Structure - Activity Relationships of Polymyxin Antibiotics

1898 J. Med. Chem.
2010, 53, 1898–1916

DOI: 10.1021/jm900999h
Structure-Activity Relationships of Polymyxin Antibiotics
Tony Velkov,*,†,‡ Philip E. Thompson,‡ Roger L. Nation,§ and Jian Li*,§

†
School of Medicine, Deakin University, Pigdons Road, Geelong 3217, Victoria, Australia, ‡Medicinal Chemistry and Drug Action and
§
Facility for Anti-infective Drug Development and Innovation, Drug Delivery, Disposition and Dynamics, Monash Institute of
Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia
Received July 7, 2009
1. Polymyxins, a Last-Line Therapy against Gram-Negative

“Superbugs”
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The world is facing an enormous and growing threat from the

emergence of bacteria that are resistant to almost all available
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antibiotics.1 Multidrug resistance is a significant public health

issue in regard to both Gram-positive and Gram-negative
bacteria, but the problem is arguably most grave for the latter
class of bacteria. In recent years, virtually no novel drugs
targeting multidrug-resistant (MDRa) Gram-negative bacteria,
in particular Pseudomonas aeruginosa, have been developed. As
described in the “Bad Bugs, No Drugs” paper published by the
Infectious Diseases Society of America (IDSA),1,2 “as antibiotic
discovery stagnates, a public health crisis brews”. Therefore,
there is an urgent need for new antibiotics, particularly those
active against Gram-negative “superbugs”, such as P. aerugi-
nosa, Acinetobacter baumannii, and Klebsiella pneumoniae.1-4
It is precisely this mismatch between increasing multidrug
resistance and the dry antimicrobial-drug development pipeline,
as highlighted in the “Bad Bugs Need Drugs” campaign,1,2
that led the IDSA to place P. aeruginosa, A. baumannii and
K. pneumoniae on a “hit list” of the six top-priority dangerous
MDR microorganisms. These pathogens have been identified as Figure 1. Chemical structures of polymyxin B and colistin. The
requiring the most urgent attention for discovery of novel functional segments of polymyxins are colored as follows: yellow,
NR fatty acyl chain; green, linear tripeptide segment; red, the polar
antibiotics. Meanwhile, the polymyxins are increasingly being residues of the heptapeptide; blue, the hydrophobic motif within the
used as last-line therapy to treat infections caused by Gram- heptapeptide ring. The amino acids positions are numbered in
negative bacteria that are resistant to essentially all other accordance to references in the text.
currently available antibiotics.5-9
Polymyxins were discovered more than 50 years ago.10 (1) and PMB2 (2), the main components of PMB, are N-
Polymyxin B (PMB) and colistin (polymyxin E) are secondary terminally acylated by (S)-6-methyloctanoic acid and (S)-6-
metabolite nonribosomal peptides produced by the soil bac- methylheptanoic acid, respectively. Similarly, the two main
terium Bacillus polymyxa. PMB and colistin share a common components of colistin, colistin A (7) and colistin B (8),
primary sequence, the only difference being at position 6 which are acylated by (S)-6-methyloctanoic acid and (S)-6-methyl-
is occupied by D-Phe in PMB and D-Leu in colistin (Figure 1). heptanoic acid, respectively. Because the early clinical experi-
Pharmaceutical grade preparations of PMB and colistin are ence, before the 1970s, with parenteral administration
composed of a mixture of closely related components. PMB1 of PMB and colistin (or its nonactive prodrug colistin metha-
nesulfonate11) led to concern over the potential for nephro-
*To whom correspondence should be addressed. For T.V.: phone, toxicity and neurotoxicity, their clinical use waned.7,9,12-14
þ61-3-9903 9539; fax, þ61-3-9903 9582; e-mail, Tony.Velkov@deakin.
edu.au or Tony.Velkov@pharm.monash.edu.au. For J.L.: phone, þ61- Since the mid-1990s, there has been a greatly renewed
3-9903 9702; fax, þ61-3-9903 9629; e-mail, Jian.Li@pharm.monash. interest in their clinical use due to prevalent MDR Gram-
edu.au. negative bacteria (especially P. aeruginosa, A. baumannii and
a
Abbreviations: PMB, polymyxin B; LPS, lipopolysaccharide; Dab, K. pneumoniae) and lack of novel antibiotics.5-9,12,14 Even
L-R-γ-diaminobutyric acid; Dap, 2,3-diaminopropionic acid; DPPA,
diphenylphosphorylazide; FA, fatty acyl; MDR, multidrug-resistant; though there is still a dearth of knowledge on how to use them
XDR, extremely drug-resistant; PyBOP, benzotriazole-1-yl-oxy-tris- optimally, polymyxins are mostly being used as last-line anti-
pyrrolidinophosphonium hexafluorophosphate; Boc, tert-butyloxycar- biotics for otherwise untreatable serious infections. Although
bonyl; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; ivDde,
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl; Mtt, 4-meth- the incidence of resistance to polymyxins is currently relatively
yltrityl; 2-ClZ, 2-chlorobenzyloxycarbonyl. low, resistance can emerge rapidly in vitro in P. aeruginosa,
pubs.acs.org/jmc Published on Web 10/29/2009 r 2009 American Chemical Society

Perspective Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1899
Figure 2. Chemical structure of E. coli lipid A. The amino-4-deoxy-L-arabinose modification of the phosphate groups observed in polymyxin-
resistant strains is shown in gray.
A. baumannii, and K. pneumoniae.6,15,16 More worrying, poly- 2. Mechanisms of Polymyxin Activity and Bacterial Resis-
myxin resistance in hospitalized patients has been increasingly tance
reported.6,15,17 As noted above, there is only one amino acid
difference between colistin and PMB and, not surprisingly, 2.1. Mechanism of Antibacterial Activity of Polymyxins. A
cross resistance exists.7 The emergence of extremely drug- key role of the outer membrane (OM) of Gram-negative
resistant (XDR) P. aeruginosa, A. baumannii, and K. pneumo- bacteria is to act as a permeability barrier.21,22 The initial
niae, including resistance to polymyxins, is posing the most target of polymyxins is the LPS component of the mem-
serious therapeutic challenges.17-20 In essence, resistance to brane. An understanding of the mechanism of polymyxin
polymyxins implies a total lack of antibiotics for treatment of activity therefore requires knowledge of LPS structure. LPS
life-threatening infections caused by these Gram-negative “su- is composed of three domains, a conserved inner core 2-keto-
perbugs” and highlights the urgency to develop new antibiotics. 3-deoxyoctonoic acid (Kdo) bound to lipid A and a variable
In view of the generally good antimicrobial activity of the O-antigen composed of repeating units of various polysac-
naturally occurring polymyxins and relatively low prevalence charides.22 The consensus structure of lipid A consists of a
of resistance that exists, it is perhaps not surprising that there β-10 -6-linked D-glucosamine (GlcN) disaccharide that is
has been substantial effort into discovery of polymyxin ana- phosphorylated at the 1- and 40 -positions (Figure 2). Lipid
logues with improved microbiological, pharmacological, and A usually contains six acyl chains (designated A-F in
toxicological profiles. In the early part of this paper, we review Figure 2). Four acyl chains attached directly to the glucosa-
the current understanding of the mechanisms of microbiolo- mine sugars are β-hydroxyacyl chains (usually C12 and C14
gical activity and bacterial resistance. The synthesis of poly- in length), while two secondary acyl chains are often attached
myxins is next reviewed, followed by a comprehensive treatise to the β-hydroxy group. Lipid A acts as a hydrophobic
on the structure-activity relationships of the polymyxins and anchor with the tight packing of the fatty acyl chains helping
the various analogues that have been investigated. In under- to stabilize the overall OM structure.22
taking this review, a literature search was conducted using Most of the investigations to elucidate mechanisms of
PubMed (NLM) with keywords of polymyxin and structure- action have been conducted using PMB. Polymyxins exert
activity relationships. their antimicrobial action by permeabilizing the bacterial
1900 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 Velkov et al.
although partly speculative, is consistent with available data

