Activity and Predicted Nephrotoxicity of Synthetic Antibiotics Based on Polymyxin B

Article abstract of DOI:10.1021/acs.jmedchem.5b01593

The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately, their utility is compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have conducted a systematic activity-toxicity investigation by varying eight of the nine polymyxin amino acid free side chains, preparing over 30 analogues using a novel solid-phase synthetic route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened for in vitro cell toxicity. Promising compounds underwent additional testing against primary kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of the new compounds possessed equal or better antimicrobial potency compared to polymyxin B, and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and human primary kidney cells. These initial structure-activity and structure-toxicity studies set the stage for further improvements to the polymyxin class of antibiotics.

Full text of DOI:10.1021/acs.jmedchem.5b01593

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Article
Activity and Predicted Nephrotoxicity of
Synthetic Antibiotics Based on Polymyxin B
Alejandra Gallardo-Godoy, Craig Muldoon, Bernd Becker, Alysha G Elliott, Lawrence H.
Lash, Johnny Xiao Huang, Mark Butler, Ruby Pelingon, Angela M Kavanagh, Soumya
Ramu, Wanida Phetsang, Mark Arnold Thomas Blaskovich, and Matthew A. Cooper
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01593 • Publication Date (Web): 06 Jan 2016
Downloaded from http://pubs.acs.org on January 14, 2016
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Activity and Predicted Nephrotoxicity of Synthetic Antibiotics
Based on Polymyxin B
Alejandra Gallardo-Godoy1, Craig Muldoon1, Bernd Becker1, Alysha G. Elliott1, Lawrence H.
Lash2, Johnny X. Huang1, Mark S. Butler1, Ruby Pelingon1, Angela M. Kavanagh1, Soumya
Ramu1, Wanida Phetsang1, Mark A.T. Blaskovich1* and Matthew A. Cooper1*.
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1)
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland
4072 Australia. E-mail: m.cooper@uq.edu.au, m.blaskovich@uq.edu.au, Fax +61-7-3346-2090,
Phone +61-7-3346-2044
2)
Department of Pharmacology, Wayne State University, School of Medicine, 540 East
Canfield Avenue, Detroit, MI 48201, USA
* co-corresponding authors
ABSTRACT
The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for
infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately their utility is
compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have
conducted a systematic activity-toxicity investigation by varying eight of the nine polymyxin
amino acid free side chains, preparing over thirty analogs using a novel solid-phase synthetic
route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened
for in vitro cell toxicity. Promising compounds underwent additional testing against primary
kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of
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the new compounds possessed equal or better antimicrobial potency compared to polymyxin B,
and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and
human primary kidney cells. These initial structure-activity and structure-toxicity studies set the
stage for further improvements to the polymyxin class of antibiotics.
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KEY WORDS: polymyxin, colistin, antimicrobial resistance, multidrug-resistant bacteria,
antibiotics, nephrotoxicity
INTRODUCTION
Bacterial sepsis kills millions of people every year. In the United States, patients hospitalized for
septicemia or sepsis are more than eight times as likely to die during their hospitalization
compared to those hospitalized for other diagnoses,1 with over 200,000 deaths in 2008.2 Over one
third of patients with sepsis treated in an intensive care unit die in the hospital.3 Sepsis is the
most expensive condition treated in U.S. hospitals, costing more than $20 billion in 2011.4 Sepsis
is most commonly caused by Gram-positive (G+ve) Staphylococcus aureus and Streptococcus
pyogenes and Gram-negative (G-ve) Klebsiella spp., Escherichia coli, and Pseudomonas
aeruginosa.5 Unfortunately there has been an ominous rise in highly drug-resistant G-ve bacteria
that are able to overcome almost every known antibiotic, threatening a return to a ‘pre-antibiotic’
era if new therapies are not discovered. The current clinical pipeline is mainly populated by
G+ve drug candidates.6 One possible approach to antibiotic development is to revisit antibiotics
discovered in the ‘golden age’ of antibiotic discovery (the 1950-70s) to see if they can be
optimized or improved.
