Structure-function studies of polymyxin B lipononapeptides

Article abstract of DOI:10.3390/molecules24030553

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there h

Full text of DOI:10.3390/molecules24030553

molecules
Article
Structure-Function Studies of Polymyxin
B Lipononapeptides
Alejandra Gallardo-Godoy , Karl A. Hansford * , Craig Muldoon, Bernd Becker,
Alysha G. Elliott , Johnny X. Huang , Ruby Pelingon, Mark S. Butler
Mark A. T. Blaskovich * and Matthew A. Cooper *
,
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia;
a.gallardogodoy@uq.edu.au (A.G.-G.); cmouldy@yahoo.com.au (C.M.); bbeckerr@gmail.com (B.B.);
a.elliott@imb.uq.edu.au (A.G.E.); johnny.xiao.huang@gmail.com (J.X.H.); r.pelingon@imb.uq.edu.au (R.P.);
m.butler5@uq.edu.au (M.S.B.)
* Correspondence: k.hansford@uq.edu.au (K.A.H.); m.blaskovich@uq.edu.au (M.A.T.B.);
m.cooper@uq.edu.au (M.A.C.); Tel.: +61-7-3346-2107 (K.A.H.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 December 2018; Accepted: 27 January 2019; Published: 2 February 2019
Abstract: The emerging threat of infections caused by highly drug-resistant bacteria has prompted a
resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort
therapies. Given the emergence of resistance to these drugs, there has also been a renewed
interest in the development of next generation polymyxins with improved therapeutic indices
and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides
structurally characterised by an exocyclic FA-Thr2-Dab3 lipodipeptide motif instead of the native
FA-Dab1-Thr2-Dab3 tripeptide motif found in polymyxin B, removing one of the positively charged
residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase
synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position
2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria,
including polymyxin-resistant strains. Pairwise comparison of N-acyl nonapeptide and decapeptide
analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly
influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights
that antimicrobial potency may be retained upon truncation of the N-terminal L-Dab-1 residue of the
native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of
next generation polymyxins.
Keywords: polymyxin; antibiotic resistance; antibiotics; nonapeptide; lipopeptide
1. Introduction
The polymyxins (Pmx) are natural product polycationic lipodecapeptides produced by
Paenibacillus polymyxa (Figure 1), exemplified by polymyxin B
as colistin) [ ]. First discovered in 1947, with subsequent studies reporting the isolation and
characterization of additional Pmx derivatives from natural product sources [ ], PmxB and
colistin have been part of the clinical antibiotic repertoire for over 50 years, albeit approved
for human use in an era with less stringent regulatory requirements compared to contemporary
standards. However, toxicity issues, in particular nephrotoxicity [ 10], led to their gradual
replacement with safer alternatives. The past decade has seen increasing application of ‘last-resort’
antibiotics due to the ominous rise of infections caused by extended-spectrum -lactamase- (ESBL)
1 (PmxB) and E 2 (also known
1–4
5–
8
1
2
9
,
β
and carbapenemase-producing strains of Acinetobacter baumannii, Pseudomonas aeruginosa and
Enterobacteriaceae, with some strains now exhibiting multidrug resistance to practically all known
Molecules 2019, 24, 553; doi:10.3390/molecules24030553
www.mdpi.com/journal/molecules

Molecules 2019, 24, 553
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antibiotics [11–14]. New treatments are urgently needed, but scientific [15] and economic [16] hurdles
have slowed progression of the antibiotic clinical pipeline, particularly for Gram-negative therapies [17].
This has prompted a resurgence in the use of PmxB and colistin in spite of their toxicity, as well as
renewed interest in the creation of improved analogues, with significant effort focused on optimising
dosing strategies [18,19], developing a deeper understanding of structure-toxicity relationships [20,21],
and investigating the clinical implications of the increasing prevalence of Pmx-resistance, which has
come to the fore in recent years [22].
Figure 1. Structures of polymyxin B 1 and polymyxin E (colistin) 2.
The polymyxins act initially by binding to lipid A, the membrane-anchoring component of
lipopolysaccharide (LPS), which decorates the outer membrane of Gram-negative bacteria. The anionic
nature of LPS facilitates electrostatic interaction with the pentacationic polymyxins. This initial
interaction then leads to disruption of bacterial outer membrane permeability barrier through
destabilisation of the LPS layer, and hydrophobic insertion of the fatty acyl chain of polymyxin into
the lipid domain of lipid A. Subsequently, cytoplasmic membrane disruption and potential additional
intracellular interactions lead to cell death [23]. In this context, an important structural feature of the
polymyxins is the presence of multiple positively charged L-α-γ-diaminobutyric acid (Dab) side chains,
which interact with the phosphate groups on lipid A [24]. However, this interaction alone is insufficient
to kill bacteria, as polymyxins lacking a fatty acyl tail are poor antibiotics. For example, polymyxin B
nonapeptide (PMBN), which contains an N-terminal Thr2-Dab3 dipeptide motif lacking a fatty acyl
tail, is not antibacterial [25]. Thus, effective bacterial killing requires the concomitant interaction of
LPS and the bacterial outer membrane with the Dab side chains and the lipid tail of polymyxin.
