Propargylglycine-based antimicrobial compounds are targets of TolC-dependent efflux systems in Escherichia coli

Article abstract of DOI:10.1016/j.bmcl.2019.126875

A library of novel L-propargylglycine-based compounds were designed and synthesized with the goal of inhibiting the growth of Gram-negative bacteria by targeting LpxC, a highly conserved Gram-negative enzyme which performs an essential step in the lipid A biosynthetic pathway. These compounds were designed with and without a nucleoside and had varying tail structures, which modulate their lipophilicity. The synthetic scheme was improved compared to previous methods: a methyl ester intermediate was converted to a hydroxamic acid, which obviated the need for a THP protecting group and improved the yields and purity of the final compounds. Antimicrobial activity was observed for non-nucleoside compounds containing a phenyl propargyl ether tail (5) or a biphenyl tail (6). An MIC of 16 μg/mL was achieved for 6 in Escherichia coli, but inhibition was only possible in the absence of TolC-mediated efflux. Compound 5 had an initial MIC >160 μg/mL in E. coli. Enhancing outer membrane permeability or eliminating efflux reduced the MIC modestly to 100 μg/mL and 80 μg/mL, respectively. These results highlight the importance of hydrophobicity of this class of compounds in developing LpxC inhibitors, as well as the design challenge of avoiding multidrug efflux activity.

Full text of DOI:10.1016/j.bmcl.2019.126875

Journal Pre-proofs
Propargylglycine-based antimicrobial compounds are targets of TolC-depend-
ent efflux systems in Escherichia coli
Rebeca J. Roldan, Andrea O. Pajarillo, Jacob D. Greenberg, Joyce E. Karlinsey,
Mauricio Cafiero, Elaine R. Frawley, Larryn W. Peterson
PII:
S0960-894X(19)30853-4
DOI:
https://doi.org/10.1016/j.bmcl.2019.126875
BMCL 126875
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2019
26 November 2019
27 November 2019
Please cite this article as: Roldan, R.J., Pajarillo, A.O., Greenberg, J.D., Karlinsey, J.E., Cafiero, M., Frawley, E.R.,
Peterson, L.W., Propargylglycine-based antimicrobial compounds are targets of TolC-dependent efflux systems in
Escherichia coli, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126875
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Propargylglycine-based antimicrobial compounds are targets
of TolC-dependent efflux systems in Escherichia coli
Rebeca J. Roldan a, Andrea O. Pajarillo a, Jacob D. Greenberg a, Joyce E. Karlinsey b, Mauricio Cafiero a,
Elaine R. Frawley c,*, Larryn W. Peterson a,*
a
Department of Chemistry, Rhodes College, Memphis, TN 38112, USA;
Department of Microbiology, University of Washington, Seattle, WA 98195, USA;
Department of Biology, Rhodes College, Memphis, TN 38112, USA;
b
c
* Correspondence: frawleye@rhodes.edu; petersonl@rhodes.edu; Tel.: 1-901-843-3562 (E.F.); 1-901-843-3545
(L.P.).
Abstract: A library of novel L-propargylglycine-based compounds were designed and synthesized with
the goal of inhibiting the growth of Gram-negative bacteria by targeting LpxC, a highly conserved Gram-
negative enzyme which performs an essential step in the lipid A biosynthetic pathway. These compounds
were designed with and without a nucleoside and had varying tail structures, which modulate their
lipophilicity. The synthetic scheme was improved compared to previous methods: a methyl ester
intermediate was converted to a hydroxamic acid which obviated the need for a THP protecting group
and improved the yields and purity of the final compounds. Antimicrobial activity was observed for non-
nucleoside compounds containing a phenyl propargyl ether tail (5) or a biphenyl tail (6). An MIC of 16
µg/mL was achieved for 6 in Escherichia coli, but inhibition was only possible in the absence of TolC-
mediated efflux. Compound 5 had an initial MIC >160 µg/mL in E. coli. Enhancing outer membrane
permeability or eliminating efflux reduced the MIC modestly to 100 µg/mL and 80 µg/mL, respectively.
These results highlight the importance of hydrophobicity of this class of compounds in developing LpxC
inhibitors, as well as the design challenge of avoiding multidrug efflux activity.
Keywords: L-propargylglycine; lipopolysaccharide; tolC; antimicrobial; efflux pumps
Incidences of multidrug-resistant pathogens have increased on a global basis, with some bacterial
strains now exhibiting resistance to even last-resort antibiotics.1-9 In the US alone, at least 2 million cases
of multidrug resistant bacterial infections occur each year, resulting in approximately 23,000 deaths per
annum.10 Development of new antimicrobials that are not subject to existing mechanisms of resistance is
an important goal of current research.
Lipopolysaccharides (LPS) are an important structural component of the Gram-negative outer
membrane, creating both a physical and charge barrier to permeability by a wide variety of compounds
1