in the literature.
An alternative mechanism has been proposed wherein
polymyxin mediated contacts between the periplasmic leaf-
lets of the inner and outer membranes. This postulate is
based on the observation that PMB bound to anionic
phospholipid vesicles is capable of forming vesicle-to-vesicle
contacts.25,32-36 These contacts promote phospholipid ex-
change between vesicles. In the bacterial membrane, lipid
exchange between the inner and outer membrane leaflets
would result in the loss of phospholipid compositional
specificity, potentially leading to an osmotic imbalance that
leads to lytic cell death.
2.2. Mechanisms of Resistance to Polymyxins. As noted
above, a critical first step in the action of polymyxins on
Figure 3. Diagram depicting putative antimicrobial action of PMB Gram-negative bacteria is the electrostatic interaction be-
on Gram-negative bacterial outer membrane. tween the positively charged Dab residues of polymyxins and
the negatively charged phosphate groups on lipid A.23,25,37,38
OM via direct interaction with the lipid A component of Thus, many bacterial mechanisms of resistance to polymyx-
the LPS (Figure 3). The narrow spectrum of activity of ins are based on modifications to the lipid A head groups that
polymyxins for Gram-negative bacteria is coincident with reduce this initial electrostatic interaction. In Escherichia
their binding selectivity for LPS.22-24 The amphipathicity coli, Salmonella enterica serovar Typhimurium, K. pneumo-
of the polymyxins and, possibly, their ability to form pore- niae, and P. aeruginosa, modification of the phosphates of
like aggregates may be responsible for their OM permeabi- lipid A with positively charged moieties, such as 4-amino-4-
lizing action.23-27 The conserved elements in the chemical deoxy-L-arabinose and/or phosphoethanolamine, reduces
structure of polymyxins consist of two hydrophobic domains the net negative charge of lipid A, thereby increasing resis-
(the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 tance to polymyxins (Figure 2).39-44 In K. pneumoniae, the
segment) separated by segments of polar (Thr) and cationic presence of capsule may also be important for polymyxin
(L-R-γ-diaminobutyric acid (Dab)) residues. The elucidation resistance.45,46
of the three-dimensional NMR solution state structure of In many bacterial species resistance to cationic antimicro-
PMB in complex with LPS revealed the PMB molecule is bial peptides (CAP) is regulated by a two-component master
folded such that the polar and hydrophobic domains form regulatory system, PhoP-PhoQ. This system is also em-
two distinct faces, thereby conferring structural amphipathi- ployed for survival under conditions of low Mg2þ that would
city (Figure 4).28-30 A detailed account of the structural destabilize the OM because of the decrease in the bridging
aspects of the polymyxin B-LPS interaction will be given in action of divalent cations between LPS molecules. PhoP-
section 3. The general model for the action of amphipathic PhoQ remains repressed in high (mM) Mg2þ environments
antimicrobial peptides involves interaction of the polar face and is activated under conditions of low (micromolar)
of the peptide with the polar lipid A head groups, while the Mg2þ.39,47 In S. enterica, the PhoP-PhoQ two-component
lipophilic face inserts into the hydrophobic fatty acyl layer of system has been well characterized.40,41,44,47,48 The PhoP-
the OM.23-27 The positive charge of polymyxins allows for PhoQ acts as a master regulator of S. enterica virulence and
accumulation at the anionic bacterial membrane. The elec- evasion of CAP killing. PhoQ is an inner membrane sensor
trostatic interaction between the positively charged poly- kinase that phosphorylates the cognate response regulator
myxin Dab residues and the negatively charged lipid A PhoP in response to low Mg2þ or sublethal concentrations of
phosphates is believed to displace divalent cations (Ca2þ CAPs. This in turn leads to activation of PmrA-PmrB which
and Mg2þ) that normally function to bridge and stabilize the confers resistance by activating genes that encode enzymes
LPS OM monolayer.22-27 This initial electrostatic inter- required for the covalent modification of the phosphate
action temporarily stabilizes the complex and brings the groups on lipid A with 4-amino-4-deoxy-L-arabinose or
N-terminal fatty acyl chain of the polymyxin molecule into phosphoethanolamine.40,41,48 This modification serves to
proximity with the OM. Insertion of the fatty acyl chain and decrease the net negative charge and reduces repulsion
6 7 6 7
D-Phe -L-Leu (PMB) or D-Leu -L-Leu (colistin) hydropho- between neighboring LPS molecules, thereby strengthening
bic domain acts to weaken the packing of adjacent lipid A the packing of the OM.22 CAPs have also been shown to
fatty acyl chains causing expansion of the OM monolayer. directly activate the PmrA-PmrB system.49
The fact that polymyxin B nonapeptide (PMBN (193),
i.e., PMB minus the N-terminal fatty acyl chain and Dab1
3. Polymyxin-LPS Complex
residue), lacks antibacterial activity highlights the impor-
tance of both the electrostatic and hydrophobic interactions From the foregoing, it is evident that an understanding
for the mechanism of polymyxin action. Following inser- of the mechanisms of action and resistance of the polymyxins,
tion, polymyxins are purported to transit the OM via a and of their SAR, requires knowledge of the interaction
self-promoted uptake mechanism.23-25,27,29-31 Subse- between polymyxins and LPS. Such knowledge is also
quently, the polymyxin molecule inserts and disrupts the central in efforts to develop polymyxin analogues with re-
physical integrity of the phospholipid bilayer of the inner duced potential to be affected by the polymyxin resistance
membrane via membrane thinning by straddling the inter- mechanisms discussed above. The PMB-LPS binding
face of the hydrophilic head groups and fatty acyl chains or interaction has been studied by several biophysical techniques
transient poration.23-25,27,29-31 This general model, including isothermal titration calorimetry (ITC),50-53
Figure 4. A color coded schematic diagram summarizing the key contacts involved in complex formation between PMB and the lipid
A component of LPS. FA = N-terminal fatty acyl chain.
displacement of fluorescent probes,54-56 surface plasmon range of TFE concentrations, whereas the remainder of the
resonance (SPR),57,58 and nuclear magnetic resonance molecule is more flexible.28,31 Simulated annealing calcula-
(NMR) spectroscopy.28-31,59,60 tions suggest that the rigidity about residues 6, 7, and 10
Thermodynamic analysis of the interaction between PMB constrains both sides of the ring, thereby limiting flexibility to
and LPS using ITC measurements indicated that binding is variations in ring pucker. The linear peptide segment of both
entropically driven (i.e., driven by hydrophobic forces) in the molecules displays a greater mobility than the heptapeptide
gel phase of the hydrocarbon chains of LPS (<30 °C) but ring. Faster motions are also seen for the side chains than the
enthalpically driven in the liquid crystalline phase (>35 °C).50 heptapeptide ring.28,31 Burch et al.28,31 purport the increased
Furthermore, the ITC binding isotherms indicated the com- mobility of the side chains allows the cationic Dab residues to
plex is stoichiometric and noncooperative with affinity in the more efficiently dock onto the anionic phosphoester groups
low micromolar range.50-53 SPR experiments showed a on the lipid A component of LPS. This flexibility would also
poorer affinity and kinetics of binding for PMBN to LPS allow the PMB molecule to bind to both mono- and divalent
compared to PMB, whereas, the binding of a Dab f Lys phosphoester ligands. In PMB, the N-terminal fatty acyl chain
substituted cyclic decapeptide PMB analogue was compar- displays a rapid, independent motion from the heptapeptide
able to that of PMB.57,58 These observations were attributed core. The authors suggest this flexibility facilitates passage of
to the amphilic properties of each peptide such that the the PMB molecule across the OM layer and into the periplas-
presence of Lys residues that contain two extra methylene mic space and accounts for some of the hydrophobic forces
groups in their side chains (compared to a single methylene in driving LPS binding, as shown by thermodynamic
the Dab side chain) gives the cyclic decapeptide higher measurements.50-53 Interestingly, all of the internal motions
amphilicity than PMBN.57,58 These data highlight the impor- were faster for PMBN compared to PMB, suggesting that
tance of amphilicity for high affinity binding to LPS. despite its independent motion, the N-terminal fatty acyl
The interaction between polymyxins and LPS at the chain somehow restricts the mobility of the heptapeptide
molecular level has been well characterized by NMR tech- ring.28,31 Burch et al.28,31 further speculated that the topolo-
niques.28-31,59,60 This structural information has proved in- gical flexibility in the higher-order structures is important for
valuable for the interpretation of available SAR data and accommodating motions that may be necessary to allow for
toward the development of novel polymyxin compounds binding to the dynamic environment of the bacterial OM.
against which bacteria cannot easily develop resistance. In Nevertheless, since PMBN also displays the same flexibility as
the ensuing discussion we will attempt to convey the current PMB, conformational flexibility per se may not be essential
consensus regarding the available structural details of the for antimicrobial activity.
polymyxin-LPS complex. Subsequently, Pristovsek and Kidric29,30 successfully
One of the early structural studies of PMB and PMBN characterized the solution-state NMR structure of PMB
utilized 2D NMR methods in combination with simu- in aqueous solution, both in the free and in the LPS-bound
lated annealing computational calculations to characterize state. Similar to the conformation observed for the free
their structure and dynamics in aqueous trifluoroethanol PMBN structure, the free PMB is in a fast exchanging
(TFE).28,31 Circular dichroism experiments indicated PMB conformational regime with a preference toward a type II0
adopts a comparable structure in aqueous TFE and when β-turn from residues 5 to 8 and a γ-turn about residue 10.
bound to phospholipid vesicles, indicating TFE does indeed Unlike the PMBN-LPS complex where both motifs are
act as a membrane-mimetic solvent. In the free state, the preserved, these structures were not evident in the LPS-bound
overall structural dynamics of both PMB and PMBN were conformation of PMB. Transferred nuclear Overhauser effect
quite similar.28,31 The peptides exhibited a differential mobi- (trNOE) NMR techniques were employed to determine the
lity over the heptapeptide ring, with the more rigid residues conformation of PMB when bound to LPS. On the basis of the
acting as pivot points. The data reveal that in both molecules, NMR structure of PMB when bound to LPS, a molecular
residues 6, 7, and 10 maintain a stable conformation over a model of the PMB-LPS complex was constructed (Figures 4
Figure 5. Molecular models of polymyxins in complex with LPS derived from NMR restraints. The LPS molecule is shown in space filling
representation, and polymyxins are shown in stick representation: (A) molecular model of the complex between E. coli LPS with PMB;29,30
(B) molecular model of the complex between E. coli LPS with PMB in a dodecylphosphocholine micelle shown in surface representation;59
(C) backbone conformation of polymyxin M;60 (D) molecular model of the complex between E. coli LPS with PMBN.61
and 5A).29,30 The modeling process took into account the hydrogen bonding to stabilize the ring conformation. In
electrostatic interactions between the Dab side chains and addition to maintaining the optimal distance between the
lipid A phosphates and maximized the reduction of solvent positive charges, the envelope-like fold of the backbone also
exposed hydrophobic area on both molecules. The model forces the two phosphoester binding sites to one face of
implies the complex is stabilized by a combination of electro- the PMB molecule. The heptapeptide ring of PMB covers
static and hydrophobic interactions. The bacterial lytic activ- the GlcN disaccharide unit core of lipid A, forming a “charge
ity of polymyxins is believed to correlate with their clamp” such that the positive charges on Dab1 and Dab5 allow
amphiphilic character. Coincidently, in the LPS-bound state bonding with the negatively charged 40 -phosphate group
the PMB backbone adopts an envelope-like fold separating of lipid A, and Dab8 and Dab9 similarly bond with the
the polar/charged residues from the hydrophobic components 1-phosphate. The complex is further stabilized by hydrophobic
giving the structure amphilicity (Figure 6A). The model contacts: the N-terminal fatty acyl chain of PMB interacts with
indicates that the N-terminal fatty acyl chain and D-Phe6- fatty acyl chains A and B of lipid A, and the side chains of
7 6
L-Leu hydrophobic motif penetrate into hydrocarbon por- D-Phe interacts with the A and C fatty acyl chains on lipid A
tion of the lipid A layer whereas the polar Thr2, Dab3, and and Leu7 with C and F (Figures 2 and 5A). In three-dimen-
Thr10 side chains are oriented to point into the hydrophilic sional space the Dab1 and Dab5 side chains are adjacent to the
environment formed by the Kdo units of the inner core N-terminal fatty acyl chain of PMB, and as such may facilitate
oligosaccharide of LPS. Similar to other cyclic peptides of its insertion into the lipid A fatty acyl layer of the OM. More
nonribosomal origin, the backbone conformation of PMB recently, this model was consolidated by another NMR study
appears to be maintained by an ordered series of intramole- which utilized chemical shift mapping data and intermolecular
cular hydrogen bonds (Figure 6B). Amide protons that are NOEs to provide additional constraints to model the PMB-LPS
least affected by changes in temperature are most likely complex in the presence of dodecylphosphocholine micelles
shielded from the solvent by participation in hydrogen bond- (Figure 5B).59 The Mares et al.59 model was largely consistent
ing.29 The temperature dependence of the amide proton with the model of Pristovsek and Kidric29,30 with the only major
chemical shifts indicated that two of the heptapeptide amide difference being polar contacts to the Kdo-C unit of the inner
protons from residues Dab4,8 participate in intramolecular oligosaccharide and the polar Thr10 residue of PMB.
tion of PMB) giving a less dramatic separation of the polar