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In 1947 two American groups independently discovered antibiotic substances produced by
Bacillus polymyxa.7-9 The same year a group in England described a new antibiotic they named
aerosporin isolated from a strain at first thought to be B. aerosporus, but later shown to be
identical to the strain B. polymyxa.10 Many examples of polymyxin derivatives have since been
isolated from nature, all of them with various degrees of activity against bacteria.11-15 A review
by Stansly published in 1949 on early human clinical use of four distinct polymyxin antibiotic
complexes A, B, C and D concluded that there is “little doubt that polymyxins have exhibited
effective therapeutic activity in certain infections of man produced by Gram-negative bacteria. It
is apparent, however that their application in this field is likely to be limited because of the
occurrence of untoward reactions elicited by material used in the clinical trials.”16 Nonetheless,
two of the polymyxins, Polymyxin B 1 and Polymyxin E (colistin) 2 entered clinical use, and in
recent years have become last-resort antibiotics for multi-drug resistant G-ve infections, fueling a
surge of interest in their use.17-22 The main limitation of the polymyxins is nephrotoxicity, with
adverse renal toxicity observed in over 50% of cases in some studies.23,24 This has led to a
systematic effort to improve dosing regimens.25,26
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Polymyxins 1 and 2 are both positively charged macrocyclic peptide antibiotics composed of
a heptapeptide ring and three amino acid exocyclic tail capped by a mixture of two fatty acids
(see Figure 1), differing only in a D-Phe/D-Leu alteration at position-6. They are highly basic,
containing six 2,4-diaminobutyric acid (Dab) residues, including one residue whose side chain is
used for lactam formation with the C-terminal Thr residue. While Polymyxin B is administered
as the parent compound sulfate salt, colistin is almost always used as a poly-methanesulfonylated
prodrug, colistin methanesulfonate sodium, which spontaneously hydrolyses to release
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Polymyxin E 2. Given that the polymyxins are natural products produced by fermentation, their
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composition does vary considerably between manufacturers.27
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Figure 1. Structures of Polymyxin B 1 and Polymyxin E (Colistin) 2
The antimicrobial activity of the polymyxins is initiated by binding to Lipid A, the
membrane-anchoring component of lipopolysaccharide (LPS) found in the outer membrane of
G–ve bacteria.17,28,29. Displacement of membrane-stabilizing divalent cations and insertion of the
fatty acyl chain expands and weakens the bacterial outer membrane, causing cell leakage and
allowing polymyxin entry.17,30,31 Once inside the cell, further modes of action have been
postulated, including the generation of hydroxyl radical production32 and inhibition of the
respiratory enzyme NADH-quinone oxidoreductase.33,34
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Unfortunately, resistance to the polymyxins is appearing and has been identified in most
major pathogens, including A. baumannii, K. pneumoniae and P. aeruginosa.17 The main
mechanisms of resistance involve alterations to LPS, in particular via addition of
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phosphoethanolamine and/or L-4-amino-arabinose to Lipid A, which lowers the binding affinity
with the Dab residues of the polymyxins due to reduction of negative charge.17,35,36 This increase
in resistance, coupled with the growing importance of polymyxins in treating highly drug-
resistant G-ve infections, has led to a number of efforts in recent years to prepare polymyxin
analogs to understand structure-activity relationships, improve potency, reduce nephrotoxicity
and overcome resistance. Despite their length of time in use, there have been relatively few
structure-activity relationship (SAR) studies conducted to date compared to other classes of
antibiotics, with most publications focusing on the readily modified exocyclic tail (Pfizer,37-39
Novacta Biosystems Ltd/Cab-Tab Anti-Infectives Ltd,40-42 Northern Antibiotics,43-45 Sakura et
al.46) or selective/nonselective modifications of the multiple Dab side chains (Northern
Antibiotics,43-45 Pfizer,37-39,47 and Schering-Plough48). Biosource Pharm Inc/Cubist developed a
method for fatty acid deacylation and linear tail amino acid hydrolysis, followed by
reconstruction of the linear chain.49-54 From this study, CB182,80455 entered Phase I clinical
trials, but development was discontinued in 2009. In one of the few studies to investigate changes
within the polymyxin ring system, Li et al. at Monash University patented a set of polymyxin
derivatives that retain activity against polymyxin-resistant strains; this group centered their
efforts on the variation of hydrophobicity at positions 6 and 7 (numbering as in Figure 1).56-58
Early efforts at polymyxin SAR were summarized by Storm et al. in 1977,59 while more recent
developments were reviewed by Velkov et al. in 201030 and Vaara in 2013.60
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A set of novel polymyxin derivatives developed by Northern Antibiotics, with the exocyclic
Dab-1 residue removed/substituted and the fatty acid tail altered, were found to be less toxic than
polymyxin B61 based on cytotoxicity testing in the porcine renal proximal tubular cell line LLC-
PK1. It was proposed that this was due to a reduction in the number of positive charges coupled
with alteration of hydrophobicity of the fatty acid. Based on these results, and the improved
activity of the Monash compounds, we decided to investigate the effects of alterations at other
positions within the polymyxins, with a particular focus on modifying the basic Dab residues
contained within the ring to alter their basicity. Given that the Dab residues are proposed to be
integral to binding to Lipid A, and that mutation of Lipid A to introduce phosphoethanolamine is
a key polymyxin resistance mechanism, we also wanted to examine whether introducing
zwitterionic residues or substituents capable of hydrogen-bonding at these positions might help
overcome resistance.