Over the years, several groups have attempted to develop next generation polymyxins with
improved safety profiles. New compounds have been reported by Cubist [26], Pfizer [27], Cantab
Anti-Infectives [28], MicuRx [29], University of Barcelona [30] and Northern Antibiotics [31]. Most
strategies have focused on developing analogues designed to include the native lipodecapeptide
structure of Pmx, as both the cyclic heptapeptide ring and the exocyclic lipotripeptide sequence are
generally required for optimal antimicrobial activity. In contrast, the team led by Vaara at Northern
Antibiotics have demonstrated antimicrobial potency for nonapeptide variants of Pmx in which the
exocyclic fatty acyl-diaminobutyryl-threonyl-diaminobutyryl (FA-Dab1-Thr2-Dab3) linear tripeptide
segment of Pmx was substituted with a truncated fatty acyl-dipeptide motif (FA-Thr2-XX3, where
XX = D-Thr or D-Ser), exemplified by their compound NAB739 [31]. More drastic changes have
been reported, including des-fatty acyl derivatives [32
antimicrobial activity, and instead act as membrane sensitizers that potentiate the activity of other
antibiotics. PMBN is an archetypal example [25 34]. Spero Therapeutics have advanced a related
,33], but such compounds usually lack intrinsic
,
analogue SPR-741 (formerly known as NAB741) into Phase I clinical trials; it maintains the PmxB
heptapeptide ring, but incorporates an exocyclic fatty acyl-dipeptide motif Ac-Thr2-D-Ser3 [35].

Molecules 2019, 24, 553
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We recently reported a systematic activity-toxicity study of PmxB, with a predominant focus on
analogues maintaining the lipodecapeptide structure but with variations at every position [36]. As an
extension of this study, we sought to examine the effect of N-terminal Dab-1 truncation, removing one
of the positive charges purported to be associated with nephrotoxicity, leading to nonapeptide variants
bearing an exocyclic lipodipeptide motif FA-aa2-aa3 (aa = amino acid) instead of the native tripeptide
motif FA-Dab1-Thr2-Dab3 of Pmx (Figure 2). Herein we report the synthesis and biological evaluation
of 36 unique Pmx nonapeptide analogues, alongside comparative biological data for 10 compounds
that have been reported previously [33,36–39]. In the new nonapeptide series, we examined the effect
of altering the fatty acid component, as well as the influence of variations at positions P2, P3 and P4
(numbering based on original Pmx decapeptide scaffold, with P4 the diamino acid residue involved
in peptide cyclisation). Collectively the data has enabled a side-by-side comparison of truncated
Pmx lipononapeptides versus their lipodecapeptide counterparts, in turn providing insight into the
relative contribution of Dab-1 to antimicrobial potency. PmxB
1 and colistin 2 remained the most
potent of all the compounds tested, but some nonapeptide analogues possessed similar potency to their
decapeptide counterparts, contingent upon the fatty acyl component. Selected analogues also showed
moderate activity toward a polymyxin-resistant clinical isolate of P. aeruginosa without appreciable
cytotoxicity against human proximal tubular epithelial cells (HK-2).
O
O
FA-1
FA-2
O
FA-3
O
H2N
FA-aa2-aa3
O
FA-4
O
O
O
P2
HN
H
NH
N
N
H
NH
N
P4
O
O
FA
H
O
O
O
FA-8
FA-9
Cl
P3
n
O
HN
NH
O
O
H
N
NH2
HN
NH
O
N
O
OH
O
FA-5
FA-7
NH2
FA-6
O
FA-11
OH
O
O
FA-10
Figure 2. General structure of polymyxin B nonapeptides and fatty acyl tails used in this study.