and hydrophobic side chains. These conformational differ-
ences are focused in the region of the hydrophobic motif
within the heptapeptide ring and are most likely a result of the
amino acid differences in this segment between the two
molecules, PMB (D-Phe6-L-Leu7) and PMM (D-Leu6-L-Thr7),
whereas the rest of the two molecules are identical. The recent
chemical shift mapping NMR study by Mares et al.59 sug-
gested that PMM binds LPS in a similar manner to PMB, with
subtle differences at the interaction interface involving the
region of the inner oligosaccharide core of LPS. In the
PMM-LPS complex, polar interactions between the Dab
residues and the GlcN-B and Kdo-C,D carbohydrate units
dominate, whereas in the PMB-LPS complex these polar
interactions are not prominent. Since PMM displays compar-
able antimicrobial activity to PMB,59 the additional polar
contacts between the polar side chains of PMM and the Kdo
units of the inner core of LPS may help compensate for the
loss of binding energy between the hydrophobic domains due
to the Leu7 f Thr substitution.
Bhattacharjya et al.61 examined the structure of PMBN in
aqueous solution both free and in the LPS bound state using
trNOE NMR and molecular dynamics techniques. Both the
free and LPS bound structures of PMBN are characterized by
a type II0 β-turn centered around D-Phe6-L-Leu7 and an
inverse γ-turn at Thr10. The linear dipeptide segment (Thr2-
Dab3) and the side chains of the residues forming the hepta-
peptide ring displayed a reduced mobility in the LPS bound
state. The proposed model indicates a different complex
topology from that of the PMB-LPS complex (Figure 5D).
Although the PMBN molecule straddles the GlcN disacchar-
Figure 6. (A) Conformation of the cyclic heptapeptide backbone of ide of lipid A similar to PMB, the PMBN molecule sits on top
PMB when bound to LPS.29,30 The structure is shown in two of the lipid A molecule and does not appear to make the
different views from the top left orientation by 90° rotation about hydrophobic contacts seen in the PMB complex. This model
the x- and y-axis. (B) NMR structure of PMB when bound to LPS can be used to contrast the OM permeabilizing and antibac-
showing predicted intramolecular hydrogen bonds. terial activities of PMB, to PMBN which possesses OM
permeabilizing activity only.62 In an analogous manner to
As mentioned above, covalent modification of the lipid PMB, PMBN binds lipid A by displacing the divalent cations
A phosphoester groups with positively charged, amine-con- that bridge adjacent head groups, thereby partially disrupting
taining moieties such as amino-4-deoxy-L-arabinose or the membrane packing and creating gaps in the polar sur-
phosphoethanolamine is a mechanism bacteria employ for face that allow hydrophobic antibiotics to penetrate into the
attaining resistance to cationic antimicrobial peptides such as fatty acyl layer.22 However, unlike PMB, PMBN lacks the
polymyxins.41,43,44,48 The aforementioned model implies that N-terminal fatty acyl chain and therefore cannot penetrate the
an amino-4-deoxy-L-arabinose modification on the lipid A 10 OM; i.e., PMBN lacks the ability to promote its own uptake
and 40 -phosphate groups would destabilize the complex by which is a requirement for antibacterial activity.
blocking electrostatic interactions with the positively charged The conformation of a lysine substituted PMB analogue in
amino groups on the side chains of the Dab residues.41,43,44,48 dimethyl sulfoxide has been determined by 2D NMR techni-
The structure of polymyxin M (23) (PMM; syn. mattacin ques and molecular dynamics simulations.63 The backbone of
syn. polymyxin A) bound to LPS has also been characterized the analogue adopts a rectangular shape stabilized by intra-
using trNOE NMR techniques.60 Similar to PMB, the back- molecular hydrogen bonds. Similar to PMB, the overall
bone of the LPS-bound PMM molecule displays a chairlike structural fold separates the polar and hydrophobic domains,
conformation wherein the side chains of Dab5,8 point in the giving the structure amphilicity.
opposite direction from those of Leu6 and Thr7, giving Collectively, the binding and structural data support a two-
the structure amphilicity (Figure 5C). Similar to PMB, the stage mechanism of the interaction between PMB and LPS,
temperature dependence of the amide proton chemical shifts wherein the first stage electrostatic interactions between the
indicated the amide protons of Dab5,8 are most likely involved positive charges of PMB and the negative LPS head groups
in intramolecular hydrogen bonding to stabilize the ring stabilize the complex. In the second stage, hydrophobic inter-
conformation. However, on closer inspection it is clear the actions between the hydrophobic domains of each molecule
backbone conformation of PMM is different from that facilitate penetration of PMB into the OM. The NMR models
determined for PMB. In the PMB structure the envelope-like of the PMB-LPS complex clearly demonstrate that the back-
fold of the backbone is so dramatic that the polar and bone of PMB adopts an envelope-like fold that separates the
hydrophobic residues in the ring are well separated, whereas polar and hydrophobic domains of the molecule, giving the
in PMM, the backbone is less contorted (i.e., PMM displays a structure amphilicity that is essential for its antimicrobial
chairlike as opposed to the envelope-like backbone conforma- action.29,30,59,60 Most importantly, the model demonstrates
a mode of binding that would disrupt the supramolecular

structure of the LPS packing in the bacterial OM, ultimately
resulting in cell death.
4. Complete Synthesis of Polymyxins

