Antibacterial Small Molecules That Potently Inhibit Staphylococcus aureus Lipoteichoic Acid Biosynthesis

Article abstract of DOI:10.1002/cmdc.201900053

The rise of antibiotic resistance, especially in Staphylococcus aureus, and the increasing death rate due to multiresistant bacteria have been well documented. The need for new chemical entities and/or the identification of novel targets for antibacterial

Full text of DOI:10.1002/cmdc.201900053

Accepted Article
Title: Antibacterial small molecules that potently inhibit Staphylococcus
aureus lipoteichoic acid biosynthesis
Authors: Herman O. Sintim, George A. Naclerio, Caroline W. Karanja,
and Clement Opoku-Temeng
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10.1002/cmdc.201900053
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Antibacterial small molecules that potently inhibit
Staphylococcus aureus lipoteichoic acid biosynthesis
George A. Naclerio1, Caroline W. Karanja1, Clement Opoku-Temeng1,2, and Herman O. Sintim1*
1
Mr George A. Naclerio, Ms Caroline W. Karanja, Dr Clement Opoku-Temeng, Prof Herman O. Sintim (0000-0002-2280-9359)
Chemistry Department
Institute for Drug Discovery
Purdue University
West Lafayette, IN USA 47907
Email: hsintim@purdue.edu
2
Graduate Program in Biochemistry
University of Maryland
College Park, MD USA 20742
Abstract: The rise of antibiotic resistance, especially in
staphylococcus aureus, and the increasing death rate due to
multi-resistant bacteria have been well documented. The
need for new chemical entities and/or the identification of
novel targets for antibacterial drug development is high.
Lipoteichoic acid (LTA),
a membrane attached anionic
polymer, is important for the growth and virulence of many
Gram-positive bacteria and interest has been high in the
discovery of LTA biosynthesis inhibitors. Thus far only a
handful of LTA biosynthesis inhibitors have been described
with moderate (MIC = 5.34 µg/mL) to low (MIC = 1024 µg/mL)
activities against S. aureus. Here we describe the
identification of novel compounds that potently inhibit LTA
biosynthesis in S. aureus, displaying impressive antibacterial
activities (MIC as low as 0.25 µg/mL) against methicillin-
resistant S. aureus (MRSA). Under similar in-vitro assay
conditions, these compounds are 4X more potent than
vancomycin and 8X more potent than Linezolid against
MRSA.
The rise of antibiotic resistance and the increasing
death rate due to infections with multidrug-resistant
bacteria and Staphylococcus aureus (S. aureus) have
been well documented. S. aureus,
a Gram-positive
bacterial pathogen, is one of the leading causes of
community- and hospital- acquired bacteremia[1]. The rise
of antimicrobial resistance strains partly contributes to the
increasing death rate associated with S. aureus infection[2].
Methicillin-resistant S. aureus (MRSA) bacteremia is
accompanied by higher mortality rates compared to methicillin-
sensitive S. aureus (MSSA) bacteremia[3]. In 2013, the Center
for Disease Control and Prevention (CDC) estimated that
bacterial infections kill at least 23,000 annually in the US alone
with MRSA being responsible for nearly half of the mortalities[4].
Vancomycin, a glycopeptide antibiotic, is used for the treatment
of severe MRSA infections. However, emergence of vancomycin
intermediate and resistant S. aureus (VISA/VRSA) strains
further limits therapy [5]. The discovery of antimicrobial agents
with a novel mode of action is vital for the successful treatment
of S. aureus infections.
Figure 1. HSGN-189 has potent antibacterial activity and inhibits LTA
biosynthesis in S. aureus. (A) The biosynthesis of LTA takes place at the cell
membrane. UDP-Glc is produced by the conversion of glucose-6-phosphate to
glucose-1-phosphate by the α-phosphoglucomutase PgcA, followed by
activation of UTP:α-glucose-1-phosphate by uridyltransferase GtaB. The
glycosyltransferase YpfP transfers two glucose molecules from UDP-Glc to
diacylglycerol (DAG), generating the glycolipid Glc2-DAG. Glc2-DAG is then
displaced to the outer membrane by LtaA. LtaS uses glycerol phosphate as a
substrate to repeatedly transfer glycerol phosphate to the Glc2-DAG anchor,
producing LTA. (B) Previous LTA biosynthesis inhibitors include Compound
1771 and the probe-like molecule Congo Red. These molecules exhibit
moderate to low antimicrobial activity with MIC values of 5.34 µg/mL and 1024
µg/mL against S. aureus respectively. (C) We previously identified F6-15 as a
weak antibacterial agent against MRSA.11 With further optimization, HSGN-
189 was indentified to be a potent anti-MRSA agent (MIC = 0.25 µg/mL) and
LTA biosynthesis inhibitor.