2. Results and Discussion
2.1. Chemistry
A total of 36 compounds were synthesized in this study (ten of which have been reported
previously [33 36 39]), with the structures presented in Tables 1–3 and Table S1 (Supporting
,
–
Information). All compounds possessed >95% purity, as determined by LCMS analysis using both
ELSD and UV (210 nm) detection. The compounds were prepared by solid phase peptide synthesis
(SPPS) (Scheme 1). Several SPPS strategies to construct the polymyxin scaffold have been reported,
starting from the C-terminal Leu-7 [27] or Thr-10 [40,41] residue attached to the resin through the
carboxyl terminus. Both strategies require a subsequent solution phase cyclisation step. On the other
hand, on-resin cyclisation has been reported using Dab-9 as the anchoring point, which was attached
to the resin via the Nγ-amino group of the side chain [42]. In the present study, which utilizes an
on-resin cyclisation strategy, the polymyxin scaffold was constructed by anchoring the side chain
β
-hydroxy group of the C-terminal Thr-10 residue onto a dihydropyran DHP HM resin, as previously
reported (Scheme 1) [36]. The C-terminal carboxylic acid of Thr-10 and the Nγ-amino group of Dab-4
were masked as allyl ester and carbamate protecting groups, respectively, allowing for orthogonal
deprotection in the presence of the remaining Boc-protected Dab side chain amino groups en route
to on-resin cyclisation. Thus, the synthesis was initiated using resin-bound Fmoc-L-Thr-CO2Allyl
3
(Scheme 1) [36]. The peptide sequence was constructed using SPPS with sequential Fmoc deprotection
(30% piperidine in DMF) followed by Fmoc-amino acid coupling (HCTU, DIPEA in DMF), leading to

Molecules 2019, 24, 553
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the intermediate heptapeptide construct
4. On-resin cyclisation was effected by in situ deprotection
of both the C-terminus of Thr10 and the Dab-4 side chain amine using Pd(PPh3)4 and PhSiH3,
generating 5, which was then treated with DPPA and DIPEA in DMF overnight at room temperature
to give the cyclised resin-bound intermediate
Dab-4 -amino Fmoc group followed by the sequential addition of the linear exocyclic tail residues
Fmoc-L-Dab(Boc)-OH and Fmoc-L-Thr(tBu)-OH to give the penultimate resin-bound precursor
Polymyxin B nonapeptide (PMBN) was prepared from by sequential Fmoc removal from Thr-2
followed by treatment with TFA/Et3SiH/H2O (95:1:4), which liberated the peptide from the resin
with concomitant side chain deprotection. Analogues 10 12 14 16 28 30 44 and 46 47 were also
prepared from by sequential Fmoc removal from Thr-2 followed by acylation with the appropriate
6. The synthesis was then completed by removal of the
α
7.
7
3
–
,
–
,
–
,
–
–
7
fatty acid, and cleavage/deprotection using TFA/Et3SiH/H2O (95:1:4). All compounds were purified
by rp-HPLC and isolated as their TFA salts.
NHBoc
NHBoc
O
O
O
H
N
H
i
FmocHN
ii
FmocHN
N
O
O
N
H
N
N
H
H
O
O
HN
Fmoc SPPS
O
O
O
HN
O
NHBoc
O
O
3
O
4
BocHN
O
NHBoc
NHBoc
O
O
O
O
O
NH
HN
H
N
H
N
FmocHN
FmocHN
iii
i
P4
N
H
N
N
NH
O
H
H
O
O
O
HN
HO
O
NH
HN
NH2
NHBoc
O
O
O
NH
O
HN
NHBoc
5
O
NHBoc
6
BocHN
H2N
P2
O
OH
O
OtBu
O
O
O
O
H
H
N
NH
N
HN
HN
N
N
N
iv or v
R
H
N
P4
H
H
FmocHN
H
P4
O
O
NH
O
O
NH
O
O
NH2
NH
NHBoc
NH
HN
O
HN
P3
O
H
N
NH
O
NH
O
HN
NHBoc
NH2
O
O
OH
7
O
PMBN (R = H)
8 (R = nC7H15CO)
NHBoc
NH2
20 (R = nC7H15CO-L-Dab; P3 = D-Dab)
Scheme 1. General on-resin cyclisation synthetic route to polymyxin nonapeptides, exemplified by the
a
synthesis of PMBN, compound
8
and compound 20 a
.
Reagent and conditions: (i) (a) 30% piperidine,
DMF; (b) Fmoc-amino acid, HCTU, DIPEA, DMF; (c) repeat; (ii) Pd(PPh3)4, PhSiH3; (iii) DPPA, DIPEA,
DMF; (iv) (a) 30% piperidine, DMF; (b) TFA/Et3SiH/H2O (95:1:4); (v) (a) 30% piperidine, DMF;
(b) octanoic acid, HCTU, DIPEA, DMF; (c) TFA/Et3SiH/H2O (95:1:4).