The polymyxin structure possesses elements atypical of
peptides in general, such as the NR fatty acyl chain and
multiple nonproteogenic L-Dab amino acids, one of which
(Dab4) participates in an intramolecular cyclic lactam with the
carboxyl terminal L-Thr10, an element not readily amenable to
solid-phase synthesis. The challenge of synthesis has been no
small hindrance in the development of polymyxin research.
Total synthesis of a polymyxin was first achieved by Vogler
et al.64-68 using a solution-phase segment condensation ap-
proach. However, the efficient generation of analogues has
ultimately depended upon the development of solid-phase
synthetic methods and specifically the development of ortho- Figure 7. Generalized schemes for peptide syntheses of polymyxin
gonal protection strategies around the L-Dab residues. Such peptides. From Sharma et al.:70 O = Sasrin resin, 0 = Dde
protection provides a mechanism to yield a partially protected protecting group, 4 = Boc/tBu protection, FA = fatty acid; (i)
linear precursor amenable to cyclization (Figure 7). This has hydrazine and then 1% TFA; (ii) DPPA; (iii) 95:5 TFA/H2O. From
been somewhat challenging, as protected L-Dab residues are Tsuberry et al.:72 O = 2-ClTrt resin, 0 = Mtt protecting group, 4
not so readily accessible, but in recent years examples such = Boc/tBu protection, FA = fatty acid; (i) 1% TFA; (ii) PyBOP;
(iii) 95:5 TFA/H2O (PMBN only). From Sakura et al.73 and Vaara
as Fmoc-L-Dab tert-Bbutyloxycarbonyl (Boc),69 Fmoc-L- et al.:74 O = Wang resin, 0 = Boc protecting group, 4 = Bz/Cbz
Dab 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde),70 protection, FA = fatty acid; (i) 95% TFA; (ii) PyBOP or DPPA;
Fmoc-L-Dab 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-me- (iii) HF or hydrogenation.
thylbutyl (ivDde),71 Fmoc-L-Dab 4-methyltrityl (Mtt),72 and
Fmoc-L-Dab 2-chlorobenzyloxycarbonyl (2-ClZ)73 have been Kenner’s safety catch sulfonamide resin with Dab4 protected
described. as an ivDde derivative for chain construction and then
In the first reported solid-phase method, Sharma et al.70 changed to the Mmt group for linker activation. Treatment
employed standard Fmoc-based methods but utilizing Dde with diisopropylethylamine effected intramolecular cycliza-
protection for Dab7 and synthesis on Sasrin resin to yield the tion releasing the protected peptide which was then fully
key partially protected precursor peptide after treatment with deprotected with TFA. Tsuberry72 also reported an on-resin
2% hydrazine and cleavage with 1% trifluoroacetic acid approach using a carbamate linkage through Dab9 side chain
(TFA). The cyclization was effected with diphenylphospho- but found it less efficient than the solution based methods.
rylazide (DPPA), and the cyclic peptide was then fully depro- Given the challenge of synthesis, the replacement of the
tected with 95% TFA. lactam bridge by something more synthetically facile is a
Similarly, Tsubery et al.72 prepared the polymyxin nona- logical progression, although the consequences on the cyclic
peptide using Mtt protection for Dab4 and synthesis on peptide conformation of such changes have the potential to
2-chlorotritylresin, yielding the partially protected peptide diminish activity. Synthetic analogues where a cystine disul-
under mild acidic conditions. Cyclization was best achieved fide replaces the Dab-Thr linkage have been reported by
with (benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium Clausell et al.35,36 (211-213) and Rustici et al.75 (153-155).
hexafluorophosphate) (PyBOP) and again full deprotection The synthesis under these circumstances becomes relatively
with TFA. In the same report, Tsubery et al.72 investigated the trivial with a linear sequence prepared and then oxidized
use of Fmoc methods for synthesis on Wang resin with Boc under standard conditions.
protection for the Dab4 but utilizing the TFA-stable 2-(ClZ)
protecting group for the other Dab residues. In this way, 5. SAR of Polymyxins
cleavage with TFA yielded the partially protected peptide. This review attempts to provide a comprehensive treatise on
Cyclization was performed again using PyBOP, and final the current state of development of polymyxin analogues.
deprotection was achieved with catalytic hydrogenation. Medicinal chemistry strategies for improving the antibacterial
Most recently, Sakura et al.73 and Vaara et al.74 extended activity and toxicity of polymyxins have included modifica-
this method for synthesis including the use of OBzl protecting tions of the length and size of the N-terminal fatty acyl chain
groups for the Thr residues. In this way, cleavage with TFA moiety (section 5.1), the Dab side chains and amino acid
yielded the partially protected peptide. Cyclization was per- substitutions (section 5.2), the D-Phe6-L-Leu7 hydrophobic
formed again using DPPA or PyBOP, and final deprotection motif (section 5.3), the size of the cyclic peptide ring (section
was achieved with hydrogen fluoride (HF) or hydrogenation. 5.4), and the length of the N-terminal linear tripeptide seg-
These methods have allowed for the synthesis of approxi- ment (section 5.5). Other strategies have involved conjugation
mately 20 peptides in each of these studies. (section 5.6), linearization (section 5.7), and generation of
Total solid phase synthesis of polymyxin peptides via on- mimetic compounds (section 5.8). Hereon in we shall review
resin cyclization is rendered a challenge because of the need for each of these targeted modifications in detail, and where
an accessible C-terminus and the absence of more obvious side activity data are available, structure-activity correlations will
chain linkage points (e.g., Asn or Gln residues). However, the be made with reference to the three-dimensional models of the
use of a “safety catch” methodology was described by de PMB-LPS complex.29,30 This section makes extensive refer-
Visser et al.71 Standard Fmoc synthesis was performed on ence to the chemical structures documented in the Supporting
Information, and as such, it is intended to be read in tandem generate PMB1-6 (1-6).70,82 Interestingly, the N-terminal
with the body of the text. In addition the supplementary variation across the naturally occurring PMB1-6 structures
section documents minimum inhibitory concentrations did not markedly affect antimicrobial activity (MICs against
(MICs) and LPS binding affinity data where available. E. coli of 0.5-1.0 μM). In contrast, the LPS binding assay
5.1. N-Terminal Fatty Acyl Analogues. Functionally the N- results indicated that the more hydrophobic N-terminal
terminal fatty acyl chain is purported to penetrate the OM substituents such as nonanoyl (PMB5) possess a greater
and disrupt the packing of the lipid A fatty acyl chains.29,30 LPS binding affinity compared to the octanoyl (PMB3)
Isothermal titration calorimetry studies have shown that the and heptanoyl (PMB4) substituents.82,94,95
hydrophobic contribution from the N-terminal fatty acyl Chihara et al.96,97 were the first to attempt a SAR char-
chain is the predominant energetic driving force for acterization of the NR fatty acyl segment (37-58). This early
PMB-LPS complexation.50-53 Therefore, the N-terminal work employed colistin nonapeptide as starting material; NR
fatty acyl chain is crucial for the antimicrobial activity of acylation was performed in a buffered aqueous solvent
polymyxins. without any protection of the Dab Nγ-amino side chain
The importance of the N-terminal fatty acyl segment for groups.96,97 The activities of N-terminal fatty acyl derivatives
the antimicrobial properties of polymyxins first became containing C9-C14 unbranched fatty acyl chains showed
evident when polymyxin nonapeptides were characterized.62 that the longer fatty acyl derivatives were more active against
Polymyxin nonapeptides are composed of the cyclic hepta- polymyxin-resistant strains and Gram-positive bacteria,
peptide and the N-terminal Thr2-Dab3 dipeptide compo- whereas the C10 and C12 derivatives were most effective
nents. PMBN (193) and colistin nonapeptide are generated against the polymyxin-susceptible strains.
by proteolytic release of the N-terminal fatty acyl-Dab1 The most comprehensive NR SAR data have been deliv-
segment with either ficin or papain proteases.62 Although ered by Sakura and colleagues.82,94,95 The group utilized a
PMBN and colistin nonapeptide lack any direct antimicro- semisynthetic approach across these studies, wherein pur-
bial activity, they retain the ability to bind LPS with high ified PMB or colistin were chemically converted to nonapep-
specificity and efficiently perturb the OM to sensitize Gram- tides by treatment with S-ethyl trifluorothioacetate and used
negative bacteria to hydrophobic antibiotics that are nor- as starting materials. The key step in this successful semi-
mally impermeable to the OM.62 More importantly, PMBN synthetic approach is the preparation of Nγ-protected non-
and colistin nonapeptide display a significantly reduced apeptide by trifluoroacetylation (Tfa) or with the
toxicity profile compared to their parent compounds (LD50 trichloroethoxycarbonyl (Troc) protecting group, followed
of 43 mg/kg for PMBN vs 9 mg/kg for PMB in mice).76 by chemical cleavage with methanesulfonic acid to remove
Thus, the toxicity of polymyxins can partly be attributed to NR-alkanoyl-Nγ-[Tfa/Troc]-Dab1-OH, yielding the tetrakis-
the N-terminal fatty acyl segment.62,74,77 Accordingly, a (Nγ-protected)nonapeptide.
number of medicinal chemistry programs have focused on Following on from the early work of Chihara,96,97 a
generating NR analogues with the goal of developing poly- complementary study from Sakura et al.95 also examined
myxins with improved toxicity profiles. A number of differ- the effect of NR fatty acyl chain length on antimicrobial
ent synthetic routes were employed across these reports and activity (59-63). The study employed the aforementioned
will be reviewed briefly preceding each synopsis. semisynthetic approach. All products were HPLC purified,
Across the naturally observed PMB peptide components and their identity was confirmed by MS analysis. It was
the type of fatty acyl chain linked to the N-terminal Dab found that N-terminal fatty acyl chains intermediate in
residue is restricted to (S)-6-methyloctanoyl (PMB1) (1), length (octanoyl) (63) are optimal, whereas longer
6-methylheptanoyl (PMB2) (2), octanoyl (PMB3) (3), hepta- (myristoyl, C14) (61, 62) and smaller acetyl-PMBN (59, 60)
noyl (PMB4) (4), nonanoyl (PMB5) (5), and 3-hydroxy-6- analogues displayed reduced antimicrobial activity com-
methyloctanoyl (PMB6) (6).62,78-82 The naturally occurring pared to PMB. Interestingly, the acetyl-PMBN analogues
polymyxins also display variation of the methyl substitu- displayed poor antimicrobial activity against E. coli, and
tion at the 6 and 7 positions of the fatty acyl chain.62,78-81 S. enterica serovar Typhimurium, however, retained potent
The confirmed synthesis of the naturally occurring 6- and activity specifically against P. aeruginosa. It is noteworthy
7-methyloctanoyl isomers of colistin (7, 17) indicated that that the potent activity against P. aeruginosa displayed by the
the one carbon migration of the methyl group does not compounds (59, 60)95 is not consistent with the observed
substantially affect antimicrobial activity.71 The naturally correlation between the length of the NR fatty acyl chain and
occurring octapeptin subclass of polymyxin peptides are antimicrobial activity reported elsewhere.96,97 This observa-
N-terminally substituted with long β-hydroxy fatty acyl tion can be potentially construed in terms of the differences
moieties, including the straight chain β-hydroxydecanoyl in the lipid A fatty acyl chain composition across these
and the branched chain 3-hydroxy-8-methylnonanoyl or bacteria which in turn confers different OM properties.95,98
3-hydroxy-8-methyldecanoyl (24-36).83-92 In their more recent work Sakura et al.94 described a
Early studies of the NR fatty acyl SAR of PMB and colistin comprehensive series of NR analogues were derived by
component peptides isolated from cultures of producer acylation of the tetrakis(Nγ-Troc)-PMB or colistin nonapep-
strains were not sufficiently comprehensive to provide any tides with various hydrophobic acids with aliphatic or
meaningful SAR data.93 This is in large part due to the hydrophobic ring structures (64-79). The NR analogues
difficulties associated with separation of such closely related were tested for LPS binding affinity and antimicrobial