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Twenty-six new analogs were prepared and assayed. In addition, six key compounds from the
literature were also prepared to provide a systematic SAR comparison assessed under common
conditions. Some of the compounds retained better antimicrobial activity against polymyxin-
resistant strains compared to polymyxin B and colistin . Most importantly, some of the most
promising compounds showed less cytotoxicity than polymyxin B and colistin when tested in
mammalian cell lines, and less toxicity than polymyxin B, colistin and gentamicin in a
cytotoxicity assay potentially predictive of nephrotoxicity as measured by LDH and GGT release
from human primary kidney cells.
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RESULTS AND DISCUSSION
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Chemistry. A total of 32 compounds were synthesized (six of these have been reported
previously46-48,53,54,57,58) with the structures presented in Tables 1 and S1 (Supporting
Information). All compounds possessed > 95% purity, as determined by LCMS analysis using
both ELSD and UV (210 nm) detection. The synthetic routes towards the final compounds in this
library are summarized in Schemes 1, 2 and S2 (Supporting Information). The first solid-phase
total synthesis of polymyxin B1 was developed in 1999 by Sharma62 using Fmoc-Thr(tBu)-
SASRIN resin, with the linear peptide cleaved from the resin and cyclized in solution. In the
current study we present a new synthetic route to the polymyxins employing an on-resin
cyclization that allows for alteration of any substituent position other than Thr-10 (see Scheme 1
for a representative synthesis of 10). The first step towards this library of compounds was the O-
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allyl ester protection of the C-terminal amino acid Fmoc-L-Thr-OH 3 (Thr10 in the final
product). The side-chain alcohol of the protected amino acid 4 was then loaded onto a
dihydropyran DHP HM resin (3,4-dihydro-2H-pyran-2-yl-methoxymethyl polystyrene). The
peptide sequence was constructed by standard solid-phase peptide synthesis (SPPS) using
sequential Fmoc deprotection followed by amino acid attachment using a systematic double
coupling protocol, including a capping step with acetic anhydride and pyridine (50:50 v/v) to
avoid the formation of deletion products due to unreacted free amino groups. On-resin
cyclization was conducted following incorporation of the Fmoc-Dab(Alloc) residue by in situ
deprotection of both Thr10 allyl ester and the Dab-4 side chain amine Nɣ-Alloc protecting groups
(Scheme 1, step vii), followed by overnight cyclization using DPPA and DIPEA in DMF at room
temperature. Fmoc deprotection of the cyclized product then allowed completion of the exocyclic
linear tail by further amino acid couplings. Following each step, an analytical sample of resin
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was cleaved with TFA/Et3SiH/H2O (95:1:4) followed by LCMS analysis to confirm the coupling
with the corresponding amino acid. The final coupling capped the peptides with the appropriate
fatty acid. Cleavage of the cyclic decapeptides from the resin with concomitant side chain
deprotection was achieved with the addition of a solution of TFA/Et3SiH/H2O (95:1:4) for 30
min at room temperature, providing crude compounds that were then HPLC-purified to afford the
final polymyxin analog TFA salts as white powders; total yields were generally between 5-30%
based on the initial resin loading.
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Scheme 1. General on-resin cyclization synthetic route exemplified by synthesis of 10.
Reagent and Conditions: (i) Cs2CO3, MeOH, H2O, pH 7-8; (ii) Allyl bromide, dry DMF; (iii)
DHP polystyrene resin, PPTS, dry DCE; (iv) 30% piperidine, DMF; (v) Solid-phase peptide
synthesis (SPPS) with corresponding amino acid (Table S3), HCTU, DIPEA (vi) Acetic
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anhydride, pyridine; (vii) Pd(PPh3), PhSiH3; (viii) DPPA, DIPEA, DMF; (ix) 4-Ph-PhCO2H,
HCTU, DIPEA; (x) TFA/Et3SiH/H2O (95:1:4).
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For some syntheses, an orthogonal protecting group was employed for one of the multiple
Dab residues, allowing for selective modification of individual Dab side chains. Thus, side-chain
ivDde-protected Dab was used at positions P5, P8 or P9 (see Scheme 2 for generic route; Scheme
S1 for explicit synthesis of 18 and 19). The P4 Alloc-protected Dab was deprotected and used for
cyclization as before, with the fully protected resin-bound peptide treated with 2% hydrazine to
selectively unmask the desired Dab residue. This was then derivatized by coupling with Boc-
Arg(Pbf)-OH or Boc-Glu(OtBu)-OH, with global deprotection and purification as previously
described.
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Scheme 2. General on-resin cyclization synthetic route with selective Dab modification used
for synthesis of compounds 18, 19, 22, 23, 31 and 32. Reagent and Conditions: (iv) 30%
piperidine, DMF; (v) Solid-phase peptide synthesis (SPPS) with corresponding amino acid
(Table S3), HCTU, DIPEA (vi) Acetic anhydride, pyridine; (vii) Pd(PPh3), PhSiH3; (viii) DPPA,
DIPEA, DMF; (ix) n-C7CO, HCTU, DIPEA; (x) 2% H2NNH2 in DMF; (xi) peptide coupling
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with corresponding amino acid: Boc-
(xii) TFA/Et3SiH/H2O (95:1:4).
