Hybrid Antibiotic Overcomes Resistance in P. aeruginosa by Enhancing Outer Membrane Penetration and Reducing Efflux

Article abstract of DOI:10.1021/acs.jmedchem.6b00867

Therapeutic interventions to treat multidrug-resistant (MDR) Pseudomonas aeruginosa infections are severely limited and often require the use of colistin as drug of last resort. The major challenges impeding the development of novel antipseudomonal agents

Full text of DOI:10.1021/acs.jmedchem.6b00867

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Article
Hybrid antibiotic overcomes resistance in P. aeruginosa by
enhancing outer membrane penetration and reducing efflux
Bala Kishan Gorityala, Goutam Guchhait, Sudeep Goswami, Dinesh M.
Fernando, Ayush Kumar, George G Zhanel, and Frank M. Schweizer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00867 • Publication Date (Web): 13 Aug 2016
Downloaded from http://pubs.acs.org on August 14, 2016
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Hybrid antibiotic overcomes resistance in P.
aeruginosa by enhancing outer membrane
penetration and reducing efflux
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Bala Kishan Gorityala,† Goutam Guchhait,† Sudeep Goswami,† Dinesh M. Fernando,# Ayush
Kumar,#,§ George G. Zhanel,§ and Frank Schweizer*†
†Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
#Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2,
Canada.§Department of Medical Microbiology, University of Manitoba, Winnipeg, MB R3T
1R9, Canada.
Keywords: antibiotics, drug design, Pseudomonas aeruginosa, antimicrobial resistance
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Abstract
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Therapeutic interventions to treat multidrugꢀresistant (MDR) Pseudomonas aeruginosa
infections are severely limited and often require the use of colistin as drug of last resort. The
major challenges impeding the development of novel antipseudomonal agents are the lack of cell
penetration and extensive efflux. We have discovered a tobramycinꢀmoxifloxacin hybrid core
structure which enhances outer membrane permeability and reduces efflux by dissipating the
proton motive force of the cytoplasmic membrane in P. aeruginosa. The optimized hybrid
protects Galleria mellonella larvae from the lethal effects of MDR P. aeruginosa. Attempts to
select for resistance over a period of 25 days resulted in a 2ꢀfold increase in the minimal
inhibitory concentration (MIC) for the hybrid while moxifloxacin or tobramycin resulted in a
16xꢀ and 512xꢀfold increase in MIC. Although the hybrid possesses potent activity against MDR
P. aeruginosa isolates the activity can be synergized when used in combination with other
classes of antibiotics.
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Introduction
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The world is facing an enormous and growing threat from the emergence and dissemination of
Gramꢀpositive and Gramꢀnegative bacteria that are resistant to almost all currently available
antimicrobials.1 But the problem is arguably most serious for Gramꢀnegative bacilli, which are
frequently multidrugꢀresistantꢀMDR (resistant to ≥ 3 different antimicrobial classes) and for
which no novel drug (or drug class) has been developed in over 30 years.2 Among Gramꢀ
negative pathogens, Pseudomonas aeruginosa is the leading cause of nosocomial infections and
chronic lung infections in cystic fibrosis patients with mortality rates ranging from 30ꢀ50%.3 In
the United States, P. aeruginosa is the most common Gramꢀnegative pathogen causing
nosocomial pneumonia and it is frequently implicated in hospitalꢀacquired urinary tract and
bloodstream infections.4,5 The Infectious Disease Society of America includes P. aeruginosa in
its list of “ESKAPE” pathogens that pose the greatest public health threat due to a combination
of increasing prevalence and ineffectiveness of existing antibacterial agents.6 Infections caused
by P. aeruginosa are particularly difficult to treat since the organism is both intrinsically
resistant and capable of acquiring resistance to most antibiotics. This is in large part the result of
the low permeability of its outer membrane which is 12ꢀ100 times less permeable than that of E.
coli owing to its selective porins.7 The intrinsic resistance mechanisms are part of the genetic
makeꢀup of P. aeruginosa, leading to very high baseline minimal inhibitory concentrations
(MIC) and rendering many common antibiotics ineffective against P. aeruginosa. The reduced
penetration of antibiotics across the outer membrane in P. aeruginosa enables secondary
adaptive resistance mechanisms to function more efficiently.8 These mechanisms include efflux
as a result of intrinsic or induced expression of efflux pumps, and enzymatic antibiotic
modifications (e.g. βꢀlactamases). Besides intrinsic and adaptive resistance, P. aeruginosa, can
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further reduce susceptibility to antibacterials by the acquisition of inheritable traits such as
horizontal transfer of genetic element and mutational resistance generally referred to as acquired
resistance. As a result of intrinsic, adaptive and acquired resistance the antibiotic arsenal to treat
P. aeruginosa infections are limited to select penicillins (e.g., piperacillin/tazobactam),
cephalosporins (e.g. ceftazidime), carbapenems (e.g. imipenem), fluoroquinolones (e.g.