structures with the available technology at the time. In more activity against E. coli, S. enterica serovar Typhimurium,
recent times, a few researchers have reported successful and P. aeruginosa. It was found that cyclohexylbutanoyl-
separation techniques; however, these studies did not include (69, 71), 4-biphenylacetyl- (65, 67), and 1-adamantaneacetyl-
any microbiological data for the isolated components.78-81 (64, 66) NR analogues displayed comparable activities to
Subsequently, the NR SAR of the PMB component peptides the parent compounds (PMB and colistin), with improved
was confirmed using a solid-phase synthesis approach to LPS binding affinity. Generally, the entire series displayed
comparable activity to PMB against P. aeruginosa, whereas evidence of the purity and identity of the compounds is
marked differences in activity against E. coli and S. enterica documented.
serovar Typhimurium were evident across the series.94 de Visser et al.71 synthesized a series of NR PMB analogues
Clausell et al.36 reported a PMB analogue that incorpo- with short (<C7) unbranched fatty acyl chains. This study
rates a pyrene group NR substituent. The compound was employed a solid-phase synthetic approach based on clea-
employed for examining membrane association and inter- vage-by-cyclization using a safety-catch sulfonamide linker
membrane contact formation, and no microbiological data resin.71 The hexanoyl (C6) (91) and 1-adamantane (83) NR
were reported. The same compound was reported by Sakura analogues displayed potent antimicrobial activity (albeit less
et al.94 (72, 74) as part of their comprehensive NR SAR study; active than PMB), whereas the pentanoyl (C5) (92) and
the analogue displayed a reduced antimicrobial activity butanoyl (C4) (90) fatty acyl derivatives were 10- to 20-fold
compared to PMB against E. coli and S. enterica serovar less active than PMB. It is noted that, unless the MIC of
Typhimurium, and comparable activity to PMB was ob- PMB was measured in each study as a control, care is
served against P. aeruginosa. required when comparing the MICs across different series
The Pfizer group99 explored the effect of increasing the of analogues.
length of the N-terminal fatty acyl chain on antimicrobial Tsubery et al.101 adopted a total synthetic approach
activity of PMBN analogues (80-82). In this study, PMBN by employing a combination of solid-phase linear chain
prepared from commercial PMB by papain treatment was elongation (γ-fluorenylmethoxycarbonyl (Fmoc) strategy)
employed as the starting material. The Dab Nγamino side and subsequent cyclization after release. Side chain Dab
chain groups were blocked with a protecting group. The Nγ-amino protecting groups were tert-butyloxycarbonyl
NR-amino was not selectively protected prior to reaction (tBoc) and benzyloxycarbonyl (Cbz). Two pairs of NR
with the protecting group, as it was purported that the Dab PMBN and PMB decapeptide analogues were synthesized
Nγ-amino groups, being more electron-rich and less steri- (93-96). In one pair the N-terminus of PMBN was substi-
cally hindered, would preferentially react with the protecting tuted with [Ala]3 and [Ala]6 oligoalanyl (93, 95). Because of
group 1-(Boc-oxyimino)-2-phenylacetonitrile (Boc-ON). the long hydrophobic chain, [Ala]6-PMBN (95) was expected
The reaction was performed over a short period (<20 min to possess potent antimicrobial activity; however, both
at 23 °C) and quenched to avoid complete reaction to the oligoalanyl NR analogues did not significantly improve
penta-Boc byproduct. NR analogues with unbranched ocat- activity compared to the control compound PMBN. This
noyl (C8) fatty acyl chains (80) displayed significantly en- could potentially be attributed to the extensive methyl side
hanced antimicrobial activity (up to 8-fold based on MICs) chain branch points along the oligoalanyl structure. The
compared to PMB (both commercial and synthetically N-terminus of the other pair was substituted with the hydro-
sourced). The nonanoyl (C9) (81) and decanoyl (C10) (82) phobic Fmoc group (94, 96). The hydrophobic Fmoc NR
fatty acyl NR derivatives showed decreased activity. The substitution significantly enhanced in vitro antimicrobial
findings of this study suggest that NR fatty acyl chains activity and reduced toxicity in a mouse acute toxicity model.
>C8 in length and branching such as the 6-methyl moiety This indicates that the hydrophobicity of the NR substituent
in PMB act to decrease antimicrobial activity, potentially by influences both antimicrobial activity and acute toxicity.
sterically hindering OM insertion of the fatty acyl moiety. Compared to the Fmoc-PMBN (94), the Fmoc-PMB
It is interesting to note that in comparison, only marginal decapeptide (96) was significantly more active against
differences in activity were evident between the (S)-6-methyl- P. aeruginosa and K. pneumoniae while comparable against
octanoyl- (PMB1) (1) and unbranched octanoyl- (PMB3) (3) E. coli.
NR native PMB peptides.82 However, considering that the Collectively, these experiments underscore the important
Pfizer compounds are PMBN analogues, this discrepancy contributions made by the N-terminal fatty acyl chain to the
simply emphasizes the important role of Dab1 for regulating binding interactions with LPS and consequent antibacterial
antimicrobial activity. Other reports have also shown longer activities of polymyxins. A comparison across all NR analo-
NR-terminal fatty acyl substituents such as decanoyl reduce gues documented indicates antimicrobial activity appears to
antimicrobial activity compared to the corresponding octa- correlate with the length and bulkiness of the NR substituent.
noyl substituted analogues.74 A similar trend was reported The optimal fatty acyl chain length appears to be C7-C9 (as
for a series of octapeptins where a decrease in activity was per the native peptides), as longer or shorter chain NR
observed with compounds that possess NR fatty acyl chains analogues display reduced antimicrobial activity. LPS bind-
longer than C8.100 ing affinity appears to correlate with the length of the NR
There are noticeable discrepancies in the literature with fatty acyl chain.82,95,99 By reference back to the molecular
reported NR analogue SAR data. The Sakura82 and Pfizer model of the PMB-LPS complex (Figure 5A), it is likely that
groups99 reported octanoyl- and myristonyl-PMB analogues hydrophilic, long (>C9 fatty acyl chain), bulky, or exten-
with potent antimicrobial activity, whereas the same com- sively branched NR substituents sterically hinder OM inser-
pounds were reported 30 years ago by Chihara et al.96,97 to be tion. Therefore, the packing of the lipid A fatty acyl chain
20 times less active. This discrepancy is most likely due to layer is not sufficiently disrupted to produce antibacterial
contamination of the products reported by Chihara et al.96,97 activity.
with Nγ acylated nonapeptides, as the acetylation reaction 5.2. Dab Side Chain Derivatives and Substitutions. Even at
was conducted without protection of the Dab Nγ-amino side the very beginnings of polymyxin research, it was understood
chain groups. In comparison, the more recent efforts from that the positive charges of the Dab residues are important
Sakura et al.82 and Pfizer99 employed protecting groups for antimicrobial activity.102 This was first evidenced by the
for the Dab Nγ side chains prior to NR acetylation. More- inactive sulfomethyl PMB (97).102 Sulfomethyl PMB is a
over, the products were purified by HPLC prior to testing derivative of PMB where the free amino groups are blocked
for antimicrobial activity. Thus, SAR data obtained from by sulfomethylation, yielding a compound that is inactive in
earlier reports should be viewed with caution unless clear vitro.102 In this section we shall discuss the numerous
synthetic or semisynthetic modifications targeted at the Dab than commonly observed.7 In light of the other PMB analo-
positions in an effort to improve microbiological activity and gues with hydrophobic modifications on the Dab side chains
minimize potential for toxicity. reviewed herein, it appears that the introduction of lipophilic
Early attempts to neutralize the untoward effects of the groups acts to broaden the antibacterial spectrum. Although
Dab residues mostly involved simple chemistries for derivi- this appears promising, one concern is the potential for
tization of the Dab Nγ-amino side chain groups.102-107 Most hemolytic activity against the host associated with such
of these studies used commercial preparations of PMB lipophilic peptides.109
(sulfate) or colistin (sulfate) as the starting material and The inception of solid-phase peptide synthesis in 1963110
did not employ protecting groups; as such, the amine cou- has allowed chemists to more readily explore amino acid
pling reactions were performed in a nonspecific manner. substitutions in bioactive peptides. In an effort to circumvent
Chemical modifications to the Dab Nγ-amino groups the potential toxicity, many medicinal chemistry programs
through amide linkages or Schiff base formation have in- have employed solid-phase synthesis to substitute the non-
cluded acetylation (98-102),103,105 deamination (103),103 proteogenic Dab residues with neutral or basic proteogenic
formylation (104-108),103 dinitrophenylation (110),103,107 amino acids.
guanidation (113),103 cabamylation (115-130),107 and reac- The earliest report of an all Dab f Lys substituted colistin
tions with 5-amino-2,4-dinitrofluorobenzene (111),103 pyri- analogue (152) was from Kurihara et al.111 The microbiolo-
doxal phosphate (114),103 or dimethylaminonaphthalene-5- gical data reported for the compound indicated a compar-
sulfonyl (dansyl) (205).108 Most of these compounds were able antimicrobial activity to native colistin. Rustici et al.75
inactive; with exception, an early report from Srinivasa and synthesized a series of des-NR-acyl-PMB analogues with Lys
Ramachandran103 described a set of Dab Nγ-formylpoly- replacing all of the Dab positions, L-Phe replacing D-Phe,
myxin B derivatives (105-108) that displayed potent anti- and cyclization via an intramolecular disulfide bridge
microbial activity (except 108). Interestingly, triformyl-PMB (153-156). The compounds displayed poor antimicrobial
(106) was as active as PMB, and diformyl-PMB (105) was activity, albeit 154-156 possessed appreciable binding affi-
70% more active than PMB.103 In stark contrast, another nity for LPS. Once again this study demonstrated that the
early study reported that Dab Nγ mono-, di-, and triacyl ability to bind LPS does not necessarily correlate with
derivatives (98, 100, 102) retained only partial activity, antimicrobial activity. Acute toxicity testing in mice showed
whereas the tetra- and pentaacyl derivatives (99, 101) were these analogues were less toxic than PMB.
inactive.105 Witzke and Heding107 reported a series of PMB In their patent descriptions, Porro et al.112-115 described a
and colistin N-benzyl derivatives (115-130) prepared from large series of polymyxin analogues wherein each Dab was
Schiff bases that displayed an increased activity against the substituted with a basic amino acid (Lys, Arg, or His). All of
Gram-positive test strain, Staphylococcus aureus ATCC the peptides displayed a high affinity for LPS (even better
6538P. The reduction of cationic character through substitu- than PMB) and sensitizing activity toward hydrophobic
tion of Dab side chains with lipophilic groups appears to antibiotics against the Gram-negative bacterial strains
increase activity against Gram-positive bacteria (131, 133, tested, namely, S. Typhimurium, Haemophilus influenzae,
138, 140, 142).106,107 and Vibrio cholerae.
Weinstein et al.106 employed tetra-Boc PMB as starting A recent study from the Sakura group73 reported a series
material to prepare a series of analogues selectively modified of PMB alanine scanning analogues aimed at examining the
at the Dab1 and Dab9 Nγ-amino side chain groups contribution of each amino acid position to antimicrobial
(131-151). Monofunctionalization of PMB at the Dab1 activity and LPS binding (158-167). The [Dab5 f Ala]PMB
and Dab9 positions was achieved by protection of the most (161) analogue displayed the most significant reduction in
basic Dab Nγ-amino groups (Dab1 and Dab9) by proto- activity. In addition, it was found the Dab positions within
nation with strong acids while introducing protecting Boc the heptapeptide ring (Dab5,8,9) were more important for
groups on the remaining Dab Nγ-amino groups. PMB antimicrobial activity than the Dab residues in the linear