L-Arg(Pbf)-OH or Boc-L-Glu(OtBu)-OH, HCTU, DIPEA;
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Table 1. Compound Structures.
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Cmpd
FA
P1
P2
P3 P4
P5
P6
P7
P8
P9
6Me-C7CO/
6Me-C6CO
6Me-C7CO/
6Me-C6CO
nC7CO
Dab
Thr
Dab
Dab
Leu
Dab
Dab
1 Pmx-B
D
D
-Phe
Dab
Thr
Dab
Dab
Leu
Dab
Dab
2 Colistin
-Leu
11a
12b
13c
10
14
15d
16
17
18e
19
20
21
22
23
24
25
26
27
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
Dab
-
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Bip
Dab
Dab
Dab
Dab
nC7CO
2-ClPhNHCO
Ph-4-PhCO
PhO-4-PhCO
4-Ph-PhNHCO
2-ClPhNHCO
2-ClPhNHCO
nC7CO
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
D
-Dab
Dab
Dab
D
-Ser
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab(Arg)
Dab(Glu)
4NH2Phe
Arg
nC7CO
Dab
nC7CO
Dab
nC7CO
Dab
nC7CO
Dab(Arg)
Dab(Glu)
Hser
Dab
nC7CO
Dab
nC7CO
Dab
nC7CO
4NH2Phe
Arg
Dab
nC7CO
Dab
nC7CO
Dab
Dab
DL-
OctGly
Leu
28f
D
-Phe
nC7CO
nC7CO
nC7CO
Dab
Dab
Dab
Thr
Thr
Thr
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
D
-Bip
29
30g
DL-
OctGly
Leu
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
nC7CO
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
-
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Dab
Dab
Dab
Dab
Dab
Dab
Dab(Arg)
Dab(Glu)
Hser
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
D
D
D
D
D
D
D
D
D
D
D
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
-Phe
31
32
33
34
35
36
37
38
39
40
41
4NH2Phe
Arg
Dab
D
-Dab
Dab
D
-Ser
Gly
Gly
Gly
Thr
Dab
Dab
Dab
D
-Dab
Gly
Dab
a
-isomers unless otherwise indicated. 11 = Polymyxin B3. 12 = compound
b
All amino acids are
L
5 in O’Dowd et al.47 c 13 = compound 5 in Leese et al.54 d 15 = compound 18 in Leese et al.53
e 18 is similar to compound 24 (retains PmxB mixture of fatty acids) in Weinstein et al.48 f 28 =
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g
-Ade compound 2 in Li et al.57
30 = DL-OctGly version of D-Ade
3
4
5
DL- OctGly version of
L
compound 1 in Li et al.58
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
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Biological activity. The 32 compounds, along with vancomycin, gentamicin, colistin and
polymyxin B, were assessed against a panel of predominantly G-ve bacteria, with S. aureus used
as a representative G+ve strain. Five antibiotic-sensitive and resistant ATCC reference strains
covering the G-ve ESKAPE pathogens (E. coli, K. pneumoniae, A. baumannii, P. aeruginosa)
were used, along with polymyxin-resistant clinical isolates of K. pneumoniae, A. baumannii, and
P. aeruginosa (Table S2). Notably, compounds that were resyntheses of those previously
reported demonstrated similar activity to the earlier literature values (footnotes in Tables 2,3).
Counter-screening for general cytotoxicity was conducted using human liver hepatocellular
carcinoma cells (HepG2). Our previous studies have demonstrated that cytotoxicity
measurements against these immortalized cells were not predictive of nephrotoxicity,63,64 but
they do provide an indication of potential general cell toxicity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Alterations to the fatty acid tail (Table 2) had minimal effect on activity against the reference
strains, but biphenyl or biphenyl ether substituents (10, 14 and 15) did promote improved activity
against polymyxin-resistant strains, albeit accompanied by greater cytotoxicity. In the linear
exocyclic peptide tail (Table 2), replacement of Dab-3 with
D-Dab (36, 16) or Gly (38) had
minimal effect. Analog 38 is particularly interesting since it retained MIC activity among the
reference strains (0.5-2 µg/mL for most strains) with low cytotoxicity seen in HepG2 cells (CC50
> 300 µM). This is the only polymyxin B example where Dab-3 has been replaced by a nonbasic
amino acid, where the amino acid replacement also potentially impacts on the final structural
geometry. The effects of substitution of Dab-3 with
heptanoic acid 37 activity was slightly decreased (vs 11), but with o-chlorophenylurea 17 most