ciprofloxacin), aminoglycosides (e.g. tobramycin) and colistin, but resistance to these agents is
steadily increasing and there are few novel antipseudomonal agents in clinical or preclinical
development.6,9
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Two strategies, multicomponent antibiotic adjuvants10ꢀ12 and single componentꢀbased
antibacterial polypharmacology11 are currently investigated to combat bacterial resistance. Both
strategies have in common to exploit multiple modes of action. There is strong evidence that
antibacterials need to interact with multiple targets and induce pleiotropic effects on the bacterial
cell in order to be successful antibiotics as observed for βꢀlactams, fluoroquinolones and
aminoglycosides.12,13 Multitargeting antibiotics are anticipated to limit the frequency of
spontaneous resistance that can arise from mutation in the target gene.13 For instance, it is quite
likely that the rate of a spontaneous chromosomal mutation conferring reduced susceptibility or
resistance to an antibiotic would be ~ 1×10ꢀ6– 1×10ꢀ9 per target, depending on the concentration
of the agent used. If the antibiotic is active against two independent targets in the organism, the
chances of two independent spontaneous mutations occurring would be 1×10ꢀ6 (ꢀ9) × 1×10ꢀ6(ꢀ9)
,
meaning the chances of resistance being conferred versus both targets through spontaneous
mutation would be dramatically less likely occur. The expectation would be that one of the
targets would still be active. Our hypothesis would be that even if an antibiotic acts on more than
one target (eg. 2 different unrelated targets), that an adaptive resistance mechanism (e.g., efflux)
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may confer reduced susceptibility or resistance to the multitarget antibiotic assuming that the
efflux pump recognizes this new molecular entity and effluxes it out of the cell. Several
approaches to develop multitargeting antibacterials have been utilized. These include the
development of agents which target the bacterial cell membrane, and the incorporation of
additional binding sites into existing classes of antibiotics. Molecules which target bacterial
membrane function intrinsically possess multimodal antibacterial properties.12 This can be
achieved through the interaction of a lipophilic moiety with the bacterial membrane causing
disruption of membrane architecture and functional integrity, through steric or functional
inhibition of membraneꢀembedded proteins and/or through alteration of the proton motive force
which drives flagellar motility, protein excretion, export of toxic metabolites and efflux.12 In
spite of this, traditional discovery efforts have not focused on the membrane as a target for
antibiotic development possibly owing to concern over the potential for these agents to disrupt
the mammalian plasma membrane and the lack of knowledge regarding chemical optimization of
such molecules to attain pathogen selectivity. However, the successful use of membraneꢀactive
antibiotics such as daptomycin, televancin, oritavancin and dalbavancin to treat Gramꢀpositive
infections indicate that bacterial specificity is achievable.14 Another approach to design
multitargeting antibacterials involves the covalent attachment of two different pharmacophores
that inhibit dissimilar targets in the bacterial cell generating hybrid antibiotics.15 Over the past 40
years more than 25 hybrid antibiotics containing fluoroquinoloneꢀ, aminoquinoloneꢀ,
vancomycinꢀ, rifamycinꢀ, oxazolidinone, βꢀlactam and aminoglycosideꢀmoieties have been
described. To date four of them a βꢀlactamꢀfluoroquinolone hybrid ROꢀ23ꢀ9423, a
fluoroquinoloneꢀoxazolidinone hybrid MCBꢀ3827, a rifamycinꢀfluoroquinolone hybrid CBRꢀ
2092 and a vancomycinꢀcephalosporin hybrid TDꢀ1792 entered clinical phase studies with TDꢀ
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1792 being the only one advancing to phase III.15 Despite their composition of at least one
pharmacophore entailing excellent inherent Gramꢀnegative activity, all clinicallyꢀevaluated
hybrid antibiotics are devoid of potent Gramꢀnegative activity. The generally observed poor
Gramꢀnegative activity of hybrid antibiotics against Gramꢀnegative bacteria and P. aeruginosa in
particular has been attributed to reduced penetration of the outer membrane due to increased
molecular weight.13,15,16 There is mounting evidence that the Achilles’ heel of synthetic
compounds as new antibiotic drugs is their inability to penetrate readily the cell envelope of
Gramꢀnegative bacteria, which comprises inner and outer membrane, porins and complex
carbohydrate polymers, coupled with their susceptibility to active efflux by membraneꢀ
associated pumps.17 Recently, we have shown that tobramycinꢀciprofloxacin hybrids devoid of
meaningful antibacterial activity nullify resistance (restore susceptibility) to fluoroquinolone
antibiotics in multidrugꢀresistant (MDR) P. aeruginosa isolates.18 Mode of action studies
indicated that the function of the hybrid is linked to outer membraneꢀdestabilizing effects in P.