monohydrochloride prepared by dissolving PMB in metha- tripeptide segment (Dab1,3). This can be fully appreciated
nol containing acetone, and HCl (1 equiv) was reacted with from the model of the PMB-LPS complex, which shows that
di-tert-butyl dicarbonate to yield tetra-Boc[Nγ-Dab1]PMB the Dab positions in the heptapeptide provide the critical
(15%) and tetra-Boc[Nγ-Dab9]PMB (36%). The tetra-Boc electrostatic contacts that anchor the whole PMB structure
products were used to prepare Nγ-alkylated and Nγ-acylated to the lipid A phosphate groups (Figures 4 and 5A). All of the
derivatives. Only derivatives with positively charged or polar Ala substitution analogues displayed a reduced LPS binding
side chain substituents displayed better antimicrobial activ- affinity compared to PMB.
ity than PMB. Notably, the arginyl-Dab9 (149) derivative In an attempt to reduce the nephrotoxicity associated with
displayed a significantly improved therapeutic index deter- the cationic nature of polymyxins, Vaara et al.74 recently
mined acutely in male CF1 mice (LD50, 15 mg/kg in- reported a series of polymyxin analogues carrying only three
travenously)/protective dose (PD50, 0.25 mg/kg subcuta- positive charges, all within the heptapeptide ring (174-192).
neously) compared to PMB (LD50, 9 mg/kg intravenously/ Analogues substituted with Thr, Ser, or aminobutyryl
PD50, 4.5 mg/kg subcutaneously). Across the entire series, groupat the Dab1,3 positions within the tripeptide linker all
the Dab9 position appeared to be more important for anti- displayed potent antimicrobial activity (gPMB). Moreover,
bacterial activity, as the Dab9 derivatives were 4-fold less irrespective of whether they had any direct antimicrobial
active than the corresponding Dab1 derivatives. The more activity, all of the analogues displayed a potent sensitizing
lipophilic R-aminoacyl (131, 133, 138, 140, 142) derivatives action toward hydrophobic antibiotics. Substitution of the
displayed a broader antibacterial spectrum, with potent Dab positions in the cyclic heptapeptide with neutral
activity against Gram-positive pathogens. Interestingly, the aminobutyric acid residues resulted in a complete loss
geometric mean MIC of PMB against the Gram-positive test of antimicrobial activity. The compounds displayed a mark-
strain (S. aureus) was 2.8 μg/mL, which is surprisingly lower edly reduced affinity for isolated rat kidney brush border
membranes, which is purported to be indicative of reduced proper spatial distribution of the positive charges for elec-
nephrotoxicity.74 trostatic interactions with the phosphates of lipid A. To date,
Tsubery et al.72,116 described a series of PMBN analogues attempts to substitute or derivatize the Dab positions have
where the Dab residues were selectively substituted with met with variable success. In general, the Dab residues,
basic amino acids with longer or shorter side chains com- particularly within the cyclic heptapeptide, are indispensable
pared to Dab (i.e., Lys; ornithine; 2-amino-4-guanidinobu- for the antimicrobial activity of polymyxins.
tyric acid; 2,3-diaminopropionic) (194-204). The SAR data 5.3. D-Phe6-L-Leu7 Hydrophobic Motif Analogues. Poly-
garnered from this study clearly showed that in addition to myxins possess two hydrophobic domains, the NR-terminal
the cationic character, the length of the amino acid side chain fatty acyl moiety and the D-Phe6-L-Leu7 segment in the
alkyl arm is also crucial for antimicrobial activity. The heptapeptide ring. Functionally, the hydrophobic side
optimum side chain length appears to be two methylene chains of the D-Phe6-L-Leu7 motif are believed to insert
groups as per the native Dab residue. PMBN analogues with into the bacterial OM and stabilize LPS complexation via
side chains that varied by four (Lys), three (ornithine), and hydrophobic interactions with the fatty acyl chains of lipid
one (2-amino-4-guanidinobutyric acid) methylene groups in A.29,30 Molecular dynamics and NMR structural studies
length were all found to display a reduced sensitizing activity. indicate that PMB adopts a type II0 β-turn centered around
In line with these findings, the model of the PMB-LPS the D-Phe6-L-Leu7 motif in the free state.28-30 A type II0
complex (Figures 4 and 5A) indicates that a two methylene β-turn is commonly found in many nonribosomal cyclic
side chain length provides the ideal bridging distance be- peptides.120-122 This structural feature is believed to
tween the Nγ-amino side chain groups and the lipid A allow the backbone of the cyclic peptide to adopt the
phosphates. biologically active conformation when binding to its target
Interestingly, as noted above, a number of studies receptors.120-122 The position i þ 1 of the type II0 β-turn is
have indicated that the antimicrobial activities and LPS generally populated by a D-amino acid or Gly that serves as
binding affinities of polymyxins and their analogues are the β-turn forming element. Across the naturally occurring
not parallel.73,94,95 A polymyxin analogue can display potent polymyxins, position 6 (about which the type II0 β-turn is
antimicrobial activity and concomitantly only moderate LPS centered) is commonly populated by D-Phe (PMB) or D-Leu
binding affinity and vice versa.73,94,95 This is possibly related (colistin). In PMB (1-6) and colistin (7, 8, 15-17, 19)
to the biophysical nature of the assay system employed to position 7 is occupied by L-Leu. Moreover, [Ileu7] (12, 18),
measure LPS binding affinity. The most commonly used [Thr7] (20-23), [NorVal7] (10, 13), and [Val7] (11, 14) pep-
assay is the N-dimethylaminonaphthalene-5-sulfonyl-PMB tides have been identified in PMB and colistin minor
(dansyl-PMB) (205, 230) fluorometric displacement assay. components.80-82 By employment of complete synthesis to
The dansyl moiety is an environmentally sensitive fluoro- generate defined products, substitution of Leu7 f Ileu or Thr
phore that has a low quantum yield in the high dielectric in PMB or colistin has been shown not to noticeably affect
environment of the aqueous buffered assay solution. antimicrobial activity.82,123
Whereas in the low dielectric hydrophobic environment The contribution of the D-Phe6-L-Leu7 segment was first
formed by the LPS binding surface, it is highly fluorescent. evaluated in PMBN by substitution of D-Phe6 with D-Trp or
7
Therefore, the assay measures the enhancement in fluores- D-Tyr (206-209) and substitution of the L-Leu position with
124
cence emission of the dansyl moiety upon binding to the L-Phe (206) or L-Ala (207). PMBN was employed as the
hydrophobic LPS molecule. Titration with unlabeled poly- control compound such that the contribution of the D-Phe6-
7
myxin results in a quenching of fluorescence by competitive L-Leu motif could be evaluated without the influence of the
R
displacement of dansyl-PMB from the LPS binding surface. N fatty acyl chain.124 The shortcoming of this approach is
Dansyl-PMB is usually prepared from commercial prepara- that only relative sensitizing activity can be measured, as
tions of PMB by reaction of the Dab Nγ-amino side chain PMBN lacks any antimicrobial activity. Substitution of
groups with dansyl chloride in solution.108 The reaction either D-Phe6 or L-Leu7 with more polar amino acids such
products are usually heterogeneous. Our recent unpublished as Ala or Tyr significantly reduced OM permeabilizing
data show mixtures consisting of mono-, di-, tri-, tetra-, and activity. Replacing the D-Phe6 with L-Phe resulted in an
pentalabeled products, which are inactive, yet bind LPS. The almost complete loss of OM permeabilizing activity.124
original report108 described a dansyl-PMB product that Together, these results possibly reflect an impairment of
retained potent antimicrobial activity; however, this pre- the stability of the type II0 β-turn or the inability of the polar
paration was not purified, and therefore, the observed anti- residues to insert into the fatty acyl layer formed by lipid A in
microbial activity is probably attributable to unreacted the bacterial OM. The D-Trp6 (209) analogue displayed
PMB. The ideal approach is to employ a structurally well- marginally reduced permeabilizing activity relative to
defined monodansylated [dansyl-Gly1]PMB (230) generated PMBN, consistent with the conservative nature of these
via solid-phase synthesis.82 The [dansyl-Gly1]PMB retains substitutions.
almost complete antimicrobial activity as PMB; therefore, The recent work of the Sakura group73 thoroughly exam-
the assay more faithfully measures competitive binding for ined the effect substitutions at D-Phe6-L-Leu7 on antimicro-
the same sites on the LPS molecule.82 bial activity and LPS binding. PMB analogues substituted at
The critical involvement of the cationic Dab residues in position 6 with D-Trp (168), D-Ala (167), and L-Phe (172) or
conferring the antimicrobial activity of polymyxins has been at position 7 with L-Ala (162) and L-Trp (173) retained both
well documented.56,102,117-119 In summary, the key features potent antimicrobial activity and LPS binding. These find-
of the Dab residues that are important for antimicrobial ings would suggest that neither a loss of hydrophobicity
activity and LPS binding affinity include (a) the cationic within the D-Phe6-L-Leu7 motif nor the D-configuration of
character of the side chain groups, (b) the two-methylene the position 6 amino acid was essential for antimicrobial
length of the Dab side chain, and (c) the specific order of the activity. These results are somewhat at odds with the find-
Dab residues within the primary sequence that confers the ings of Tsubery et al.124 and the line of thought that the
D-configuration of the position 6 amino acid is necessary for (in which all L-amino acids are replaced by their optical
the formation of the type II0 β-turn. However, the findings of D-isomers) lacks the ability to sensitize Gram-negative bac-
Sakura et al.73 cannot be directly compared to that of teria toward hydrophobic antibiotics. Interestingly, both
Tsubery et al.,124 as the latter study examined PMBN enantiomers displayed comparable LPS binding affinity,
analogues. Nevertheless, the discrepancy between the two again suggesting that LPS binding and OM permeabilizing
studies indicates the NR fatty acyl chain is more than capable activity are not coincident. In another study, Fridkin and
of compensating for a loss of hydrophobicity within the colleagues described the synthesis of a series of PMBN
6 7 6 7
D-Phe -L-Leu motif. The [Gly -Gly ]PMB (170) double analogues wherein the ring size was varied from 20 to 26
substitution analogue was completely inactive; this is most atoms (193-200).72,116 All of the ring variants displayed a
likely a result of the complete loss of hydrophobicity or a significantly reduced OM permeabilizing activity compared
dramatic conformational change in the heptapeptide ring.73 to PMBN, indicating that the native 23 atom ring provides
The position 7 substitution analogues [L-Ala7]PMB (163), the optimal structural configuration.
[Gly7]PMB (171), and [L-Trp7]PMB (173) displayed margin- Taken together, these SAR data demonstrate it is the
ally reduced antibacterial activity and LPS binding com- precise combination of topographic chemical features and
pared to PMB. the 23 atom ring structure that are essential for efficient
Clausell et al.35 described an analogue that incorporated a binding to LPS and subsequent OM permeabilizing activity.
permutation where the D-Phe6-L-Leu7 motif was disrupted Thus, the 23-atom size of the native PMB ring provides the
by an intervening Dab residue (212). Although no micro- most ideal structural configuration for potent antimicrobial
biological data were reported, the analogue was shown to activity.
lack selectivity for lipids that exchange through vesicle-to- 5.5. Linear Tripeptide Segment Analogues. The heptapep-
vesicle contacts and had a significantly lower permeabilizing tide cyclic core of the polymyxin molecule is bridged to the
activity toward artificial vesicle membranes. Another analo- fatty acyl chain by a linear tripeptide segment (Dab1-Thr2-
gue containing D-Phe6 f D-Trp substitution, comparable to Dab3) (Figure 1). Although the isolated fatty acyl-tripep-
PMB, showed binding to vesicles and formation of vesicle- tide moiety (6-methyloctanyl fatty acyl-Dab1-Thr2-Dab3-
to-vesicle contacts. COOH) lacks both direct antimicrobial activity and OM
de Visser et al.71 reported a series of analogues where the permeabilizing activity,93 the structure represents a key
6 7
