobutylalu-
minum hydride, TFA=trifluoroacetic acid.
[*] T. L. Harris, Dr. R. J. Worthington, Prof. Dr. C. Melander
Department of Chemistry, North Carolina State University
Raleigh, NC, 27695 (USA)
E-mail: ccmeland@ncsu.edu
[**] The authors thank the DOD DMRDP program (W81XWH-11-2-
0115) for support of this work. The DMRDP program is adminis-
tered by the Department of Army; The U.S. Army Medical Research
Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-
5014 is the awarding and administering office. The content of this
manuscript does not necessarily reflect the position or the policy of
the Government, and no official endorsement should be inferred.
NARSA isolates were obtained through the Network on Antimicro-
bial Resistance in Staphylococcus aureus (NARSA) program, sup-
ported by NIAID/NIH.
an Iberian MRSA clone when co-dosed at sub-MIC levels.[13]
Herein, we report that this compound exhibits similar activity
against a USA300 MRSA clone. Analogue synthesis resulted
in the identification of a significantly more active derivative
that reduced oxacillin MICs up to 512-fold, thus taking the
MIC significantly below the breakpoint for clinical resistance.
Screening this compound against a number of USA300
MRSA mutant strains indicates that the VraSR two-compo-
nent system (TCS) plays a role in the activity of this
compound.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206911.
11254
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 11254 –11257

Angewandte
Chemie
The MIC of compound 1 against a USA300 MRSA strain
(ATCC BAA-1556) was determined to be 50 mm
(23.7 mgmLÀ1) using the CLSI broth microdilution proto-
col.[14] We subsequently determined the oxacillin MIC in the
absence and presence of 40% of the MIC of compound
1 (20 mm, 9.5 mgmLÀ1). Compound 1 lowered the oxacillin
MIC by eight-fold at this concentration, from 32 mgmLÀ1 to
4 mgmLÀ1. With the aim of developing a compound with
increased activity, a series of analogues of compound 1, in
which substituents were placed at the N1-position of the
2-aminoimidazole, were synthesized using an approach that
we had previously developed.[15]
a marked increase in activity, lowering the oxacillin MIC by
128-fold, 64-fold, and 64-fold, respectively. The ability of the
pilot library to suppress oxacillin resistance in another CA-
MRSA USA300 strain (JE2) was next investigated (Table 1)
and activity for each compound against this strain was
virtually identical to that of BAA-1556.
Compound 7d, which possesses a 4-butylbenzyl substitu-
ent, was selected as the lead compound for further study,
owing to the considerably lower concentration at which it
displayed activity compared to 7k and 7l (5 mm compared to
40 mm and 20 mm respectively). A control compound, 8, which
possesses only the N1 substituent of compound 7d was
synthesized in the same manner as the pilot library and shown
to be devoid of both bactericidal activity (MIC > 200 mm) and
resistance suppression activity at concentrations as high as
50 mm. Time-kill curves were constructed for strain JE2
cultured in the presence of combinations of oxacillin and
compound 7d (Figure 1). Compound 7d, when dosed alone at
The reaction of a-amino ester 4[8] with N-(2-azidoethyl)-4-
pentylbenzamide[13] under Huisgen cycloaddition conditions
followed by deprotection delivered key intermediate 5
(Scheme 1). Diversity was introduced through reductive
amination of 5 with a variety of commercially available
benzaldehydes. Boc-protection (Boc = tert-butoxycarbonyl)
of the resulting secondary amines and subsequent conversion
of the ester to the corresponding Weinreb amide generated
intermediates 6a–n. Finally, each Weinreb amide was reduced
to the corresponding a-amino aldehyde with diisobutylalu-
minum hydride (DIBAL-H) which, following Boc-deprotec-
tion and cyclization with cyanamide, afforded the 1,5-
substituted 2-aminoimidazole/triazole conjugates 7a–n.
After purification, each compound was converted to the
corresponding HCl salt for biological screening.