activity was lost. The inversion of -Dab-1 to -Dab-1 in 40 abrogated most activity, a much
larger effect than the inversion use of -Dab-3 to
D-Ser depended on the fatty acid; with the
L
D
L
D
-Dab-3 in 36/16. Finally, replacing the linear
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chain Dab-Thr-Dab sequence with Gly-Dab-Thr in 41 was also detrimental to antibacterial
5
6
activity.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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52
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Table 2. Summary of MIC against a panel of sensitive and resistant bacteria and cytotoxicity (CC50) against HepG2 cells for a set of
5
6
2
compounds with diversity in the exocyclic chain and fatty acid tail.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Vancomycin
Gentamicin
Colistin 2
>32
0.25
0.25
1
>32
>32
8
>32
>32
>32
32
>32
1
>32
32
>32
>32
128
128
>32
>32
>32
>32
>32
1
2
>300
0.25
0.5 >300
>128 >300
>64 >300
Controls
0.25 0.25 0.25 0.25
0.5
0.5
>32
32
>64 >128
64 64
64
64
Polymyxin B 1
Fatty Acid
nC7CO
1
0.5
0.25 0.25
Sample
11a
12b
13c
10
Position
0.5
1
2
1
1
2
0.5
2
0.5
2
1
0.5
0.5
1
>32 >128 >64 >128 >128 >128 >300
nC7CO
P1=None
>32
>32
8
>32
>32
>32
32
>32
>32
8
>32
>32
>32
>32
16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32 >300
>32 >300
2-ClPhNHCO
Ph-4-PhCO
PhO-4-PhCO
Ph-4-PhNHCO
2-ClPhNHCO
2-ClPhNHCO
nC7CO
1
2
2
4
1
0.5
0.5
2
0.5
0.5
2
1
0.5
0.5
1
0.5
0.5
1
>32
>32
32
298
287
316
0.5
2
1
8
16
14
15d
1
16
8
16
P3=D-Dab
1
0.5
32
32
>32
0.5
>32
0.5
0.5
0.5
16
8
1
2
0.5
8
16
>32
>32
>32
>32
>32
>32
32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32 >300
>32 >300
>32 >300
>64 >300
>64 >300
>32 >300
>32 >300
>32 >300
16
P3=D-Ser
32
16
>32
0.5
4
>32
16
32
0.5
>32
4
>32
>32
>64
1
>32
>32
>32
>32
>32
>32
>32
17
P1=Gly, P2=Dab; P3=Thr
P1=D-Dab, P3=Gly
P3=Gly
2
41
nC7CO
8
16
2
40
nC7CO
1
38
nC7CO
P1=None, P3=Gly
P3=D-Dab
>32
1
>32
1
16
2
39
nC7CO
4
36
nC7CO
P3=D-Ser
4
2
16
1
2
>32
37
3
All amino acids are L-isomers unless otherwise indicated.
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7
8
9
4
5
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7
8
9
ND: Not determined. PmxR: Polymyxin resistant. CI: Clinical isolate. MSSA: Methicillin resistant S. aureus. MICs were determined
visually after 18 hours of incubation at 37°C, with the MIC defined as the lowest compound concentration at which no bacterial
growth was visible. MIC in ꢀg/mL. 2-Chlorophenyl carbamoyl (2-ClPhNHCO), 4-biphenyl-benzoyl (Ph-4-PhCO), 4-phenoxybenzoyl
(PhO4-PhCO), 4-biphenyl carbamoyl (Ph-4-PhNHCO), octanoyl (nC7CO).
a11 is Polymyxin B3. Literature MIC E. coli = 0.5 and P. aeruginosa = 1.0 nmol/mL. E. coli IFO12734, and P. aeruginosa IFO3080
from the Institute for Fermentation, Osaka, Japan.46
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
10 b12 is compound 5 in O’Dowd et al..; MIC E. coli = 0.03, P. aeruginosa = 4, S. aureus = >16 ug/mL.47
11 c13 is compound 5 in Leese et al.; MIC90 P. aeruginosa (100 strains) = 2, A. baumannii (81 strains) = 4, E.coli (80 strains) = 2, K.
12 pneumoniae (81 strains) = 4 µg/mL.54
13 d15 is compound 18 in Leese et al.; MIC E. coli = 2.5, P. aeruginosa ATCC 27853 = 2.5 µg/mL; MIC values were determined by
14 serial twofold broth dilution method.53
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For alterations within the heptapeptide ring system (Table 3), replacement of D-Phe/D-Leu-6 or
5
6
7
8
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Leu-7 with the more lipophilic residues DL-Ade or Bip/D-Bip (27-30) replicated the results of
Velkov et al.,56 where these substitutions restored activity against polymyxin-resistant strains.
However, these changes also caused increased cytotoxicity against HepG2 cell lines. The effects of
altering the ring Dab residues varied significantly depending on which Dab was altered.
Replacement of Dab-5 with homoserine (Hse), Arg, or 4′-amino-Phe (33-35), or acylation with Arg
or Glu (31, 32), almost completely abolished activity (16 - > 32 µg/mL against all tested strains).