D-Phe -L-Leu motif was substituted with dipeptide functional feature of polymyxins. Functionally, this segment
mimics: (a) δ-aminovaleric acid (84), (b) the extended con- contributes two positive charges toward the binding inter-
formation inducer 4-phenyl-4-carboxymethylpiperidine (85), action with LPS. Moreover, the molecular model of the
(c) m-aminomethylbenzoic acid (86), (d) the β-turn inducing PMB-LPS complex indicates hydrogen bonds (a) between
amino acid (S)-3-amino-1-carboxymethylcaprolactam (87), the amide nitrogen of Dab3 and the hydroxyl side chain of
(e) N-carboxymethylpiperazine (88), and (f) trans-4-amino- Thr2 and (b) between the main chain carbonyl of Dab4 and
methylcyclohexane (89). Unfortunately none of these analo- the amide nitrogen of Thr2, which bends the tripeptide
gues were active against E. coli. toward the heptapeptide core (Figure 6B). This configura-
In summary, the D-Phe6-L-Leu7 segment in the polymyxin tion ensures that the amphipathic structure of the molecule
heptapeptide ring forms a hydrophobic domain that is highly remains well organized into the respective hydrophobic and
conserved across the naturally occurring polymyxins and hydrophilic domains.
appears to be important. However, it is not indispensable for A number of medicinal chemistry studies have ex-
antibacterial activity and LPS binding. plored the SAR of the linear tripeptide segment by
5.4. Cyclic Peptide Ring Modifications. Many nonri- examining the effects of amino acid deletions and/or
bosomal antimicrobial peptides display a cyclized back- substitutions.64,74,76,82,98,127-129 A variety of prepara-
bone.120,121,125 Studies with deacylated polymyxins clearly tive techniques including enzymatic, semisynthetic, and
demonstrated that the major structural requirement for complete solid-phase synthesis methods have been employ-
direct antimicrobial activity is the cyclic heptapeptide struc- ed.64,74,76,82,98,127-129 Both fatty acyl-PMB and des-fatty
ture. In the polymyxin structure, the Nγ-amino side acyl-PMB analogues have been employed as the parent
chain of Dab4 is deacylated by the C-terminal Thr10 to form structure across these studies.
a 23-membered lactam ring. The molecular model of the The very first such study was from Vogler et al.64,66 A pair
PMB-LPS complex (Figures 4 and 5A) indicates it is the of ring modified PMB analogues were synthesized which also
precise 23-atom size of the native heptapeptide ring that acts featured Dab deletions within the tripeptide segment (214,
as a scaffold for electrostatic and hydrophobic LPS contact 215). Both analogues displayed significantly lower antimi-
points. This has been well accepted, and only a few attempts crobial activity compared to PMB1. Nevertheless, the re-
have been made to examine modifications to the native ring duced activity cannot unequivocally be attributed to the
size on antimicrobial activity.64,72,116 tripeptide segment modifications due to altered cyclic core.
Vogler et al.64,66 were the first to describe a PMB analogue Sakura.et al.82 described three fatty acyl-PMB analogues
with an extended ring structure (214, 215) generated by with truncated tripeptide segments, octanoyl fatty acyl-PMB
the insertion of an additional Dab residue. The resultant nonapeptide (octanoyl FA-PMBN) (231), octanoyl fatty
compounds were significantly less active than PMB against acyl-PMB octapeptide (octanoyl FA-PMBO) (232), and
E. coli. octanoyl fatty acyl-PMB heptapeptide (octanoyl FA-
The Fridkin group72,76,101,116,124,126 have been very active PMBH) (230). Octanoyl FA-PMBN (231) displayed com-
in the area of polymyxin SAR. Most notably, the group parable activity to PMB, whereas the double deletion analo-
demonstrated that the sensitizing activity of PMBN is gue octanoyl FA-PMBO (232) was 6 times less active against
stereospecific and highly dependent on the overall structural E. coli. The complete tripeptide deletion analogue, octanoyl
orientation of the heptapeptide.126 Compared to the native FA-PMBH (230), was devoid of antimicrobial activity. In
L-enantiomeric PMBN, the D-enantiomeric PMBN analogue addition, a corresponding set of des-fatty acyl tripeptide
analogues were synthesized.82 The nona-, octa- (234), and segment of octanyl-PMB analogues described by the
heptapeptide (235) des-fatty acyl analogues were devoid of Vaara group.74 Across the naturally occurring polymyxins,
direct antimicrobial activity, whereas the des-fatty acyl Thr2 f Ser polymyxin E4 (16), Dab3 f Ser (19), polymyxin
decapeptide analogue with the intact tripeptide (des- D1 (20), polymyxin D2 (21), polymyxin S1 (22), substitu-
FA-[Dab1-Thr2-Dab3]-PMB) (233) retained antibacterial tions are observed within the tripeptide linker, whereas the
activity. The activity of these fully synthetic analogues is Dab1,3 positions are strictly conserved.131-134 This conser-
consistent with the antimicrobial activity reported for corre- vative substitution does not appear to affect antimicrobial
sponding analogues derived enzymatically, as discussed activity.131-134
below.128,129 The PMB-LPS structural model shows an electrostatic
The importance of the tripeptide side chain for OM interaction between Dab1 and the 40 phosphate of lipid
permeabilizing activity was examined in the early work of A (Figures 4 and 5A). However, a few studies have indicated
Vaara,128,129 which compared the OM permeabilizing activ- that the Dab1 position is dispensable for the LPS binding
ity of PMBN to PMB decapeptide (PMBD) (233), PMB and antimicrobial activity of PMB.73,82,98 [Ala1]PMB
octapeptide (PBMO) (234), and PMB heptapeptide (PMBH) (158) and [dansyl-Gly1]PMB (229) analogues both displayed
(235). The compounds were prepared by proteolytic diges- antimicrobial activity comparable to PMB.73,82 Moreover,
tion of commercial PMB with polymyxin acylase (yielding the semisynthetic fatty acyl-[Thr2-Dab3]PMB (231) analo-
PMBD), nagarse (yielding PMBO), or bromelain (yielding gue82,95 displayed comparable activity to PMB, again sug-
PMBH). All products were purified by reversed-phase gesting the Dab1 position is dispensable for antimicrobial
HPLC. PMBO displayed comparable sensitizing activity to activity. Tsubery et al.101 reported a NR Fmoc-PMB deca-
PMBN against E. coli and S. enterica serovar Typhimurium. peptide (Fmoc-PMBD) (96) and nonapeptide (Fmoc-
In comparison, a 3-fold greater concentration of PMHP PMBN) (94) analogue pair. The Fmoc-PMBD displayed
(236) was required to achieve the same sensitizing effect. an approximately 2-fold greater activity than the Fmoc-
PMBD was a slightly more effective OM permeabilizing PMBN, suggesting Dab1 provides a significant contribution
agent than PMBN.129 In contrast to PMBN, at high con- to antimicrobial activity and LPS binding. In one of their
centrations (30 μg/mL) PMBD displayed direct antimicro- recent studies, Sakura and colleagues described a series of
bial activity. The report by Kimura et al. is in remarkable des-fatty acyl-PMB analogues substituted at Dab1 with
contrast to the report by Ito-Kagama and Koyama127,130 various polar or hydrophobic amino acids (des-FA-[X1]-
that showed an approximate 100-fold difference in OM PMB) (216-228).98 The des-FA-[Ser, 2,3-diaminopropionic
permeabilizing activity of colistin nonapeptide and colistin acid (Dap), Arg, Phe, Trp1]PMB (218, 221, 222, 226, 227)
heptapeptide. This discrepancy may be a result of the analogues displayed comparable antimicrobial activity as
different preparative and purification procedures employed the control compound des-FA-[Dab1]PMB (220) against
by the two groups. most of the test strains. The des-FA-[Lys, Ala, Leu1]PMB
Naturally occurring polymyxins with amino acid deletions (216, 224, 225) analogues displayed a reduced antimicrobial
within the tripeptide segment have been documented.83-92 activity relative to the control compound (220). Not surpris-
The octapeptins are naturally occurring nonribosomal ingly, the introduction of a negatively charged residue
peptides that display the same heptapeptide primary struc- generated an inactive compound des-FA-[Glu1]PMB (223),
ture as PMB but lack the N-terminal tripeptide segment presumably because of electrostatic repulsion with the nega-
(24-36).83-92 Instead the heptapeptide core is linked to a tively charged phosphate groups of lipid A. All of the
single D-Dab or D-Ser residue attached to a long β-hydroxy compounds exhibited a reduced affinity for LPS compared
fatty acyl chain. In a structural perspective, it would appear to PMB. Collectively, these data would imply that the Dab1
the loss of the Dab1-Thr2 segment (which interacts with the position plays more of a fine-tuning as opposed to a critical
40 -PO4 of lipid A in the PMB-LPS complex, cf. Figures 4 and role toward the binding interaction.
5A) is compensated by the additional hydrophobic and polar The Sakura group also described a series of N-terminal
contributions from the longer chain and secondary hydroxyl tripeptide PMB analogues (217, 219, 228).98 The des-
groups of the fatty acid. Despite their structural similarities FA-[Dab-Dab-Dab1]PMB (228) and des-FA-[Arg-Arg-
at the primary level, the octapeptins and polymyxins display Arg1] PMB (219) analogues showed potent antimicro-
marked differences in their antimicrobial spectrum.83-92 bial activity against P. aeruginosa but were poorly active
The octapeptins are active against polymyxin-resistant against E. coli and S. enterica. Moreover, both comp-
strains.64,83-85,89 For example, the MIC of octapeptin ounds displayed comparable LPS binding affinity to PMB.
EM49β (24) for the polymyxin-resistant strain E. coli The des-FA-[Ala-Ala-Ala1]PMB (217) analogue was
SC9253 was 0.3 μg/mL whereas the MIC of PMB inactive across all three test strains and displayed poor
was >200 μg/mL.64,83-85,89 Moreover, octapeptins are ac- LPS binding. The authors’ purport that the selective anti-
tive against Gram-positive bacteria as well.64,83-85,89,100 microbial activity of these compounds against P. aeruginosa
Sakura et al. examined the contribution of each amino can be attributed to structural differences between the
acid position in the tripeptide by employing alanine scanning lipid A of P. aeruginosa compared to that of E. coli and
substitutions into synthetic fatty acyl-PMB peptides S. enterica. The lipid A of E. coli and S. enteric are similar,
(158-166).73 The Thr2 position appears to be more impor- both display four C14 and two C12 fatty acyl chains.
tant compared to the Dab1,3 positions, as the FA-[Ala2]PMB In comparison, the lipid A of P. aeruginosa possesses
(159) analogue displayed significantly reduced antimicrobial four C12 and two C10 fatty acyl chains. Consequently,
activity and LPS binding compared to the corresponding the OM of P. aeruginosa is less tightly packed because of
positions 1 and 3 Ala substituted analogues. Similarly, a the reduced hydrophobic interactions between the lipid A
decrease in antimicrobial activity was observed with neutral fatty acyl chains. Thus, given the presence of a strong ionic
or hydrophobic amino acid substitutions (Ala or 2-amino- bonding potential between the polymyxin analogue and
butyric acic (Abu)) (174, 178, 179, 185) in the tripeptide LPS, the hydrophobic interaction from the side chains of
6
D-Phe -L-Leu7 is sufficient to cause disordering of the OM
that leads to cell death.
Tsuebery et al.76 described the synthesis of PMB and
colistin tripeptide analogues where a short chemotactic
peptide consisting of [Met-Leu-Phe] was linked to Thr2
(236-239). Although the analogues lacked any direct anti-
bacterial activity, they retained the ability to sensitize the
OM to hydrophobic antibiotics and displayed binding to
LPS. Upon binding to the bacterial OM, the analogues acted
as potent opsonins to promote bacterial destruction by
phagocytic cells.76 This novel design strategy was primarily
aimed at exploiting the PMB structure as a delivery vector to
target chemotactic peptides to the bacterial cell surface.
In summary, the available SAR data relating to the
tripeptide segment demonstrate that it represents an integral
functional feature of the polymyxin structure. Two main
principles can be drawn from the SAR data: (a) the tripeptide
segment can only be truncated by one amino acid position
from the N-terminus with a negligible loss of antimicrobial
activity; (b) only conservative amino acid substitutions
appear to be tolerated.
5.6. Linear Polymyxin Analogues. To date a number of
attempts have been made to generate linear polymyxins (156,
240-243). Again, these efforts have been underpinned by the Figure 8. Polymyxin B pharmacophore model. Red location