and antibiotics.11-15 Each LPS molecule consists of three sections: an O-antigen, a core oligosaccharide,
and lipid anchor embedded in the membrane. UDP-(3-O-((R)-3-hydroxymyristoyl))-N-
a
A
acetylglucosamine deacetylase, or LpxC, is responsible for the first committed step of lipid A synthesis and
is essential in Escherichia coli.16-18 Inhibition of LpxC prevents LPS formation and enhances antibiotic
sensitivity.19-21 LpxC is conserved across Gram-negative phyla and shares no homology with mammalian
enzymes, making it an attractive target for antimicrobial treatment.22-24
Our analysis of the crystal structure of the Aquifex aeolicus LpxC active site25 found three target areas
for ligand binding: a Zn2+ ion, a polar region, and a hydrophobic passage (Figure 1).26-27 The LpxC natural
substrate consists of a nucleoside, a diphosphate group, a glucosamine, and a long hydrocarbon tail. The
strongest potential interactions can occur with the Zn2+ ion via ionic binding.28 Among the LpxC inhibitors
found in literature all share a hydroxamate head group (replacing the diphosphate group of the natural
substrate), which binds strongly to the Zn2+ ion29 forming a thermodynamically favorable five-membered
ring.30 Most inhibitor design has focused on optimizing the tail moiety, and few inhibitors include the
nucleoside for binding in the polar region.31-32 In this work, potential inhibitors of LpxC capable of targeting
two or more regions in the LpxC active site were designed. The structures differ in inclusion of a nucleoside
and in type of hydrophobic tail (Figure 2).
Zn2+
Zn2+
Hydro-
phobic
pocket
Hydro-
phobic
pocket
Polar-
Polar-
binding
region
binding
region
Figure 1. (Left) Compound 2 (ball and stick) optimized in the A. aeolicus LpxC active site (M062X/6-31G) showing the
molecule interacting with the binding site via the polar binding region, the Zn2+ ion, and the hydrophobic pocket.
(Right) Compound 4 (ball and stick) optimized in the A. aeolicus LpxC active site (M062X/6-31G) showing the
molecule interacting with the binding site via the Zn2+ ion, and part of the hydrophobic pocket.27
2

Efficacy of antimicrobial compounds depends not only on their ability to bind and inhibit a specific
molecular target, but also to accumulate within bacterial cells. Sufficiently lipophilic compounds can
diffuse across the outer and inner membranes while other compounds enter bacterial cells via channels
and porins that are selective according to size and other properties such as hydrophilicity or charge.33-34
Once inside the cell, many antimicrobial compounds are the target of various efflux systems including
those from the resistance-nodulation-division (RND), major facilitator (MF), ATP-binding cassette (ABC),
small multidrug resistance (SMR) and multidrug and toxic compound extrusion (MATE) families. Of these
groups, the RND family transporters have the widest range of substrates and account for the majority of
intrinsic resistance in Gram negative bacteria.15 It remains challenging to predict whether a compound
will be targeted for efflux by RND transporters but transport does correlate positively with
hydrophobicity.15, 35
Herein, we report the synthesis of a new nucleoside-containing compound 2 and the design and
synthesis of novel propargylglycine-based compounds designed to inhibit LpxC activity (4-6, Figure 2).
Additionally, a new synthetic procedure, which obviated the need for the THP-protecting group, was
implemented to reduce the number of steps and improve the yield of the final hydroxamic acids. Finally,
the minimal inhibitory concentrations (MICs) for compounds 1-6 were established for P. aeruginosa and
E. coli. Improved antibacterial activity was observed in E. coli strains with either increased permeability or
lacking TolC-dependent efflux.
O
O
NH
NH
O
N
N
N
N
N
N
O
N
O
N
O
O
N
H
R
H2N
HN
O
O
R
HN
O
O
OH
HN
HN
OH OH
OH OH
OH
OH
4
5
6
R = H
R = OCH2CCCH3
R = Ph
2
3
R = OCH2CCCH3
R = Ph
1
OH
O
NH2
H
O
N
H
N
OH
N
H
N
OH
O
H
O
O
O
N
CHIR-090
Piizzi-1
Figure 2. The structures of the propargylglycine-based compounds 1-6, CHIR-090 and Piizzi-1.
3