The Gram-positive bacteria cell envelope consists of a
membrane and a peptidoglycan cell wall with anchored anionic
polymers (teichoic acids). Teichoic acids include wall teichoic
acids (WTA), which are covalently linked to the peptidoglycan,
and lipoteichoic acids (LTA), which are anchored together via a
glycolipid[6].
1
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Both polymers are vital components of the cell envelope
involved in bacterial growth, replication, colonization and
virulence[7]. LTA in S. aureus, is composed of a 1,3-glycerol
phosphate polymer linked by
a diglucosyl diacylglycerol
glycolipid anchored to the membrane[6b]. The LTA varies greatly
amongst Gram-positive bacteria. Yet, several Gram-positive
pathogens, including Bacillus subtilis, Enterococcus faecalis,
and Listeria monocytogenes, produce the same polyglycerol
phosphate polymer as S. aureus [6b, 7a]. LTA is synthesized by
lipoteichoic acid synthase (LtaS) from phosphatidylglycerol.
Depletion of ltaS (gene for LtaS) and LTA in S. aureus results in
growth arrest, cell wall envelope and cell division defects[8]. The
essential nature of LTA in S. aureus, along with the fact that it is
not present in eukaryotic cells, makes LTA an ideal antimicrobial
target.
Thus far, there have been efforts to develop potent LTA
biosynthesis inhibitors with antibacterial activity by few groups.
However, the compounds developed to date are significantly
less potent than vancomycin. For example, the first LTA
biosynthesis inhibitor, Compound 1771 possessed a minimum
inhibitory concentration (MIC) of 5.34 µg/mL against S. aureus.[9]
Compound 1771 contains an ester moiety, a potential liability
due to esterase hydrolysis in blood. In a more recent publication
Walker et al. demonstrated that Congo red inhibits LtaS
activity[10], however exhibited very low antimicrobial activity (MIC
of 1024 µg/mL) against S. aureus[10]
.
Due to the essential nature of LTA, we have been
interested in developing antibacterial agents that inhibit LTA
biosynthesis. Our group has demonstrated that N-(1,3,4-
oxadiazol-2-yl)benzamides are potent antibacterial agents with
MIC values of 2 µg/mL against MRSA[11]. Here, we report a new
generation of N-(1,3,4-oxadiazol-2-yl)benzamides, exhibiting
MIC values as low as 0.25 µg/mL against MRSA and are more
potent than frontline antibiotics used for MRSA infections (4X
more potent than vancomycin and 8X more potent than linezolid).
Figure 3. Series of 1,3,4-oxiadizolyl-based compounds synthesized for study.
moieties typically found in other clinical compounds. Several
compounds containing the 1,3,4-oxadiazolyl unit have
demonstrated interesting biological activities, as exemplified by
the drugs such as furamizole[12] (antibacterial), nesapidil[12]
(antiarrhythmic), raltegravir (HIV antiviral)[13] and zibotentan
(underwent clinical trials for prostate cancer)[14] (see Figure 2).
Figure 2. .Clinical compounds containing 1,3,4-oxadiazole unit.
Our group has embarked on the generation of proprietary
compounds for evaluation against drug resistant bacteria. As a
strategy to increase the chances of advancing a hit molecule to
the clinic, we have prepared a library that is enriched with
2
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We previously discovered that compound F6-15 displayed
weak antibacterial properties with a MIC of 32 µg/mL against S.
aureus. Remarkable enhancements in the activity of the lead
compound were obtained upon strategic methyl group
substitution (the methylation effect)[15]. The installment of the
3,5-dimethyl groups on the piperidine ring gave rise to F6, which
displayed MIC of 2 µg/mL[11] Notably F6 was well tolerated in
mice and capable of reducing bacterial burden in a wound
infection model[11]. While a MIC of 2 µg/mL is respectable, we
desired to further optimize this compound by the synthesis of
new analogues, which were initially screened for their ability to
inhibit the growth of S. aureus at 16 µg/mL (ESI, Figure S1).