The same modifications at Dab-9 (18-21) gave more variable results, retaining moderate levels of
activity (4-16 µg/mL for most strains). The Dab-8 position was most tolerant of alterations, with
even neutral Hse 24 and zwitterionic Dab(Glu) 23 retaining good activity (1-8 µg/mL for most
strains). Unfortunately, the hypothesis that a zwitterionic substituent might overcome Lipid A
resistance modifications was not correct, with all of the Dab(Glu) substitutions inactive against the
polymyxin-resistant strains. The Dab-8 replacement with Arg-8 26 (Table 3) had the best activity of
all the analogs made, and showed some activity against the polymyxin-resistant strains. While not
as potent as the Arg-8 26 analog, the 4′-amino-Phe-8 analog 25 also showed good activity, this is an
interesting result due to the aromatic nature of the replacement for the otherwise aliphatic amino
acid Dab. Further investigation to elaborate on the SAR trends observed with these type of
compounds is in progress in our laboratory.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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46
47
48
49
50
51
52
53
54
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57
58
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34
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Table 3. Summary of MIC against a panel of sensitive and resistant bacteria and cytotoxicity
(CC50) against HepG2 cells for a set of compounds with diversity in the heptapeptide ring.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Vancomycin
Gentamicin
Colistin 2
>32
0.5
>32
>32
8
>32
>32
>32
32
>32
1
>32
32
>32
>32
128
128
>32
>32
>32
>32
>32
1
2
>300
>300
0.25
0.5
Controls
0.25 0.25 0.25 0.25 0.25
0.5
0.5
>32
32
>64 >128
64
64
>128 >300
>64 >300
0.25
1
0.5
0.25 0.25
64
64
Polymyxin B 1
Sample
31
Position
P5=Dab(Arg)
P5=Dab(Glu)
P5=Hser
32
>32
16
>32
16
1
32
>32
>32
>32
>32
4
16
>32
>32
>32
32
2
16
32
32
>32
32
1
32
32
>32
>32
>32
32
>32
>32
>32
>32
>32
16
>32
>32
>32
>32
>32
16
>32
>32
>32
>32
>32
8
>32
>32
>32
>32
>32
32
>32
>32
>32
>32
>32
32
>32 >300
>32 >300
>32 >300
>32 >300
>32 >300
>32
>32
>32
>32
0.5
32
33
P5=4-NH2-Phe
P5=Arg
34
35
P6=D
-Bip
1
8
16
8
119
254
117
116
ND
29
30a
P6=DL-OctGly
P7=Bip
2
2(4)
4
1
1
1(2) 2(1)
4
8
16
16
16
1
2
1
1
2
16
16
32
32
>32
32
27
28b
22
P7= DL-OctGly
P8=Dab(Arg)
P8=Dab(Glu)
P8=Hser
1
2(1)
4
2
1
1(1) 2(4)
16
8
8
32
8
0.5
1
0.5
4
1
4
4
2
8
>32
>32
>32
32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
8
4
>32 >300
>32 >300
23
1
>32
8
2
2
8
4
24
P8=4-NH2-Phe
P8=Arg
0.5
0.25
32
4
1
2
4
8
>32
>32
ND
ND
25
4
0.5
8
0.5
8
0.5
16
16
>32
32
1
32
26
18c
P9=Dab(Arg)
P9=Dab(Glu)
P9=4-NH2-Phe
P9=Arg
32
32
8
>32
>32
>32
>32
>32
>32
>32
>32
>32 >300
>32 >300
>32 >300
>32 >300
16
4
4
19
16
16
>32
>32
16
8
16
4
32
1
20
21
37
38
39
40
41
42
43
44
45
46
47
All amino acids are L-isomers unless otherwise indicated. ND: Not determined. PmxR: Polymyxin
resistant. CI: Clinical isolate. MSSA: Methicillin resistant S. aureus. MICs were determined
visually after 18 hours of incubation at 37°C, with the MIC defined as the lowest compound
concentration at which no bacterial growth was visible. MIC in ꢀg/mL. 2-Chlorophenyl carbamoyl
(2-ClPhNHCO), 4-biphenyl-benzoyl (Ph-4-PhCO), 4-phenoxybenzoyl (PhO4-PhCO), 4-biphenyl
carbamoyl (Ph-4-PhNHCO), octanoyl (nC7CO).