Being informed by our previous computational work,26 we focus here on synthesizing and testing
antimicrobial activity of promising compounds. Compound 1, which does not contain a hydrophobic tail,
had the most negative interaction energy at -710 kcal/mol in a solvated model.26 However, as many key
human enzymes, such as matrix metalloproteinases and zinc-dependent histone deacetylases, contain
Zn2+ ions in their active sites, the inclusion of the hydrophobic tail should provide greater specificity for
LpxC.
Incorporation of a hydrophobic tail to bind in the active site offers both design and synthetic
challenges. The amino acid sequence in these tunnels differs across many Gram-negative strains, resulting
in varying passage shape.36 In order to achieve broad spectrum activity against different Gram-negative
species, the tail must be able to fit within a variety of hydrophobic passages and resolve any differences.
Many research groups have found that more linear tails can account for slight variations in the
hydrophobic tunnel, resulting in a wide variety of tails with aromatic groups bound by acetylene or
diacetylene.37 However, many inhibitors including CHIR-090 (Figure 2) showed low aqueous solubility as
well as in vitro cytotoxicity.37 In place of the diacetylene and acetylene tails, a flexible propargyl ether
group was added which could specifically inhibit P. aeruginosa LpxC.38 Piizzi-1 (Figure 2), with a propargyl
ether tail, showed an IC50 value of 0.004 M against wild-type P. aeruginosa LpxC and MIC90 of 2 g/mL in
multidrug resistant (MDR) P. aeruginosa, as well as good aqueous solubility with no cytotoxic effects.38
Due to this success, the propargyl ether tail was incorporated into our inhibitors.
The incorporation of a tail moiety allows for further modulation of the lipophilicity and solubility of
the compounds, which can be observed in the calculated log P values (Table 1), giving a relative idea of
the hydrophobicity of the compounds. The log P and ClogP of CHIR-090 and Piizzi-1 were also determined
as a comparison. Compound 1, which does not contain a hydrophobic tail, has the most negative log P
value. The other nucleoside-containing compounds (2 and 3) are still fairly hydrophilic, despite compound
3 containing the biphenyl tail group. Although the nucleoside provides additional points for interaction in
the active site, the polarity of the nucleoside decreases the relative lipophilicity, possibly providing
challenges for getting into the cell and also potentially having a larger desolvation penalty when binding
in the active site. Therefore, we decided to also investigate compounds that lack the nucleoside.
Compounds 4-6, with just the propargylglycine scaffold and various tails, have higher log P values and are
more lipophilic.
4

Table 1. LogP and CLogP values1 of synthesized compounds
Compound
Piizzi-1
CHIR-090
Log P
0.44
1.40
-3.52
-1.69
-0.01
0.61
1.29
2.28
ClogP
0.651
1.412
-5.683
-2.921
-2.183
-0.227
0.923
1.661
1
2
3
4
5
6
1 Values taken from ChemDraw Professional 18.2.
Compound 6 was initially prepared using an intermediate in the synthesis of compound 3.
Commercially available Fmoc-L-propargylglycine was converted from a carboxylic acid to a
tetrahydropyran (THP)-protected hydroxamic acid in the presence of N,N’-dicyclohexylcarbodiimide (DCC)
coupling agent to afford the alkyne 7 in a 78% yield.26 The Fmoc group was then removed using piperidine
and biphenyl carboxylic acid was immediately coupled using DCC to afford 8 in a 68% yield (Scheme 1).
Final deprotection of the THP-protected hydroxamic acid 8 was accomplished using p-toluenesulfonic
acid39 to yield the final product 6 in a 72% yield (Scheme 1). However, due to difficulty in purification
resulting from low solubility of 6, this last step underwent several attempts at optimization, but did not
give improved results.
Scheme 1
1. piperidine, CH2Cl2,
5 min, 0oC
THP-O-NH2, DCC,
Fmoc
O
Fmoc
O
2. Biphenyl-CO2H, DCC, HOBt,
DIPEA, DMF, CH2Cl2, rt, 24 h
N
H
N
H
CH2Cl2, rt, 26 h
78%
NH
OH
O
THP
68%
7
O
O
O
TsOH, MeOH
rt, 18 h
NH
HN O
O
N
THP
H
HN
OH
Ph
72%
Ph
8
6
Up until this point, we had routinely used a THP-protecting group after converting the carboxylic acid
to a protected hydroxamic acid to result in the alkyne derivative.26 However, the removal of the THP-
5