30
31
32
33
35
1
0.25
1
0.5
0.25
2
4
2
8
8
4
8
4
2
8
8
4
8
2
1
4
8
2
4
4
4
1
0.5
0.25
36, HSGN-
189
0.25
37
0.5
8
1
8
2
1
1
2
16
>16
4
16
>16
4
8
>16
2
Table 1. MIC (µg/mL) of HSGN-94, HSGN-189, analogs, vancomycin, and
linezolid against a panel of Gram-positive bacterial pathogens. Experiments
were done in triplicate and in all replications the same MIC values were
obtained.
38
39
2
S.
MRSA
ATCC
33592
E.
VRE
ATCC
51575
L.
40
2
8
4
4
aureus
ATCC
25923
faecalis
ATCC
29212
monocytogenes
ATCC 19115
Compounds
Vancomycin
Linezolid
1
2
>128
2
1
2
2
2
F6-15
32
2
32
2
16
4
64
4
32
4
F6
1
4
4
16
64
16
4
8
8
For compounds that showed inhibitory activity, we
determined the MIC (Table 1). For synthesis of compounds see
ESI Figure S1. Four types of compounds were made (series 1-4,
Figure 3). The compounds contained four rings (labeled rings A,
B, C and D, see Figure 3). Series 1 was made up of compounds
with various substitution (halogens, CF3, CN, OMe, tetrazole,
NH2, OH, Me, hydroxyamidine) to phenyl ring D. Halogen
substitutions (especially the Cl, F or CF3 groups) resulted in the
most active compounds. Hydrophilic substituents, such as the
NH2, CN, OH and tetrazole were not active. For the halogen
substituents, the position on the ring was also important. For
example, the MIC for para-CF3 (12, HSGN-94) was 0.25 µg/mL,
whereas that for the meta analog (5) was 1 µg/mL against
MRSA (Table 1). In series 2, we investigated other
heteroaromatics, such as pyridinyl (25 and 26), chlorothiophenyl
(27), dimethylthiazolyl (28), pyrazolyl (29) as ring D. For these
compounds, the chlorothiophenyl analog 27 was the most potent
(MIC = 2 µg/mL against MRSA). Series 3 explored structure-
activity-relationships (SAR) of the sulfonamide moiety (ring A).
Here both the methyl substituted piperidine and N-substituted
aniline substituents were highly active (MIC for compounds 30,
31, 32, and 35 are 0.5, 0.25, 1 and 0.5 µg/mL respectively).
Considering that the 3,5-dimethyl piperidine sulfonamide
(HSGN-94) was one of the best compounds, we proceeded to
investigate how substitution of ring B and/or position of the 3,5-
dimethyl piperidine sulfonamide (series 4) affected antibacterial
activity. Replacement of the phenyl group with thiophenyl (37) or
pyridinyl (39) led to a small reduction in antibacterial activity
(MIC = 1 µg/mL and 2 µg/mL for compounds 37 and 39
respectively). Addition of a methyl group to the 3 position of ring
B (36, HSGN-189) did not effect activity (MIC = 0.25 µg/mL).
Changing the position of the 3,5-dimethylpiperidine sulfonamide
moiety from para to meta, (compounds 38 and 40) on ring B
resulted in reduced activity against MRSA (compare MIC of 0.25
µg/mL for HSGN-94 and HSGN-189 with 8 µg/mL and 1 µg/mL
for compounds 38 and 40 respectively).