aeruginosa while the poor antipseudomonal properties of the hybrid was caused in part by
efflux. To further optimize the antipseudomonal properties of the tobramycinꢀciprofloxacin
hybrids we now report on a new class of tobramycinꢀmoxifloxacin hybrids with potent
antipseudomonal activity against MDR, extremely drugꢀresistant (XDR) [resistant to
cephalosporins (cefepime), carbapenems (meropenem and imipenem), fluoroquinolones
(ciprofloxacin and moxifloxacin) and aminoglycosides (tobramycin and gentamicin) but
susceptible to colistin], and pandrugꢀresistant (PDR) [XDR and resistant to colistin] P.
aeruginosa isolates and low likelihood of resistance development. Moreover, we demonstrate
that the potent antipseudomonal properties of the hybrid can be synergistically potentiated with
other classes of antibiotics including fluoroquinolones thereby lowering the absolute minimal
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inhibitory concentration (MIC) of the hybrid to < 1 ꢁg/mL against a panel of MDR, XDR and
PDR isolates from Canadian hospitals while at the same time reaching susceptible CLSI
breakpoints for fluoroquinolone antibiotics.
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Figure 1. Overview of tobramycinꢀmoxifloxacin hybrids used in this study. Hybrids differ in the
linkage to tobramycin. In hybrid 1moxifloxacin is linked via a C12ꢀtether to the Cꢀ5 position of
tobramycin while hybrids 2 and 3 are linked to the Cꢀ2′′ and Cꢀ6′′ꢀpositions in tobramycin.
Results
Design and Synthesis. Design of the lead tobramycinꢀmoxifloxacin hybrids (Figure 1) was
established from the previous structure activity relationships.19 It has been reported that
attachment of lipophilic groups at Cꢀ5 position of tobramycin retain antibacterial activities in
Gramꢀnegative bacteria.19,20 The 4th generation fluoroquinolone moxifloxacin was selected as
second pharmacophore as it possesses (a) potent antipseudomonal activity (MIC = 1 ꢁg/mL)
against wildꢀtype P. aeruginosa PAO1; (b) is less prone to efflux than 2nd and 3rd generation
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fluoroquinolone antibiotics and (c) interacts as a hydrophobic fluoroquinolone more strongly
with the cytoplasmic membrane than ciprofloxacin. The secondary amino function in
moxifloxacin was selected for point of modification as alkylation of this function retains potent
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Scheme 1. Synthesis of Hybrid 1
Ring II
NH2
NHBoc
O
6'
5'
O
TBDMSO
HO
1'
4'
3'
(i) Boc2O, Et3N,
MeOH, H2O
NH2
NHBoc
O
4
BocHN
H2N
O
NH2
O
O
1
NHBoc
O
HO
HO
O
OTBDMS
OTBDMS
6''
OH
OH
2''
(ii) TBDMS-Cl,
HO
TBDMSO
N-Methyl Imidazole,
DMF (85 % Yield, 2 steps)
NH2
NHBoc
4
5
NHBoc
O
TBDMSO
Br
Br
NHBoc
O
BocHN
CH)
(
2
12
O
NHBoc
O
Moxifloxacin,
O
Br
12
OTBDMS
OTBDMS
TBDMSO
K2CO3, DMF, (62 % Yield)
Toluene, KOH, TBAB,
(85 % Yield)
NHBoc
6
NH2
5 HCl
NHBoc
O
O
NH2
H2N
HO
MeOH:HCl (6:4),
(64 % Yield)
TBDMSO
NHBoc
O
O
NH2
O
BocHN
O
O
NHBoc
O
N
O
O
12
N
OH
HO
OH
OTBDMS
OTBDMS
O
N
TBDMSO
NH2
O
N
NHBoc
N
F
N
F
HO
O
HO
O
O
O
1
7
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antibacterial activity against bacterial pathogens.21 A Cꢀ12 tether length was selected based on
previous results obtained with the tobramycinꢀciprofloxacin hybrid antibiotics that demonstrated
that this tether length is optimal for outer membrane penetration and synergism with other
classes of antibiotics in P. aeruginosa.18 We also prepared hybrids 2 and 3 bearing a tether at the
Cꢀ2′′ꢀ and Cꢀ6′′ꢀpositions to study how the nature of the tobramycin linkage affects the
antibacterial activity. The synthetic strategy for the synthesis of hybrids 1ꢀ3 is outlined in
schemes I, II and III.