protection and purification has proven problematic, and therefore, a new synthetic scheme was created
with the aim of affording the coupled product in higher yields. An approach involving converting the
carboxylic acid first to a methyl ester and then converting to the hydroxamic acid in the last step of
synthesis was attempted.38, 40 In the interest of pursuing this method, Fmoc-L-propargylglycine was
esterified using thionyl chloride in methanol41 to form methyl ester 9 in a 100% yield (Scheme 2). A similar
method was also employed with Boc-L-propargylglycine to afford 10 in a 94% yield. Although the Boc
1
group was not observed in the H and 13C after esterification, HCl in dioxane was added to ensure the
hydrochloride salt was available for coupling.
Compound 10 was coupled with benzoyl chloride to give alkyne 13 in an 86% yield (Scheme 2), which
was significantly better than the yield obtained with the Fmoc deprotection and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC)/DCC coupling previously employed. The propargyl ether
benzoic acid derivative 12 was synthesized as previously described by Piizzi and coworkers.38 EDC and
HATU
(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate) were explored to synthesize alkyne 14 (Scheme 2). Following Fmoc deprotection
of methyl ester 9 with piperidine, acid 12 was coupled to afford alkyne 14. The EDC procedure had been
previously used by our lab, and successfully produced alkyne 14 in a 57% yield.26 However, the HATU
coupling procedure was even more successful, with alkyne 14 afforded in an 81% yield. The synthesis was
carried out this way since 14 was already available, but in the future any new syntheses should start with
Boc-L-propargylglycine. The biphenyl analogue 15 was also prepared using compound 10, biphenyl
carboxylic acid and EDC coupling to produce 15 in a 62% yield.
The coupled methyl esters 13-15 were easily converted to hydroxamic acids 4-6 through treatment
with aqueous hydroxylamine38 in 85-100% yields (Scheme 2). This method resulted in significantly higher
yields and avoided the need for column chromatography purification. The structures of alkynes 4-6 were
confirmed through 1H NMR and 13C NMR and by HR-MS. The purity was confirmed by HPLC.
6

Scheme 2
1. SOCl2, MeOH.
0°C-rt 20 h
O
BzCl, TEA, CH2Cl2
HCl
H2N
O
O
Boc
N
OH
N
H
2. 4 N HCl in dioxane,
CH2Cl2, 0°C, 1 h
0°C to rt, 1 h
86%
H
O
O
O
94%
13
10
SOCl2, MeOH
60°C, 4 h
100%
Fmoc
N
OH
Fmoc
O
N
H
O
H
O
9
O
O
1. piperidine, CH2Cl2
0°C, 15 min
O
OH
N
H
Fmoc
O
N
+
2. HATU, DIPEA,
CH2Cl2, rt, 19 h
O
H
O
O
O
12
14
9
81%
O
Biphenyl-CO2H, EDC,
O
O
N
HOBt, DIPEA, CH2Cl2,
rt, 19 h
H2N
HCl
H
O
O
Ph
62%
15
10
O
O
50% NH2OH in H2O
MeOH, rt, 20 h
O
O
N
N
H
H
O
HN
R
R
OH
13
14
15
4
R = H, 85%
R = H
R = OCH2CCCH3
R = Ph
5
R = OCH2CCCH3, 100%
R = Ph, 100%
6
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) was used to synthesize triazole-linked
compounds 1 and 3 with yields from 57-76% as previously described.26 Similar methods were utilized in
the synthesis of 2 (Scheme 3). Synthesis of the azidonucleoside 18 was accomplished as described
previously.26 Azide 18 and alkyne 5 were dissolved in acetonitrile, after which copper powder was added
to the reaction flask. Following sonication, the reaction ran for 24 hours at 35°C to afford 19 in 39% yield,
which was significantly lower than yields previously obtained with the other nucleoside analogues. This
reduced yield may be in part due to the presence of the hydroxamic acid.
After purification by column chromatography, compound 19 was treated with trifluoroacetic acid
(TFA) in order to remove the ketal protecting group. The final compound was obtained through
precipitation from a methanolic solution with diethyl ether. Compound 2 was isolated in a 81% yield,
7