2
32
2
32
4
64
8
32
4
3
5
2
2
8
2
6
1
0.5
0.5
2
1
2
1
7
2
2
2
2
8
4
8
4
4
9
16
16
0.25
2
8
32
64
2
16
32
1
8
11
16
0.25
1
32
0.5
2
12 ,HSGN-94
13
14
15
16
17
20
21
22
23
24
26
27
28
29
4
2
2
4
4
4
4
4
4
4
4
4
4
4
4
4
4
32
16
2
16
8
64
32
4
32
16
2
32
16
2
1
16
0.5
8
16
0.25
8
16
2
8
16
1
1
32
64
64
32
16
32
64
32
64
128
32
32
32
16
8
16
2
32
2
16
8
8
4
3
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HSGN-189 appears more selective than HSGN-94 (see
Table 1 for comaprision of MICs against other Gram-postive
bacteria), thus we proceeded to identify its mode of action.
Traditional ways to do this are to generate bacteria that are
resistant to the compound and use global sequencing to identify
genes that are mutated in the presence of the compound or to
use affinity probes to identify binding proteins[16]. Despite many
attempts, we have been unable to generate resistant strains
towards HSGN-189 (which looks promising for the eventual
translation of this compound or analogs thereof). Given that
HSGN-189 and the known LTA biosynthesis inhibitor,
Compound 1771, both contain aryl substituted 1,3,4-oxadiazolyl
unit, we investigated the effects of selected compounds on LTA
levels in S. aureus Excitingly, when we investigated the effects
of F6-15, F6 and HSGN-189 on LTA biosynthesis in S. aureus,
following the protocol utilized by Walker and Richter, we
observed potent inhibition of LTA by these compounds (Figure 4
and ESI, Figure S2). Interestingly, the degree of LTA
biosynthesis inhibition correlated with the MIC values, strongly
hinting that LTA biosynthesis inhibition is responsible (at least in
part) for the antibacterial activities of the compounds.
Vancomycin and Congo Red were used as negative and positive
controls respectively (see Figure S2). Whereas Congo Red
reduced LTA biosynthesis in S. aureus, vancomycin increased
LTA content (see Figure S2).
In conclusion, we have identified potent inhibitors of LTA
biosynthesis. These compounds potently inhibit MRSA with MIC
values that are 4X lower than vancomycin and 8X lower than
linezolid, two antibiotics commonly used to treat MRSA
infections. However, both traditional antibiotics have many
disadvantages. For vancomycin, it is not orally bioavailable and
displays nephrotoxicity. Likewise, linezolid can cause serious
side effects like bone-marrow suppression, lactic acidosis,
peripheral and optic neuropathy, etc[17]. Thus, alternatives to
vancomycin and linezolid are needed. Future work will focus on
the activities of the potent compounds (MIC less than 0.5 µg/mL)
in mice infection models. We will also investigate which of the
many enzymes involved in LTA biosynthesis is/are the targets of
the described compounds. This work adds to the increasing
number of reports that have attempted to address the anti-
bacterial resistance issue with novel small molecules[18]
.
Figure 4. LTA biosynthesis inhibition by 1,3,4-oxadiazolyl –based compounds.
The MIC of HSGN-189 (0.25 µg/mL) is lower than F6-15 (32 µg/mL) and F6 (2
µg/mL). At lower concentrations (0.25 µg/mL), only HSGN-189 significantly
inhibited the biosynthesis of LTA. Yet, at the MIC concentration of F6-15 (32
µg/mL) and F6 (2 µg/mL), LTA biosynthesis was inhibited.
Experimental
Section:
For
experimental
procedures,
compound syntheses and characterization data see ESI.
Keywords: LTA biosynthesis inhibitor • MRSA • Antibiotic
resistance • 1,3,4-oxadiazole • antibacterial activity
4
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5
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Entry for the Table of Contents
The Gram-positive bacteria cell envelope consists of a membrane and a peptidoglycan cell wall with anionic polymers anchored onto
these structures: wall teichoic acids (WTA) and lipoteichoic acids (LTA). Both polymers are vital components of the cell envelope and
modulate bacterial growth, replication, colonization and virulence. Thus far potent inhibitors of LTA biosynthesis have been lacking.
Here, we reveal new LTA biosynthesis inhibitors that potently inhibit LTA biosynthesis in S. aureus. Some of the LTA biosynthesis
inhibitors also inhibit bacterial growth at concentrations as low as 0.25 µg/mL.
6
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