Molecules 2019, 24, 553
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Table 1. Summary of antimicrobial activity (MIC, mg/L) and cytotoxicity (CC50, µM) against HK-2 Cells for a series of polymyxin-B nonapeptide (PMBN) derivatives
acylated with different fatty acids and varying the P4 residue.
E. coli
ATCC 25922
K. pneumonia
ATCC 700603
K. pneumonia
ATCC 13833
K. pneumonia
BAA 2146
A. baumannii
ATCC 19606
P. aeruginosa
ATTC 27853
S. aureus
ATCC 25923
CC50
HK-2
Strains 1
PmxB 1
Col 2
Gent
1
0.5
0.25
8
1
0.25
0.25
>32
>32
0.25
0.25
32
0.5
0.5
>32
>32
>32
>32
0.5
2
>300
>300
>300
>300
0.25
0.25
>32
0.25
0.25
>32
Ctrls
ID
Vanc
>32
>32
FA
P1
P2
P3
P4
PMBN a
–
–
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Dab
L-Orn
L-Lys
>32
0.5
1
>32
4
8
2
1
8
32
1
2
16
>32
>32
1
2
>32
16
32
4
>32
2
1
>32
>32
>32
16
>32
0.5
2
>32
16
8
>32
0.5
2
>32
>32
>32
32
2
0.5
0.5
32
1
2
1
1
2
1
0.5
2
16
>32
>32
>32
>32
>32
>32
>32
8
>32
>32
>32
>32
>32
>32
>32
>300
>300
209
79
>300
93
PmxB3 b
8 c
FA-1
FA-1
FA-1
FA-5
FA-4
FA-6
FA-2
FA-3
FA-7
FA-7
FA-1
FA-1
FA-1
L-Dab
–
–
–
–
–
–
–
–
–
–
–
–
9 d
10
11
12
13
2
1
46
L-Thr L-Dab
2
8
8
189
182
206
>300
88
14
32
32
2
16
>32
>32
>32
>32
2
32
>32
>32
16
32
4
16
>32
>32
>32
>32
1
>32
>32
>32
15
16 e
17
18
19
88
>300
D-Dab
1
a
ATCC 700603, ESBL; BAA-2146, NDM-1 positive. PMBN = polymyxin B nonapeptide [25]; b PmxB3 = polymyxin B3 [33]; c See ref. [36 37]; d P3 = L-Thr; P2 = L-Dab; e See ref. [36
, ,39].

Molecules 2019, 24, 553
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Table 2. Summary of antimicrobial activity (MIC, mg/L) and cytotoxicity (CC50
acylated with different fatty acids and varying the P2 residue.
, µM) against HK-2 cells for a series of polymyxin-B nonapeptide (PMBN) derivatives
E. coli
ATCC 25922
K. pneumonia
ATCC 700603
K. pneumonia
ATCC 13833
K. pneumonia
BAA 2146
A. baumannii
ATCC 19606
P. aeruginosa
ATTC 27853
P. aeruginosa
FADDI PA070
S. aureus
ATCC 25923
CC50
HK-2
Strains 1
PmxB 1
Col 2
Gent
1
0.5
0.25
8
1
0.25
0.25
>32
>32
0.25
0.25
32
0.5
0.5
>32
>32
32
>32
32
>32
>32
0.5
2
>300
>300
>300
>300
0.25
0.25
>32
0.25
0.25
>32
Ctrls
ID
Vanc
>32
>32
>32
FA
P1
P2
P3
20
21
22
23
24
25
26
27
28
29
30
31
32
33
FA-1
FA-1
FA-7
FA-8
FA-9
FA-2
FA-10
FA-3
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
L-Dab L-Thr
4
0.5
16
8
4
0.5
1
1
1
16
8
8
1
0.5
4
16
32
>32
4
4
1
16
16
4
0.5
1
8
>32
>32
8
8
>32
>32
1
8
>32
8
32
2
2
2
4
4
2
2
2
4
4
1
2
4
1
>32
8
>32
2
4
8
>32
8
8
8
16
8
>32
>32
>32
4
183
289
>300
29
–
–
–
–
–
–
–
–
–
–
–
–
–
L-Thr
L-Thr
L-Thr
L-Thr
L-Thr
L-Thr
L-Thr
16
60
2
16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>300
>300
>300
>300
>300
>300
181
1
4
32
D-Dab
4
16
>32
16
4
8
32
32
>32
>32
>32
>32
>32
>32
>32
L
-Asn
32
16
8
16
>32
16
>32
>32
>32
>32
>32
>32
L-Tyr
L-Ser
L
-Hse
-Glu
L
>32
>32
>300
262
Gly
1
ATCC 700603, ESBL; BAA-2146, NDM-1 positive; FADDI PA070, Pmx-resistant.