As with 2-AIT 1, the MIC of each compound against
MRSA ATCC BAA-1556 was first established. The MIC of
oxacillin in the presence of 40% of the MIC of each
compound was then determined (Table 1). Previously, we
had shown that the introduction of an N1-substituent resulted
in increased antibiotic activity relative to the parent com-
pound.[15] As expected, this trend was followed in the
generation of this library. Most compounds exhibited a sig-
nificantly reduced or abrogated ability to lower the oxacillin
MIC. Three compounds however (7d, 7k, and 7l), displayed
Figure 1. Time-kill curves for USA300 MRSA strain JE2. Solid lines: no
2-AIT, broken lines: 5 mm 7d. Black: no oxacillin, blue: 64 mgmLÀ1
oxacillin, red: 16 mgmLÀ1 oxacillin, green: 4 mgmLÀ1 oxacillin, purple:
1 mgmLÀ1 oxacillin.
Table 1: MIC values and oxacillin resistance suppression activity against
MRSA USA300 strains.
Entry Compound 2-AIT conc. [mm][a]
Oxacillin MIC [mgmLÀ1
ATCC BAA-1556 NARSA JE2
]
1
2
3
4
5
6
7
8
–
1
32
4
32
4
32
16
32
5 mm (3.1 mgmLÀ1), is bactericidal at early time points (up to
8 h); however, bacterial growth is similar to that of the control
by the 24 h time point. When bacteria are cultured in the
presence of combinations of oxacillin and compound 7d,
a large reduction in the number of colony forming units
(CFU) is observed, as compared to treatment with oxacillin
alone. A considerable synergistic effect can be observed at the
24 h time point. At 5 mm concentration, compound 7d alone
effected a 1.08 log reduction in CFU after 24 h, and oxacillin
effected less than 0.4 log reduction at concentrations of
16 mgmLÀ1 and below. Combining 7d (5 mm) and oxacillin
resulted in log CFU reductions of 6.41, 5.54, and 4.38 for
oxacillin concentrations of 16, 4, and 1 mgmLÀ1 respectively.
Finally, we tested the ability of lead compound 7d at 5 mm to
suppress oxacillin resistance in eight additional MRSA
20
10
5
5
5
5
5
5
5
7a
7b
7c
7d
7e
7 f
7g
7h
7i
7j
7k
7l
7m
7n
8
32
32
32
0.25
32
16
32
4
16
4
0.5
0.5
32
4
0.5
32
16
32
16
32
8
0.5
0.5
16
9
10
11
12
13
14
15
16
17
5
5
40
20
10
20
50
2
32
32
[a] 2-AIT concentration is 40% of the MIC, up to a limit of 50 mm.
Angew. Chem. Int. Ed. 2012, 51, 11254 –11257
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 11255

.
Angewandte
Communications
Table 2: MIC values and oxacillin resistance suppression activity of
compound 7d against strains from the Nebraska Transposon Mutant
Library.
isolates obtained from the ATCC (see the Supporting
Information) and observed reduction in MIC values of
4-fold to 512-fold.
Entry Strain Concentration Oxacillin MIC Oxacillin MIC with 7d
As the synthesized molecules are amphipathic, we first
investigated the effect of the compounds on cell-membrane
integrity. The ability of compound 7d to permeabilize the
bacterial cell membrane was quantified using the BacLight
assay.[16] After exposure of strain JE2 to compound 7d for one
hour, the ratio of intact/damaged cells was measured and
compared to control (treated with DMSO only) bacteria. At
4 ꢁ the MIC, 96% of cells were damaged, whereas at 1 ꢁ ,
0.4 ꢁ , and 0.25 ꢁ the MIC, only 33%, 21%, and 9% of cells
were damaged, respectively. An inactive compound (7e) was
found to be comparable, with 83%, 24%, 23%, and 16% of
cells damaged at 4 ꢁ , 1 ꢁ , 0.4 ꢁ , and 0.25 ꢁ the MIC,
respectively; this suggests that cell-membrane permeabiliza-
tion is not the mechanism by which compound 7d suppresses
resistance to oxacillin. Importantly for a potential antibiotic
adjuvant, 7d exhibited little effect on eukaryotic cell mem-
branes, as determined by measuring the hemolytic activity
against mechanically difibrinated sheep blood.[9] At its active
resistance suppression concentration (5 mm), less than 1%
lysis was observed compared to triton ꢁ positive control,
while only 5.6% lysis was observed at as high as 50 mm.