which was slightly better than literature yields of 65-70% for compounds 1 and 3 (Scheme 3).26 Synthesis
of 2 was confirmed with 1H NMR and 13C NMR and HR-MS. The purity was confirmed by HPLC.
Scheme 3
O
NH
O
N
O
Cu, H2O, CH3CN
35°C, 24 h
O
N
N3
+
H
O
HN
O
OH
39%
O
O
5
18
O
N
O
N
NH
NH
N
O
N
O
O
O
N
N
TFA, H2O, CH2Cl2
rt, 20 h
N
N
O
O
O
O
N
N
O
O
OH OH
H
H
81%
HN
HN
O
OH
O
OH
19
2
Purified compounds were dissolved in DMSO and the standard microbroth dilution method was used
to determine the minimal inhibitory concentration (MIC) for each compound in the Gram-negative
bacteria Escherichia coli and Pseudomonas aeruginosa.42 The results, which were consistent across four
independent trials, are summarized in Table 2. Compounds 1-4 failed to show any growth inhibition for
either P. aeruginosa or E. coli compared to the 1% DMSO control (Supplementary Figures 1 and 2A).
Failure to inhibit growth could result from lack of activity against the intended target, but it could also be
due to a low cell permeability or intrinsic resistance via multidrug efflux systems. To investigate these
possibilities, three E. coli strains were generated.
Table 2. MICs for compounds 1-6.
P. aeruginosa
E. coli
E. coli
ΔCΔ4LFhuA
ND
E. coli
ΔacrB
ND
ND
ND
ND
80 μg/mL
32 μg/mL
E. coli
ΔtolC
ND
ND
ND
ND
80 μg/mL
16 μg/mL
Compound
1
2
3
4
5
6
ND1
ND
ND
ND
ND
ND
ND
ND
ND
ND
100 μg/mL
ND
ND
>160 μg/mL
ND
100 μg/mL
ND
1 ND indicates than inhibition was not observed and an MIC was not able to be determined.
8

A hyperpermeable strain was constructed by creating plasmid pEF107 to constitutively express a modified
version of the FhuA outer membrane porin (ΔCΔ4LFhuA) that was previously shown to enhance sensitivity
to antibiotics that otherwise exhibit low rates of permeability.43 This constitutive expression plasmid does
not require antibiotic selection for stable maintenance, thus eliminating the concern that presence of
either an inducer or a second antimicrobial compound will impact the observed MICs. The ΔacrB strain
lacks function of the AcrAB-TolC multi-drug efflux system, which is responsible for the majority of efflux-
based resistance in E. coli.44-47 The ΔtolC strain lacks the outer membrane protein utilized by resistance-
nodulation- cell division (RND) family efflux pumps and therefore eliminates the function of all multidrug
efflux systems in this family.44, 48-49 Neither the hyperpermeable strain (Supplementary Figure 2B) nor the
efflux mutants (Supplementary Figure 2C and 2D) showed any difference in sensitivity to compounds 1-4.
Though direct interactions of these compounds with LpxC were not determined, the failure to inhibit
bacterial growth suggests that the nucleoside-based structures 1-3 are not effective inhibitors of LpxC as
modeling suggested, nor does the small tail structure of compound 4 result in antimicrobial activity
despite its increased hydrophobicity relative to compounds 1-3.
For P. aeruginosa, the MIC for compound 5 was 100 μg/mL (Figure 3). For E. coli, compound 5
inhibited bacterial growth but it was not possible to determine an MIC (Figure 4A). Despite sharing 57.4%
sequence identity and 79.7% sequence similarity for LpxC, there are modest differences between the
structures of the P. aeruginosa and E. coli enzymes that could account for this difference.36 It is also
possible that differences in outer membrane permeability or efflux activity between these species account
for the observed difference. In the E. coli strain expressing ΔCΔ4LFhuA, the MIC for compound 5 was 100
μg/mL (Figure 4B) while in the ΔacrB and ΔtolC efflux mutants, the MIC was modestly reduced to 80 μg/mL
(Figure 4C and 4D). From these results we conclude that the intermediate hydrophobicity of compound 5
limited its permeability across the E. coli outer membrane but was still sufficient for compound that did
enter to be targeted by the AcrAB-TolC efflux pump. When permeability was enhanced or efflux
eliminated, compound 5 was able to prevent E. coli growth.
9