Molecules 2019, 24, 553
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Table 3. Summary of antimicrobial activity (MIC, mg/L) and cytotoxicity (CC50, µM) against HK-2 cells for a series of polymyxin-B nonapeptide (PMBN) derivatives
acylated with different fatty acids and varying the P3 residue.
E. coli
ATCC 25922
K. pneumonia K. pneumonia K. pneumonia A. baumannii
P. aeruginosa
ATTC 27853
S. aureus
ATCC 25923
CC50
HK-2
Strains 1
ATCC 700603
ATCC 13833
BAA 2146
ATCC 19606
PmxB 1
Col 2
Gent
1
0.5
0.25
8
1
0.25
0.25
>32
>32
0.25
0.25
32
0.5
0.5
>32
>32
>32
>32
0.5
2
>300
>300
>300
>300
0.25
0.25
>32
0.25
0.25
>32
Ctrls
Vanc
>32
>32
ID
FA
P1
P2
P3
34 a
35
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-1
FA-10
FA-11
L-Dab
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
D-Ser
L-Ser
D-Ser
L-Hse
L-Asn
L-Dap
L-Orn
D-Orn
L-Lys
D-Lys
L-Cit
L-Arg
L-Trp
L-His
D-His
Gly
4
8
4
16
32
1
2
1
4
1
32
2
>32
4
2
4
0.5
2
1
2
32
>32
>32
>32
1
4
4
16
4
>32
8
32
32
4
>32
1
0.5
>32
>32
>32
>32
1
8
32
16
>32
>32
>32
>32
>32
4
>32
0.5
>32
>32
16
16
16
>32
>32
0.5
4
1
8
4
32
16
>32
16
4
>32
0.5
4
1
>32
32
>32
>32
1
16
16
32
16
>32
32
>32
>32
4
>32
1
16
2
16
16
>32
>32
1
2
4
4
4
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
139
220
213
271
284
36 b
37
38
39
40
41
42
43
44
45
290
>300
>300
>300
>300
>300
>300
>300
>300
>300
163
L-Thr
>32
4
46
47
>32
16
16
>32
2
48
49 a
50
L-Dab
–
–
Gly
Gly
Gly
>300
>300
>300
51
52
16
4
8
4
4
32
1
a
ATCC 700603, ESBL; BAA-2146, NDM-1 positive. See ref. [36]; b See ref. [38].

Molecules 2019, 24, 553
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2.2. Biological Activity
All compounds synthesised in this study were assessed for their minimum inhibitory
concentrations (MIC, mg/L) by broth microdilution assay against five antibiotic-sensitive and
resistant ATCC reference strains covering the Gram-negative ESKAPE pathogens (Escherichia coli,
Klebsiella pneumoniae, A. baumannii, and P. aeruginosa) (Tables 1–3). Staphylococcus aureus was also
included as a representative Gram-positive bacterial strain. Antimicrobial profiling was also performed
against a subset of polymyxin-resistant MDR clinical isolates of K. pneumoniae, A. baumannii, and
P. aeruginosa, but most compounds were inactive (MIC > 32 mg/L), data not shown for K. pneumoniae
and A. baumannii isolates. Polymyxin B 1, colistin 2, vancomycin and gentamicin were used as positive
inhibitor comparator compounds. Compounds were counter-screened against human proximal tubular
epithelial cells (HK-2), using LDH release as a general indicator of cellular toxicity [43–45].
Polymyxins without a fatty acyl tail lack antimicrobial potency, exemplified by PMBN [25], which
was inactive against all strains except P. aeruginosa ATCC 27853 (MIC 2 mg/L) (Table 1). Interestingly,
activity against P. aeruginosa ATCC 27853 was relatively insensitive to structural changes, with most
compounds displaying MICs 1–4 mg/L despite a variety of structural modifications (Tables 1–3).
PMBN potency was restored with incorporation of a C8 tail (octanoic acid, OA), as previously
demonstrated by nonapeptide
(MIC 1–2 mg/L for most strains) [36
tripeptide motif FA-Dab1-Thr2-Dab3 of Pmx, was 2- to 4-fold more active than nonapeptide
8
, which possessed an exocyclic dipeptide motif OA-Thr2-Dab3
37]. In comparison, PmxB3 33] incorporating the native exocyclic
[36,37]
,
[
8
against most strains (Table 1). The observation that Pmx nonapeptides produced by truncation
of L-Dab1 could retain activity when substituted with an appropriate fatty acid, exemplified by
nonapeptide 8, provided impetus to explore this phenomenon further.