To further delineate the mechanism by which these 2-AIT
conjugates are able to lower the oxacillin MIC against MRSA,
we obtained a number of mutant strains belonging to the
Nebraska Transposon Mutant Library from the Network on
Antimicrobial Resistance in S. aureus (NARSA). These
mutants are all derived from JE2, thus allowing us to probe
non-essential pathways that may be involved in suppression of
oxacillin resistance. For this screen, we focused largely on
mutants of non-essential TCS. Bacterial TCS, consisting of
a membrane-bound histidine kinase and a response regulator,
regulate adaptation to environmental changes and have been
shown to play a role in resistance to certain antibiotics[17–19] as
well as being master regulators of biofilm formation.[20,21]
Furthermore, biotinylated analogues of related 2-AI anti-
biofilm compounds employed in pull down assays bind to
response regulators involved in biofilm formation (unpub-
lished work). Therefore, we posited that these 2-AIT deriv-
atives, which also have anti-biofilm activity, might also target
other response regulators involved in antibiotic resistance.
We first established the MICs of oxacillin and 7d against
each mutant strain (Table 2; see the Supporting Information
for gene descriptions). As expected, the MIC of 7d was fairly
consistent against all mutant strains (either 6.25 or 12.5 mm),
whereas a majority of the strains examined, including several
response regulator mutants (strains NE958, NE481, NE262,
and NE49), histidine kinase mutants (strains NE218, NE147,
NE618, NE873, NE820, NE116, and NE423), and a MecR1
regulatory protein mutant (strain NE839), did not exhibit
a greater than two-fold difference in oxacillin MIC compared
to the parent strain. However, three of the strains tested
exhibited considerably lower oxacillin MIC values: NE481
(an unidentified DNA-binding response regulator mutant),
NE554 (a vraR mutant), and NE823 (a vraS mutant)
exhibited oxacillin MICs that were reduced 16-fold, eight-
fold, and eight-fold, respectively. These results are in line with
of 7d [mm]
[mgmLÀ1
]
[mgmLÀ1 [a]
]
1
2
3
4
5
6
7
8
JE2
NE218 2.5
NE147 2.5
NE958
NE481
NE262
NE618
NE554 2.5
NE823 2.5
5
32
32
32
32
2
32
32
4
0.5
8
0.25
0.25
0.25
0.5
1
5
5
5
5
4
4
9
4
10
11
12
13
14
15
16
17
NE873
NE210
NE820
NE839
NE49 2.5
NE116 2.5
5
5
5
5
32
32
32
32
32
32
16
16
0.25
0.5
0.25
0.5
16
32
0.5
4
NE95
NE423 2.5
5
[a] Oxacillin MIC values recorded in the presence of 40% MIC of 7d.
previous studies which show that expression of VraSR
contributes to oxacillin resistance.[22] The ability of 7d to
lower the oxacillin MIC against the mutant strains was then
examined in an identical manner to that used for the parent
strain (at 40% of the MIC). Of the mutants that exhibited
altered oxacillin MIC values versus the parent, compound 7d
failed to lower the MIC of both the VraSR two-component
system mutant strains NE554 and NE823, which suggests that
the mode of action of the oxacillin resistance suppression
activity of compound 7d involves VraSR. Compound 7d also
failed to lower the oxacillin MIC by more than two-fold
against strains NE116 and NE49, which suggests that com-
pound 7d may have some interaction with the pathways
controlled by the disrupted genes of these two mutants.