Figure 3. Compound 5 completely inhibits growth of P. aeruginosa at 100 μg/mL. Compound 5 delays
growth of P. aeruginosa at 25 μg/mL (pink) and 50 μg/mL (dark pink) compared to 1% DMSO alone (blue)
with complete inhibition at 100 μg/mL (red).
Figure 4. Compound 5 completely inhibits growth of E. coli when outer membrane permeability is
enhanced or multidrug efflux eliminated. A) Compound 5 inhibits growth of E. coli in a dose dependent
manner compared to 1% DMSO alone (blue) but a minor amount of growth is still observed at 160 μg/mL
(red). B) Expression of the engineered outer membrane porin ΔCΔ4LFhuA results in complete inhibition
of growth by compound 5 at 100 μg/mL (red). C) The ΔacrB strain which lacks function of the AcrAB-TolC
multidrug efflux system is sensitive to compound 5 with complete inhibition of growth occurring at 80
μg/mL (orange). D) The ΔtolC mutant which lacks function of all RND-family efflux systems is no more
sensitive to compound 5 than the ΔacrB strain and also exhibits complete inhibition of growth at 80
μg/mL (orange).
For compound 6, no MIC could initially be established for either P. aeruginosa (Supplementary Figure
5) or E. coli (Figure 5A). Similarly, no MIC could be determined in the hyperpermeable E. coli strain
expressing ΔCΔ4LFhuA (Figure 5B). It was possible to determine an MIC of 32 μg/mL in the ΔacrB strain
10

(Figure 5C) and 16 μg/mL in the ΔtolC strain (Figure 5D). Expressing ΔCΔ4LFhuA in combination with either
the ΔacrB or ΔtolC mutation did not further alter the MICs (data not shown). These results demonstrate
that compound 6, the most hydrophobic compound tested, was not limited in its ability to enter E. coli
cells but rather lack of inhibition was due to the activity of multiple drug efflux systems. The AcrAB-TolC
multidrug efflux system was responsible for the majority of the intrinsic resistance of E. coli to this
compound. However, compound 6 was a substrate for other RND-family efflux systems as well since
deletion of tolC lead to a further 2-fold decrease in MIC. It is likely that we were unable to observe
inhibition of P. aeruginosa growth by compound 6 due to similar efflux mechanisms in this organism.
Intrinsic antibiotic resistance in P. aeruginosa is mediated primarily by the MexAB-OprM efflux system,
which is also an RND-family efflux pump with a broad substrate range though, as in E. coli, multiple efflux
systems are present in the genome.50-53
Figure 5. Compound 6 completely inhibits growth of E. coli when multidrug efflux is eliminated. A)
Compound 6 fails to inhibit growth of E. coli compared to 1% DMSO alone (blue), at the solubility limit of
80 μg/mL (darkest purple). B) Expression of ΔCΔ4LFhuA does not result in growth inhibition by 80 μg/mL
compound 6 (purple) compared to 1% DMSO (blue). C) A ΔacrB mutant lacking function of the AcrAB-TolC
multidrug efflux system is inhibited by compound 6 in a dose-dependent manner compared to 1% DMSO
(blue) with complete inhibition at 32 μg/mL (red). D) A ΔtolC mutant lacking function of all RND-family
efflux systems displays increased sensitivity to compound 6 with complete inhibition at 16 μg/mL.
It is notable that both compounds 5 and 6 demonstrated some degree of bacterial growth inhibition,
while compound 4 did not. While its lower hydrophobicity may have reduced its ability to enter cells, the
11