In our previous study, we also reported decapeptide 16, possessing a relatively polar
2-chorophenyl urea fatty acyl moiety [36]. This analogue displayed MICs of 1–2 mg/L across most
of the tested Gram-negative strains (Table 1). In contrast, nonapeptide 15, the truncated form of
decapeptide 16, revealed a more striking difference between the two analogues, with 15 possessing
considerably reduced activity during pairwise comparison. This contrasts with the general retention
of activity observed between compound 8 [36,37] and PmxB3 [33]. This variation suggests a greater
influence of L-Dab1 in the presence of a relatively polar fatty acid, and implies that judicious selection
of the fatty acid in nonapeptides lacking the L-Dab1 may compensate for the reduced electrostatic
component by additional hydrophobic interactions between the fatty acid and lipid A. This observation
prompted further exploration of the fatty acyl component of analogue
8. Additional alterations to the
fatty acid tail were generally disfavoured (compare 10 15), with 12 (FA = 4-hexylbenzoic acid) and 13
–
(FA = 6-phenylhexanoic acid) providing the best activities, albeit with increased cytotoxicity and some
activity against S. aureus for 12 (MIC 8 mg/L). Data from this small SAR subset suggests that a simple
N-alkyl fatty acyl chain is sufficient for good antimicrobial potency. The activity of
8 was abolished
when the L-Thr2-L-Dab3 sequence was reversed (9, L-Dab2-L-Thr3).
The size of the macrocyclic ring was examined by substituting the diamino acid L-Dab4 in
8
with the homologs L-Orn4 17 and L-Lys4 18 (Table 1). Both modifications led to reduced activity,
although the L-Orn4 variant was better tolerated, especially against E. coli and P. aeruginosa (MIC
2 mg/L against both). In contrast, reversal of the Dab4 stereochemistry to the D-configuration in 19
was highly detrimental (MIC > 32 mg/L for all strains). The data collectively suggests that optimal
antimicrobial potency is highly dependent on the number of atoms forming the heptapeptide ring and
the absolute configuration at P4.
The observations of Vaara [38] prompted an examination of the effect of reversing the stereochemistry
of L-Dab3 in nonapeptide
somewhat comparable to
8
8
to D-Dab3 in compound 21 (Table 2). Interestingly, the activity of 21 was
, highlighting the tolerance of P3 to stereochemical inversion. A similar
result was observed when the same modification was applied to PmxB3 leading to decapeptide 20
,
which was made for comparison. Furthermore, the activities of both 20 and 21, possessing exocyclic
OA-Dab1-Thr2-D-Dab3 and OA-Thr2-D-Dab3 constructs, respectively, were notably comparable,

Molecules 2019, 24, 553
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suggesting the relative contribution of L-Dab1 toward potency was less important in this pairwise
series compared to compounds 15 and 16 described earlier. Compound 21 was one of the few
analogues examined that was active against a polymyxin-resistant strain of P. aeruginosa FADDI-PA070
(MIC 8 mg/L); cytotoxicity against HK-2 cells was promising (CC50 289
result, variation of the fatty acid component of 21 was explored, providing compounds 22
(Table 2). Analogue 25, containing 6-phenylhexanoic as the fatty acid component, possessed no
observable cytotoxicity at the tested concentrations (CC50 > 300 M) with consistent activity against
µM). Encouraged by this
–
27
µ
the ATCC strains (MIC 0.5–4 mg/L), and moderate activity against polymyxin-resistant P. aeruginosa
FADDI-PA070 (MIC 8 mg/L). On the other hand, more highly lipophilic fatty acids (e.g., 23, 24) were
poorly favoured, instead leading to increased cytotoxicity with accompanying Gram-positive activity
(S. aureus MIC 4–16 mg/L), albeit with potent activity against the polymyxin-resistant P. aeruginosa
FADDI-PA070 strain (MIC 2–4 mg/L). Compound 21 was also modified to include variants at P2,
leading to analogues 28–33 (Table 2). Potency improvements were not observed when L-Thr-2 was
replaced with other hydroxylated amino acids (Tyr, Ser, Hse), nor any of the other amino acids
examined, suggesting that threonine may be optimal at position 2 in nonapeptides bearing a D-Dab3
substituent. Likewise, in the series 28–
33, inversion of the stereochemistry at position 3 (D-Dab3 to
L-Dab3) was unproductive, with all compounds possessing MICs ≥ 16 mg/L (data not shown).