NE116 is a putative histidine kinase mutant, whereas NE49 is
an AraC family response regulator mutant. AraC family
proteins are known to play a role in antibiotic resistance and
stress responses,[23] however, as these two mutant strains did
not exhibit oxacillin MICs that differed from the parent
strain, these pathways most likely do not relate to the
mechanism of oxacillin resistance suppression by compound
7d.
As compound 7d exhibited a lower MIC against a number
of mutant strains than the parent strain (and was therefore
screened for resistance suppression at a lower concentration,
2.5 mm, 1.55 mgmLÀ1), we wanted to ensure that the lack of
resistance suppression activity was not simply a result of lower
bactericidal activity of the compound. A time-kill curve was
therefore constructed for NE554 in the presence of 2.5 mm 7d
(40% MIC) and compared to the time-kill curve of JE2 in the
presence of 5 mm 7d (see the Supporting Information). The
bactericidal activity of compound 7d, was in fact slightly
higher against strain NE554 than JE2 at the concentrations
used in the resistance suppression assay, which suggests that
the lack of activity against NE554 is due to the absence of
VraR, rather than altered bactericidal activity.
11256 www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 11254 –11257

Angewandte
Chemie
[2] IDSA Policy Paper d CID 2011:52 (Suppl 5) d S397.
VraSR has been proposed to be a “sentinel” system that is
capable of sensing perturbation of cell wall synthesis and
coordinating a response involving expression of a number of
genes involved in antibiotic resistance.[22] The expression of
VraSR is induced upon exposure to cell-wall-acting anti-
biotics, including b-lactams, glycopeptides, daptomycin, and
bacitracin,[24,25] and it has been shown that VraSR mutants are
treatable with an oxacillin regimen in vivo.[26] To further
establish the disruption of VraSR signaling in the mechanism
of 7d, we tested the ability of 7d to lower the MIC of
vancomycin. The MIC of vancomycin against JE2 was
established as 1 mgmLÀ1, but in the presence of 5 mm 7d this
is lowered to 0.25 mgmLÀ1. The MIC of vancomycin is
0.5 mgmLÀ1 against both NE554 and NE823, and this
remained unchanged in the presence of 2.5 mm 7d. Further-
more, compound 7d had little or no effect on the MICs of
streptomycin or chloramphenicol (see the Supporting Infor-
mation), which are non-cell-wall-acting antibiotics that do not
activate the VraSR TCS. The fact that compound 7d did not
lower the MIC of these latter antibiotics against the parent
strain further suggests that the reduction in oxacillin MIC
brought about by this compound is not simply due to
a combined microbicidal effect, but is rather due to disruption
of the VraSR TCS pathway.
In conclusion, we have developed a compound that is able
to suppress resistance to oxacillin in diverse MRSA strains,
achieving MIC suppressions upwards of 512-fold. We have
shown that this activity is not dependent upon membrane
disruption, and preliminary screening of USA300 mutants
indicates that VraSR plays an important role in the activity of
this compound. Given the pressing need for new strategies to
deal with the threat of multi-drug resistant pathogenic
bacteria, the identification of molecules that restore the
efficacy of approved antibiotics by interfering with bacterial
TCS represents a potential avenue for the development of
antibiotic adjuvants.
[3] F. P. OꢀHara, H. Amrine-Madsen, R. M. Mera, M. L. Brown,
N. M. Close, J. A. Suaya, C. J. Acosta, Microb. Drug Resistance
2012, DOI: 10.1089/mdr.2012.0056.
[4] A. R. M. Coates, G. Halls, Y. Hu, Br. J. Pharmacol. 2011, 163,
184 – 194.
[5] E. Dolgin, Nat. Med. 2010, 16, 1054 – 1054.
[6] L. Kalan, G. D. Wright, Expert Rev. Mol. Med. 2011, 13, 1 – 17.