relatively small size of this compound should allow it to readily enter the ΔCΔ4LFhuA-expressing strain.
Lack antimicrobial activity by compound 4 in this genetic background suggests that a larger hydrophobic
tail may be required to mediate the molecular interaction, presumably with LpxC, that results in growth
inhibition. This is corroborated by the modest inhibition seen with the slightly larger tail of 5 and greatest
inhibition with the largest and most hydrophobic tail of 6. This effect may also be explained by the entropic
contributions to the Gibbs energy of binding. As has been mentioned, the active site has a large polar
region as well as a metal-binding region. This suggests that waters bound in this active site before ligand
binding are held tightly and a large desolvation energy penalty is paid when the ligands bind, greatly
reducing the enthalpic contribution to the Gibbs energy of binding.27 The entropic contribution is thus
proportionately more important. When comparing 4 with 5 and 6, we see that the latter two have
considerably more degrees of freedom in the hydrophobic tail, and thus more entropy that is lost upon
binding (assuming that the hydrophobic tail assumes a somewhat rigid position upon binding). This will
be explored further in future computational work.
Unfortunately, the increased hydrophobicity also seems to potentiate efflux. Little structural
similarity has been observed among known substrates of RND efflux systems but one common feature
54
appears to be hydrophobicity, with more hydrophobic compounds exhibiting increased efflux.35,
Development of AcrAB-TolC inhibitors is an area of significant interest since they would function as
adjuvants for a range of antimicrobial compounds but none of these inhibitors has yet entered clinical
trials.55-61 Future work must focus on developing structures that are effective inhibitors of LpxC without
being targeted as substrates for efflux.
Inhibition of bacterial growth by compounds designed to target LpxC activity is a promising method
of antibacterial treatment that may be useful against multidrug resistant bacteria. We investigated the
use of nucleoside-containing compounds 1-3 designed to mimic the natural substrate of LpxC and interact
with the polar region of the binding site, the Zn2+ ion and the hydrophobic region (in 2 and 3). Although
the nucleoside provides additional points for interaction, based on modeling, it also decreases the log P
of the potential inhibitors. Therefore, we sought to increase the lipophilicity of the potential inhibitors
and removed the nucleoside and subsequently increased the lipophilicity. Furthermore, the removal of
the nucleoside simplifies the synthetic pathway, allowing access to the compounds in higher yields and
fewer steps. Compound 2 was synthesized in 7 steps from methyl 4-hydroxybenzoate in a yield of 19%.
Compound 4 was fully synthesized in 3 steps with an overall yield of 69%. Compound 5 was fully
synthesized in 5 steps with an overall yield of 61%. Compound 6 was synthesized in in 3 steps in an overall
12

58% yield. Ultimately, the use of a methyl ester instead of the THP-protecting group resulted in higher
yields and fewer steps and purifications (58% versus 38% for compound 6).
Nucleoside-containing propargylglycine compounds 1-3 were not effective at inhibiting the growth
of E. coli or P. aeruginosa but activity was observed for other propargylglycine compounds. The non-
nucleoside compound 4 was also not effective in inhibiting the growth of either E. coli or P. aeruginosa.
The most effective activity was displayed by compound 6, which contains a large hydrophobic biphenyl
tail, while modest antimicrobial activity was observed for compound 5 containing a phenyl propargyl ether
tail. Interestingly, compound 5 and Piizzi-1 (MIC = 2 µg/mL)38 display different MICs for P. aeruginosa
despite sharing a common tail structure. They differ in the side chain of the amino acid with compound
5 having an alkyne and Piizzi-1 has a dimethylamino group which suggests that, while presence of the full
nucleoside did not improve activity, presence of a different polar group may. The ArcAB-TolC multidrug
efflux system provided significant intrinsic resistance to both of these compounds in E. coli with the
greatest efflux-mediated resistance observed for compound 6. The seeming requirement of a large
hydrophobic tail for effective growth inhibition therefore presents a significant challenge for future
inhibitor design.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial,
or not-for-profit sectors.
Acknowledgments: High resolution mass spectra were recorded on a Waters Synapt High Resolution Mass
Spectrometer housed at the University of Memphis, Department of Chemistry. Prof. Paul Simone and Mr.
Drake Williams (University of Memphis) are thanked for assistance with obtaining HRMS data. The
authors also thank Prof. Liviu Movileanu and Syracuse University for the gift of pPR-IBA1-FhuAΔC/Δ4L.
Supplementary Material: Supplementary data (full synthetic procedures, Figure S1-S3: growth curves and
Figure S4-S12: 1H and 13C NMRs of final compounds 2, 4-6) associated with this article can be found, in the
online version, at
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17

Highlights


Novel propargylglycine-based antimicrobial compounds have been synthesized.
Compounds with a phenyl propargyl ether or biphenyl tail exhibited antimicrobial
activity.

Enhancing outer membrane permeability or eliminating efflux in E. coli led to a reduced
MIC.
18