Finally, the promising activity observed for 21, containing D-Dab3, prompted further exploration
of P3 with both D- and L-amino acids, as well as glycine (Table 3). It was previously demonstrated [36
]
that Gly3 was poorly tolerated in nonapeptide 49, in contrast to the potent activity observed for
its decapeptide counterpart 50, again emphasising the influence of L-Dab1 on potency between the
nonapeptide and decapeptide series. Potency could be partially restored in 49 by substitution of the
octanoic acid tail with alternative fatty acids (compounds 51 and 52), albeit with variable activity
across the Gram-negative panel (Table 3). Substitution of D-Dab3 in 21 with other basic amino acids
(His, Lys, Orn, Dap) was generally well tolerated, even with inversion of stereochemistry, with 39
(L-Dap3) possessing the best activity (Table 3). Substitution with histidine was exceptional, with
the D-isomer possessing much more potent activity compared to the L-isomer (compare 47 and 48
Table 3). When D-Dab3 in 21 was modified to provide analogues 35 (L-Ser3) or 36 (D-Ser3), activity
was significantly reduced (MIC 16–32 mg/L for most strains except E. coli where MIC = 4 mg/L),
,
in contrast to data previously reported by Vaara (35 = NAB743, 36 = NAB739) [31
,
38]. Of note, the
decapeptide 34
[
36] possessing an exocyclic OA-Dab1-Thr2-D-Ser3 construct, was significantly more
potent than nonapeptide 36 lacking the L-Dab1 residue, again exemplifying the importance of L-Dab1
between nonapeptide and decapeptide variants. Activity across most strains was essentially abolished
for analogues containing L-Hse3 (37), L-Asn3 (38), L-Cit3 (44) or L-Trp3 (46).
In the present study, we made a series of fatty N-acyl polymyxin nonapeptides, with alterations
of the N-acyl component, and variation of the amino acids at positions P4, P3 and P2. Despite a lack of
equipotency with polymyxin B 1 or colistin 2, selected nonapeptide analogues possessed promising
antimicrobial activity against the panel of five antibiotic-sensitive and resistant Gram-negative ATCC
reference strains tested. Two analogues, 23 and 24, were also active against a polymyxin-resistant
strain of P. aeruginosa, but displayed increased cytotoxicity and activity against the Gram-positive
S. aureus. Pairwise comparison of nonapeptide and decapeptide analogues (compare
8, 9; 15, 16; 20,
21 34 36; and 49
;
,
,
50) revealed that the influence of the L-Dab1 residue on antimicrobial activity is
contingent upon the fatty acyl component; activity was lost with more polar N-acyl components,
but was retained with octanoic acid. Nonapeptides with stereochemical inversion at position 3 (i.e.,
D-Dab3 instead of L-Dab3) retained activity (e.g., 20 and 21), as did analogues possessing alternative
positively charged amino acids (e.g., 39–43). On the other hand, activity was lost upon substitution of
L-Dab3 with Gly3 in a nonapeptide framework, but could be restored in the decapeptide equivalent
due to the presence of L-Dab1 (compare 49 and 50). A similar trend was evident upon substitution of
L-Dab3 with a neutral D-Ser3 residue (compare 34 and 36). Collectively the present study demonstrates

Molecules 2019, 24, 553
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that polymyxin B nonapeptides may find utility in the design of improved polymxin analogues to
fight antibiotic resistant infections.
3. Materials and Methods
3.1. Synthesis
Experimental procedures are described in the Supporting Information. All chemicals were
obtained from commercial suppliers and used without further purification. LC-MS analyses were
conducted using Agilent Technologies 1200 Series Instrument with a G1316A variable wavelength
detector set at
XDB-Phenyl column (3
λ
= 210 nm, 1200 Series ELSD, 6110 quadrupole ESI-MS, using an Agilent Eclipse
100 mm, 3.5 m particle size, flow rate 1 mL/min, the mobile phases 0.05%
×
µ
formic acid in water and 0.05% formic acid in acetonitrile) (Agilent Technologies, Melbourne, Australia).
Compound purification was performed using an Agilent 1260 Infinity Preparative HPLC with a
G1365D multiple wavelength detector set at
λ = 210 nm coupled to an Agilent Eclipse XDB-Phenyl
column (21.2 100 mm, 5 m particle size). Identities of final products were confirmed by high
×
µ
resolution mass spectrometry (HRMS), performed on a Bruker Micro TOF mass spectrometer using
(+)-ESI calibrated to sodium formate (Bruker Daltonics, Melbourne, Australia). Final purity of
more than 95% for all compounds was confirmed by LC-MS analysis using both ELSD and UV
(210 nm) detection.