[7] S. A. Rogers, R. W. Huigens III, J. Cavanagh, C. Melander,
Antimicrob. Agents Chemother. 2010, 54, 2112 – 2118.
[8] Z. Su, L. Peng, R. J. Worthington, C. Melander, ChemMedChem
2011, 6, 2243 – 2251.
[9] R. J. Worthington, C. A. Bunders, C. S. Reed, C. Melander, ACS
Med. Chem. Lett. 2012, 3, 357 – 361.
[10] J. K. Klitgaard, M. N. Skov, B. H. Kallipolitis, H. J. Kolmos, J.
Antimicrob. Chemother. 2008, 62, 1215 – 1221.
[11] S. A. Rogers, C. Melander, Angew. Chem. 2008, 120, 5307 – 5309;
Angew. Chem. Int. Ed. 2008, 47, 5229 – 5231.
[12] S. A. Rogers, J. D. Bero, C. Melander, ChemBioChem 2010, 11,
396 – 410.
[13] Z. Su, A. A. Yeagley, R. Su, L. Peng, C. Melander, ChemMed-
Chem 2012, DOI: 10.1002/cmdc.201200350.
[14] Twentieth Informational Supplement, Clinical and Laboratory
Standards Institute: Wayne, PA, January 2010, M100-S20 Vol. 30
No. 1.
[15] T. L. Harris, R. J. Worthington, C. Melander, Bioorg. Med.
Chem. Lett. 2011, 21, 4516 – 4519.
[16] J. J. Hilliard, R. M. Goldschmidt, L. Licata, E. Z. Baum, K.
Bush, Antimicrob. Agents Chemother. 1999, 43, 1693 – 1699.
[17] S. Gardete, S. W. Wu, S. Gill, A. Tomasz, Antimicrob. Agents
Chemother. 2006, 50, 3424 – 3434.
[18] D. Kraus, S. Herbert, S. A. Kristian, A. Khosravi, V. Nizet, F.
Gçtz, A. Peschel, BMC Microbiol. 2008, 8, 85.
[19] M. Shoji, L. Cui, R. Iizuka, A. Komoto, H. Neoh, Y. Watanabe,
T. Hishinuma, K. Hiramatsu, Antimicrob. Agents Chemother.
2011, 55, 3870 – 3881.
[20] A. P. Tomaras, M. J. Flagler, C. W. Dorsey, J. A. Gaddy, L. A.
Actis, Microbiology-Sgm 2008, 154, 3398 – 3409.
[21] M. D. Parkins, H. Ceri, D. G. Storey, Mol. Microbiol. 2001, 40,
1215 – 1226.
[22] A. Belcheva, D. Golemi-Kotra, J. Biol. Chem. 2008, 283, 12354 –
2364.
[23] M. G. Lei, D. Cue, C. M. Roux, P. M. Dunman, C. Y. Lee, J.
Bacteriol. 2011, 193, 5231 – 5241.
[24] M. Matsuo, F. Kato, Y. Oogai, T. Kawai, M. Sugai, H.
Komatsuzawa, J. Antimicrob. Chemother. 2010, 65, 1536 – 1537.
[25] V. Dengler, P. Stutzmann Meier, R. Heusser, B. Berger-Bꢂchi, N.
McCallum, BMC Microbiol. 2011, 11, 16.
Received: August 26, 2012
Published online: October 9, 2012
Keywords: 2-aminoimidazoles · adjuvants · antibiotics ·
.
antibiotic resistance · MRSA
[26] D. S. Jo, C. P. Montgomery, S. Yin, S. Boyle-Vavra, R. S. Daum,
Antimicrob. Agents Chemother. 2011, 55, 2818 – 2823.
[1] E. H. Eiland, D. J. Gatlin III, Pharmacy Practice 2008, 21, 313 –
318.
Angew. Chem. Int. Ed. 2012, 51, 11254 –11257
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 11257