a values are reported MIC for compound 2 =
b values are reported MIC for compound 1 =
L
-Ade version of 28 in Li et al. WO2010/13000757
-Ade version of 30 in Li et al. WO2012/05166358
D
c18 similar to compound 24 (retains polymyxin B fatty acid) in Weinstein et al.; MIC E. coli = 0.16,
P. aeruginosa = 0.03, S. aureus = 2 µg/mL48
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Nephrotoxicity. Traditional cytotoxicity assays have been confirmed as improper indicators for
nephrotoxicity screening.63,64 Therefore, in addition to general cytotoxicity screening assessed
using HepG2 cells, we selected four compounds with promising antimicrobial potency and
reasonable cytotoxicity against the HepG2 cell lines for further assessment for toxicity using
primary kidney cells freshly isolated from human kidneys (see Table 4). This assay, measuring
LDH and GGT release, has been reported to be a better in vitro model for predicating in vivo
nephrotoxicity.63-65 We also assessed cytotoxicity against immortalized HEK293 cells. Colistin,
polymyxin B and gentamicin were employed as control nephrotoxic compounds, and clearly
showed the greatest toxicity in both primary renal cell assay readouts (Figures 2A and 2B), in
contrast to their apparent lack of cytotoxicity in the HepG2/HEK293 assays. Compounds 10 and 14,
with non-alkyl fatty acids, had shown greater cytotoxicity than polymyxin B and colistin, but
induced almost no release of LDH or GGT at 100 µg/mL in the primary kidney cells. Compound 38
was the least cytotoxic (HepG2/HEK293 assays) of the four new analogs tested, but possessed
intermediate levels of “nephrotoxicity” against primary kidney cells, as did 15 which had similar
cytotoxicity against HepG2 cells as 10 and 14. The results support our previous assessment of in
vitro predictors of nephrotoxicity, in that there is little correlation between general cytotoxicity
measurements and the human kidney results.63,64
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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Table 4. Summary of MIC, cytotoxicity CC50 data (HepG2 and HEK293 cell lines) and
nephrotoxicity CC50 data measuring LDH (A) and GGT (B) release from primary human kidney
cells using lead compounds (10, 14, 15 and 38) compared to control compounds (colistin,
polymyxin B and gentamicin).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sample
Gentamicin
1 Polymyxin B
2 Colistin
0.25
1
8
>32 >300 >300
33.5
23.2
25.4
32.6
176.6
48.5
0.5 0.25 >300 >300
0.25 0.25 0.25 >300 >300
0.5
0.5
2
1
0.5
2
0.5
0.5
1
298
287
316
297
296
>128 >128
>128 >128
10
14
15
38
>300 >128 >128
0.5
1
0.5
>300 >300 122.0 >128
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Figure 2. In vitro nephrotoxicity studies measuring LDH (A) and GGT (B) release from primary
human kidney cells using lead compounds (10, 14, 15 and 38) compared to control compounds
(colistin, polymyxin B and gentamicin).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions. A new solid-phase synthesis of the polymyxin class of antibiotics has been
developed based on attachment to the resin through the side chain of Thr-10, allowing for on-resin
cyclization, variation at any other position, and selective derivatization of the side chains of
individual Dab residues. Thirty-two analogs were prepared and tested against a panel of reference
and clinical isolates, including polymyxin-resistant MDR strains. Cytotoxicity screening was
performed against the HepG2 cell line. The results confirmed that more lipophilic groups at P6/P7
improved activity against polymyxin-resistant strains but increased cytotoxicity against mammalian
cells. Similar effects were observed by replacement of the alkyl fatty acid with biaryl substituents.
Further testing of these compounds by assessing cytotoxicity in primary human renal cells as a
potential surrogate for nephrotoxicity conversely found reduced toxicity compared to the increased
general cytotoxicity seen in an immortalized cell line. Large differences were found when varying
the five free side-chain Dab residues: Dab-5 could not be replaced or altered without loss of
activity, while alterations at Dab-9 also severely affected potency, though to a slightly less extent.
In contrast Dab-8 tolerated a range of basic, neutral or zwitterionic replacements. Similarly, Dab-3
could be replaced by non-basic Gly or Ser with minimal effects, while inversion of the
configuration of Dab-1 abolished activity. Further studies of this class of compounds will
investigate combinations of promising substituent alterations, particularly those that reduce the
overall basic character, improve activity against polymyxin-resistant bacteria, and demonstrate
reduced potential for nephrotoxicity in human kidney cells.
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EXPERIMENTAL SECTION
5
6
7
8
9
Synthetic Material and Method. Experimental procedures are described in the Supporting
Information. All chemicals were obtained from commercial suppliers and used without further
purification. LC-MS analysis were conducted using Agilent Technologies 1200 Series Instrument
with a G1316A variable wavelength detector set at λ= 210 nm, 1200 Series ELSD, 6110 quadrupole
ESI-MS, using an Agilent Eclipse XDB-Phenyl (3 × 100mm, 3.5 ꢀm particle size, flow rate 1
mL/min, the mobile phases 0.05% formic acid in water and 0.05% formic acid in acetonitrile).