need to improve the toxicity profile while maintaining potent spheres indicate positive charge property. Hydrophobic property
is represented by cyan location spheres. Hydrogen bond donor
antimicrobial activity.75,77,111 Across most reports it was
vectors are shown in purple. The polymyxin backbone is shown in
found the loss of the cyclic structure completely abrogates ball and stick representation.
antimicrobial and OM permeabilizing activity; this indicates
that the high degree of organization imposed by the rigid
two-dimensional polymyxin SAR data in the literature com-
cyclic heptapeptide (Figures 4 and 5A) is required to main-
bined with the three-dimensional model of the PMB-LPS
tain the active conformation.
complex. A pharmacophore describes the essential, steric,
5.7. Conjugated Polymyxin Analogues. Griffin et al.135
electronic, and function-determining points necessary for an
described a large number of multivalent compounds consist-
optimal interaction with the pharmacological target.140 Phar-
ing of 2-10 PMB molecules covalently attached to one or
macophore generation was performed with the Catalyst Hy-
more linker structures (244). The compounds were intended
poGen algorithm as implemented in Accelyrs Discovery
as neutralizing agents against the septic effects of LPS and as
Studio, version 2.1.140
prophylactics for Gram-negative bacterial infections.135
The polymyxin pharmacophore indicates that the positive
Detailed microbiological or toxicological data for these
charge of the Dab1,3,5,8,9 side chains represent key features
compounds were not documented; however, it is likely that
(Figure 8, red location spheres). Hydrophobic properties are
they would not improve upon the nephrotoxicity of PMB.
key features in the regions of the NR fatty acyl chain and
5.8. Polymyxin Mimics. Many groups have abandoned
positions 6 and 7 in the cyclic heptapeptide ring (Figure 8,
making direct modifications to the polymyxin scaffold while
cyan location spheres). Amino acid positions 2-5 and 8-10
attempting to mimic the physicochemical properties of poly-
display a hydrogen bond donor capacity (Figure 8, purple
myxins. Frecer et al.136 reported a series of cyclic amphi-
vector arrows). This also explains the ability of the hydroxyl
pathic peptides (245) consisting of alternating cationic
side chain of Thr to functionally substitute at Dab side chain
(Lys) and nonpolar (Val or Phe) residues, loosely based on
positions.74
the amphipathic properties of the PMB structure. The
The pharmacophore model clearly demonstrates that the
compounds displayed potent antimicrobial activity against
polymyxin molecule can be divided into a set of polar and
E. coli, S. enterica serovar Typhimurium, P. aeruginosa,
hydrophobic domains, namely, the polar Dab and Thr residue
K. pneumoniae, and Shigella sonnei, and a high affinity
segments (Figure 4), and the hydrophobic NR fatty acyl chain
for LPS.
and D-Phe6-L-Leu7 motif. The model further emphasizes the
Other notable efforts that produced highly active com-
integral scaffolding function of the linear tripeptide and the
pounds include the spermine derivatives (246) reported by
cyclic heptapeptide for maintaining the optimal distance
David and colleagues137 and the synthetic amine and guani-
between each domain, giving the structure its amphilicity, a
dine functionalized cholic acid-derived mimics (247) re-
property that is essential for antimicrobial activity.29,30
ported by Savage and colleagues.138,139
7. Toxicological Properties of the Polymyxins
6. Polymyxin Pharmacophore The currently available polymyxins (polymyxin E (colistin)
As reviewed in preceding sections, the current understand- and polymyxin B) have the potential to cause toxicity, the
ing of polymyxin SAR is that both electrostatic and hydro- most concerning being nephrotoxicity and neurotoxicity
phobic interactions with the lipid A component of LPS which serve to be dose limiting.9,19 Toxicity was not evaluated
are essential for antimicrobial activity. Here, we have con- in the majority of previous studies of new polymyxin deriva-
ducted pharmacophore modeling based upon the collective tives. NAB7061 (174) and NAB739 (184) contain three
primary amine groups, and their affinities to isolated rat display significantly greater antimicrobial activity against
kidney brush-border membrane were substantially lower multidrug-resistant Gram-negative bacteria.
(i.e., ∼15-20%) than that of polymyxin B.74 This suggests
that they might be less nephrotoxic and better tolerated in vivo Acknowledgment. T.V. is the recipient of a Peter Doherty
than polymyxin B. The urinary recovery of both NAB Fellowship (Grant 384300) from the National Health and
compounds was substantially higher than those of colistin.141 Medical Research Council of Australia. R.L.N and J.L. are
For des-FA-[Trp1]-PMB (221) and des-FA-[Dap1]-PMB supported by research grants from the National Institute of
(222), their acute toxicity, measured by administration into Allergy and Infectious Diseases of the National Institutes of
a mouse tail vein, was much lower than polymyxin B (LD50 Health (Grants R01A1070896 and R01AI079330) and Aus-
values of 19.0 and 23.5 μM/kg, respectively, versus 4.8 μM/ tralian National Health and Medical Research Council
kg).98 Tsubery et al.101 examined the acute toxicity of a series (Project Grant 546073). The content is solely the responsibility
of PMBN derivatives by injecting them (15 mg/kg) into the tail of the authors and does not necessarily represent the official
veins of mice. Fmoc-PMBN (94) was not toxic at this dose, views of the National Institute of Allergy and Infectious
while PMB and Fmoc-PMB decapeptide (96) showed very Diseases or the National Institutes of Health. J.L. is a
similar toxicity. It was concluded that hydrophobic aromatic National Health and Medical Council of Australia and Medi-
substitutions of PMBN may significantly reduce their toxi- cal Research Council R. Douglas Wright Research Fellow.
city.101 In general, there is very little information on the
relationship between structure and toxicity (including Biographies
nephrotoxicity). Clearly, because of the potential for toxicity Tony Velkov is an Australian National Health and Medical
to be dose limiting, there is need to consider for polymyxin Research Council (NHMRC) Peter Doherty Fellow and holds
derivatives the balance between antibacterial activity and an adjunct position with the Monash Institute of Pharmaceu-
toxicity, although as noted above this has not occurred for tical Sciences, Monash University, and the Deakin University
many derivatives reviewed herein. Medical School. His research is focused in the areas of drug
design and protein biochemistry. His areas of expertise include
protein-ligand interactions, which incorporate structural biol-
8. Conclusions
ogy, medicinal chemistry, and computational chemistry.
Naturally occurring polymyxins are cationic antimicrobial Philip E. Thompson is a Senior Lecture in Medicinal Chem-
peptides with a narrow spectrum of activity mainly against istry at Monash University, and his research interests include the
Gram-negative bacteria. Multiple chemical and structural development of polymyxin analogues, peptide reagents for
properties of the polymyxin molecule are responsible for medical imaging, analogues of the neuropeptide, kisspeptin,
and inhibitors of insulin-regulated aminopeptidase. His other
LPS binding and antibacterial activity, including (a) the area of active interest is the study of cell signaling enzymes and
amphipathic and cationic distribution of charges across the inhibitors. He has 53 publications in international journals of
primary sequence, (b) the size of the lactam ring, and (c) the diverse disciplines and 5 years of experience in the biotechnology
NR fatty acyl chain. Interest in polymyxins has increased industry setting.
dramatically in recent times because of the emergence of Roger L. Nation is the Director of the Facility for Anti-
bacterial strains multiresistant to other clinically available Infective Drug Development and Innovation (FADDI) in the
antibiotics. In an attempt to ameliorate potential toxicity Monash Institute of Pharmaceutical Sciences, Monash Univer-
and improve the antimicrobial activity, a large number of sity. His research focuses on discovery, development, and
polymyxin analogues have been generated across several optimization of antimicrobial chemotherapy, particularly in-
volving polymyxins, for management of infections caused by
medicinal chemistry programs. Medicinal chemistry strategies
multidrug-resistant pathogens, in particular the Gram-negative
for improving the activity profile of polymyxins included bacterial pathogens Acinetobacter baumannii, Pseudomonas aer-
targeted modifications of the Dab side chains, modifications uginosa, and Klebsiella pneumoniae. His research has been well
to the cyclic peptide ring, and NR fatty acyl chain derivatives. supported by funding from a number of granting bodies,
SARs that can be drawn from the antimicrobial activity of including the Australian NHMRC and the National Institutes
analogues synthesized to date suggest that many of the of Health. He has more than 190 publications, and since 1999,
aforementioned modifications generate inactive compounds. his papers have been cited 1521 times.
The most common modifications reported in the literature Jian Li is an Australian NHMRC R. Douglas Wright
involve alterations that negate the positive charges of the Dab Research Fellow and a Senior Research Scientist at FADDI,
Monash Institute of Pharmaceutical Sciences, Monash
side chains, often resulting in a complete loss of antimicrobial
University. He has an internationally recognized track re-
activity. Expansion or reduction of the cyclic ring also results cord in the microbiology, pharmacokinetics, and pharmacody-
in a substantial loss of antibacterial activity. Modifications to namics of polymyxins. He first elucidated the modern pharma-
the NR fatty acyl chain indicate that the optimal chain length is cokinetics of colistin methanesulfonate and the formed colistin
between seven and nine carbons. Collectively, the SAR data in rats and humans. His main areas of research interest
clearly indicate that the unique three-dimensional architecture are polymyxin pharmacology; antimicrobial chemotherapy
of polymyxins is required for both LPS binding and antimi- against multidrug-resistant Gram-negative bacteria, particularly
crobial activity. In addition to the medicinal chemistry ap- Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella
proaches reviewed above, the recent cloning of the PMB pneumoniae; novel combinations of antimicrobials with antibac-
terial, antipathogenic, and antibiofilm effects; pharmacokinetics
biosynthetic gene cluster has opened up the prospect of
and pharmacodynamics of other antimicrobials; and discovery
generating polymyxin analogues by a recombinant approach and lead optimization of novel antimicrobials, including novel
to re-engineer the biosynthetic enzymes by module swap- polymyxin derivatives. He has 57 publications, and his papers
ping.142 have been cited more than 735 times since 2001.
In future it is hoped that the collective knowledge from the
SAR inferred from reported polymyxin analogues will be Supporting Information Available: Chemical structures of
utilized for the design of synthetic lead compounds that polymyxin analogues, some MICs, and some LPS binding
affinity data. This material is available free of charge via the (24) Hancock, R. E.; Lehrer, R. Cationic peptides: a new source of
Internet at http://pubs.acs.org. antibiotics. Trends Biotechnol. 1998, 16, 82–88.
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Structure - Activity Relationships of Polymyxin Antibiotics

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1898 J. Med. Chem.

2010, 53, 1898–1916

Structure-Activity Relationships of Polymyxin Antibiotics

Tony Velkov,,†,‡ Philip E. Thompson,‡ Roger L. Nation,§ and Jian Li,§

Received July 7, 2009

1. Polymyxins, a Last-Line Therapy against Gram-Negative

The world is facing an enormous and growing threat from the

antibiotics.1 Multidrug resistance is a significant public health

pubs.acs.org/jmc Published on Web 10/29/2009 r 2009 American Chemical Society

although partly speculative, is consistent with available data

tion of PMB) giving a less dramatic separation of the polar

a mode of binding that would disrupt the supramolecular

4. Complete Synthesis of Polymyxins

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