3.2. Minimum Inhibitory Concentration (MIC) Determination by Broth Microdilution Assay
Bacteria were either obtained from American Type Culture Collection (ATCC; Manassas, VA,
USA) or independent academic clinical isolate collections, as listed in Table S2. Bacteria were cultured
in nutrient broth (NB; Bacto Laboratories, catalog No. 234000) or Mueller Hinton broth (MHB; Bacto
◦
Laboratories, catalog No. 211443) at 37 C overnight with shaking (
∼
180 rpm). A sample of each culture
180 rpm).
was then diluted 50-fold in fresh MHB and incubated at 37 ◦C for 1.5–3 h with shaking (
∼
Compound stock solutions were prepared as 0.64 or 2.56 mg/mL in water. The compounds, at twice the
final desired concentration, were serially diluted 2-fold across the wells of 96-well plates (Polystyrene,
Corning, catalogue No. 3370). Mid log phase bacterial cultures (after 1.5–3 h incubation) were diluted
to 1
×
106 colony forming units (CFU)/mL, and 50
µL was added to each well giving a final compound
concentration range of 32 mg/L to 0.015 mg/L and a final cell density of 5
determined visually after 18 h of incubation at 37 ◦C, with the MIC defined as the lowest compound
concentration at which no bacterial growth was visible.
×
105 CFU/mL. MICs were
3.3. Cytotoxicity (Lactate Dehydragenase (LDH) Assay)
Cytotoxicity to human kidney proximal tubular epithelial cell line, HK-2 (ATCC CRL-2190,
sourced from ATCC; Manassas, VA, USA) was determined using the LDH assay as previously
described [43–45]. In brief, HK-2 cells were seeded as 2000 cells/well in black-walled clear bottom
384-well tissue culture treated plates (Corning, catalogue No. 3712) in DMEM/F12 medium (Gibco®
10565-042) containing 10% of Fetal Bovine Serum (FBS, Gibco® 10099-141) and incubated for 24 h
◦
at 37 C, 5% CO2. Compounds were then added into each well with a concentration series from
300 µM to 2.3 µM in 2-fold dilutions. Colistin and polymyxin B were used as controls and tested
at a final concentration range of 1 mM to 7.8
24 h at 37 C, 5% CO2. After the incubation, 5
µ
M. The cells were incubated with the compounds for
◦
µ
L of culture medium was added to 45 L of LDH
µ
assay buffer (Biovision, K313-500) and incubated for 30 min at room temperature. The absorbance
(ABS) was then read at 450 nm using a Polar Star Omega plate reader. The data was analysed by
Prism 6 software (GraphPad Software, La Jolla, CA, USA). Results were calculated using the following
equation: cytotoxicity %= (ABSsamples − ABSuntreated/ABS1%Triton X-100 − ABSuntreated) × 100.
Supplementary Materials: The following are available online, Table S1: Compound Structures and HRMS
Characterization; Table S2: Bacterial Strains used for Minimum Inhibitory Concentration (MIC) Determinations;

Molecules 2019, 24, 553
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Scheme S1: General On-Resin Cyclisation Synthetic Route to Polymyxin Nonapeptides, Exemplified by Synthesis
of PMBN, Compound 8
and Compound 20; Figure S1: HR-(+)-ESI-TOF-MS of the [M + 2H]2+ mass ion peak of
compound 8.
Author Contributions: Conceptualization: A.G.-G., C.M., B.B., M.S.B., M.A.T.B. and M.A.C.; Data curation:
K.A.H.; Formal analysis: K.A.H., A.G.-G., A.G.E. and M.A.T.B.; Funding acquisition: C.M., B.B., M.S.B.,
M.A.T.B. and M.A.C.; Investigation: K.A.H., A.G.-G., C.M., B.B., A.G.E., J.X.H., R.P. and M.S.B.; Methodology:
K.A.H., A.G.-G., C.M., B.B., A.G.E. and J.X.H.; Project administration: K.A.H., A.G.-G., M.A.T.B. and M.A.C.;
Writing—original draft: K.A.H.; Writing—review & editing: K.A.H., A.G.E., M.S.B., M.A.T.B. and M.A.C.
Funding: This research was funded by NHMRC grants APP1005350, APP1045326 and APP1139609, and NIH
grants R21AI098731/R33AI098731. M.A.T.B., K.A.H., and A.G.E. were supported by Wellcome Trust Seeding Drug
Discovery Award (094977/Z/10/Z) and Wellcome Trust Strategic Grant (104797/Z/14/Z). M.A.C. is a NHMRC
principle research fellow (APP1059354) and also holds a fractional professorial research fellow appointment at the
University of Queensland, with his remaining time as CEO of Inflazome Ltd., a company developing drugs to
address clinical unmet needs in inflammatory disease.
Acknowledgments: We thank Soumya Ramu, Angela M. Kavanagh, Emily Furlong, Maite Amado and Ali Hinton
for technical support with susceptibility assays.
Conflicts of Interest: The authors declare that there are no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).