Compound purification was done using a Agilent 1260 Infinity Preparative HPLC with a G1365D
multiple wavelength detector set at λ= 210 nm and an Agilent Eclipse XDB-Phenyl column 21.2 x
100mm, 5 ꢀm particle size. Ultraviolet/visible spectra were recorded with a Varian Cary 50 Bio
spectrophotometer. Identities of final products were confirmed by MS/MS spectra, obtained using
an API QSTAR™ Pulsar Hybrid LC-MS/MS System, high resolution mass spectrometry (HRMS),
performed on a Bruker Micro TOF mass spectrometer using (+)-ESI calibrated to sodium formate,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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and by H (600 MHz) and 2D NMR spectra, obtained using a Bruker Avance-600 spectrometer
equipped with a TXI cryoprobe in D2O, referenced externally with NaOAc (δH 1.90 and 8.44; 10
mg in 500 ꢀL D2O) and then internally with the HDO resonance at δ 4.77.66 Final purity of more
than 95% for all compounds was confirmed by LCMS analysis using both ELSD and UV (210 nm)
detection.
Minimum Inhibitory Concentration (MIC) determination. 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 Muller Hinton broth (MHB; Bacto Laboratories, catalog No.
211443) at 37 °C overnight with shaking (~180 rpm). A sample of each culture was then diluted 50-
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fold in fresh MHB and incubated at 37 °C for 1.5-3 h with shaking (~180 rpm). Compound stock
solutions were prepared as 0.64 mg/mL or 2.56 mg/mL in water. The compounds, at twice the final
desired concentration, were serially diluted two-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 a final concentration of 5 × 105 colony forming units (CFU)/mL, and 50 ꢀL was added to
each well giving a final compound concentration range of 32 ꢀg/mL to 0.015 ꢀg/mL (128-0.06
ꢀg/mL for colistin and polymyxin B against the polymyxin resistant clinical isolates). MICs were
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.
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Cytotoxicity Assay (Resazurin assay). Cytotoxicity to HEK293 and HepG2 cells was determined
using the resazurin assay.67,68 In brief, HEK293 and HepG2 cells were seeded as 4000 cells/well in
black-wall, clear-bottom 384-well plates (Corning, Australia) and incubated for 24 h at 37°C, 5%
CO2. Compounds were then added into each well. After 24 h incubation, 5 µM resazurin were
added per well and incubated at 37 °C for 2 h. Fluorescence intensity was then read using a
Polarstar Omega with excitation/emission 560/590. The data were then analyzed using GraphPad
Prism 6 software. CC50 values were calculated using the following equation: Percentage Viability =
(FITEST – FINegative/FIUNTREATED –FINegative)*100.
Nephrotoxicity Assay (Lactate Dehydrogenase (LDH) and Gamma Glutamyl Transferase
(GGT) release in primary cells freshly isolated from human kidneys). Primary human renal
proximal tubule epithelial (hPT) cells were isolated and used for in vitro prediction of drug-induced
nephrotoxicity63,64,69 by measuring the release of metabolism enzymes, LDH and GGT as previously
described.70
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ANCILLARY INFORMATION
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Supporting Information: Tables of structures, characterization data and bacterial strain
descriptions, detailed synthetic procedures and detailed characterization of four representative
compounds.
Corresponding Author Information: Matthew A Cooper, Institute for Molecular Bioscience,
University of Queensland, Brisbane, Australia. E-mail: m.cooper@uq.edu.au, Fax +61-7-3346-
2090, Phone +61-7-3346-2044
Acknowledgments: MAC is an NHMRC Principal Research Fellow (APP1059354). Research
conducted by AGG, MSB, AGE, AMK, SR, JXH, MATB, and MAC was supported by NHMRC
grants APP1005350 and APP1045326 and NIH grant R21AI098731/R33AI098731. MSB, JXH and
MATB were supported by a Wellcome Trust Seeding Drug Discovery Award (094977/Z/10/Z). LL
is supported by NIH grant R21AI098731/R33AI098731. We thank Wang Hengzhuang (University
Hospital of Copenhagen, Denmark), Ilias Karaiskos and Helen Giamarellou (6th Dept. of Internal
Medicine, Hygeia General Hospital, Athens, Greece), Cely S. Abboud (Instituto Dante Pazzanese
de Cardiologia, São Paulo, Brazil) and Roger L. Nation (Institute of Pharmaceutical Sciences,
Monash University, Australia) for providing the Gram-negative clinical isolates for antimicrobial
testing. The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institute of Allergy and Infectious Diseases or the National Institutes
of Health.
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Abbreviations Used: ATCC, American Type Culture Collection; Dab 2,3-diaminobutyric acid;
DIPEA, diisopropylethylamine; DMF, dimethylformamide; E. coli, Escherichia coli; GGT, γ-
glutamyltransferase; G-ve, Gram-negative; G+ve, Gram-positive; HCTU, (2-(6-Chloro-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate); HepG2, human liver
hepatocellular carcinoma cells; HEK293, human embryonic kidney 293; HK2, human kidney
proximal tubule epithelial cell line; ivDde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-
methylbutyl; LDH, lactate dehydrogenase; P. aeruginosa, Pseudomonas aeruginosa; S. aureus,
Staphylococcus aureus; S. pyogenes, Streptococcus pyogenes; SPPS, solid-phase peptide synthesis;
TFA, trifluoroacetic acid.
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