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Chemical synthesis of tobramycinꢀmoxifloxacin hybrid linked to 5 position of tobramycin
Initially, all the amino groups in tobramycin 4 were Bocꢀprotected by employing diꢀtertꢀbutyl
dicarbonate. Further, silylation of NꢀBocꢀtobramycin with excess TBDMSCl afforded partially
protected derivative 5 with free OH at 5 position in Ring I.19 Alkylation of 5 in toluene with 1,
12 dibromododecan in the presence of KOH and phase transfer catalyst (TBAB) afforded
bromoalkylated tobramycin 6. Protected hybrid 7 was synthesized by directly coupling
commercial moxifloxacin with bromoderivative of tobramycin 6 in anhydrous DMF.
Deprotection in methanolic HCl afforded analytically pure fused tobramycinꢀmoxifloxacin
hybrid 1 (Scheme 1).
Synthesis of tobramycinꢀmoxifloxacin hybrid linked to 2′′ position of tobramycin
Tobramycin upon treatment with triflylazide furnished perꢀazido derivative 8.22
Benzaldehydedimethylacetal assisted selective protection of 4′′ and 6′′ hydroxyl groups in ring
III gave tobramycin analogue 9.23 Regioselective alkylation24 at 2′′ꢀposition was achieved by
treating 9 with 1,12ꢀdibromododecane in the presence of a base to afford monoalkylated
derivative 10. In the following step, tobramycin bromoderivative 10 was conjoined with
moxifloxacin methyl ester to afford protected hybrid derivative 11.25 Deꢀesterification followed
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by deprotection of benzylidene protection group resulted in derivative 12, which was further
subjected to Staudinger reaction26 to yield final tobramycinꢀmoxifloxacin hybrid 2 (Scheme 2).
Scheme 2. Synthesis of Hybrid 2
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N3
NH2
O
6'
3'
O
HO
HO
N3
1'
4'
PhCH(OMe)2
NH2
5
N
2
H2N
3O
HO
N3
O
O
NH2
O
1
TfN3, CuSO4,
HO
O
O
p-TsOH, DMF,
(78 % Yield)
OH
6''
OH
2''
HO
OH
HO
OH
Et3N, (83 % Yield)
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N3
NH2
8
4
N3
N3
O
O
HO
HO
Br
12
(1 euiv)
Br
N3
N3
N3
N3
O
O
N3
O
N3
HO
HO
O
O
NaH, DMF,
(55 % Yield)
O
O
O
Ph
O
HO
O
Ph
O
12
Br
N3
N3
9
10
N3
O
O
N
O
F
HO
OMe
N3
N3
(i)
N3
O
N
HO
O
O
HN
O
O
Ph
O
O
CH3COOH, 80 oC
(75 % Yield)
N
N3
, K2CO3, DMF
O
O
N
N
F
(ii) LiOH, THF, ( 64 % Yield)
HO
O
N3
NH2
O
O
HO
HO
NH2
NH2
N3
N3
H2N
O
N3
O
HO
HO
O
NaOH (0.1 M)
O
OH
OH
O
O
O
OH
OH
N
O
N
PMe3, THF,
(71 % Yield)
NH2
N3
O
N
O
N
N
F
N
F
HO
O
HO
O
O
O
2
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Synthesis of tobramycinꢀmoxifloxacin hybrid linked to 6′′ position of tobramycin
Selective primary hydroxyl group protection of perꢀazido derivative 8 at 6′′ position was
achieved by TIPSCl to yield analogue 13.27 Treating this compound with pꢀmethoxybenzyl
(PMB)24 ether subsequently furnished secondary hydroxyl groups protected tobramycin
derivative 14. Further selective deprotection of silylether28 with the aid of TBAF resulted in
compound 15, which upon treatment with 1,12ꢀdibromododecane/NaOH furnished 6′′ꢀappended
bromododecyl derivative 16. Ceric ammonium nitrate (CAN)29 assisted deprotection of
secondary hydroxyl groups provided derivative 17. Protected hybrid analogue 18 was
synthesized by reaction between moxifloxacin methyl ester and corresponding tobramycin
bromo derivative 17 under basic conditions. Deꢀesterification and subsequent Staudinger
reaction finally resulted in tobramycinꢀmoxifloxacin hybrid 3 (Scheme 3).
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Scheme 3. Synthesis of Hybrid 3
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2
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N3
N3
O
PMBO
O
HO
TIPSCl, Pyr,
N3
N3
PMBCl, NaOH
N3
N3
O
N3
O
O
PMBO
N3
8
HO
O
DMAP(cat)
(74 % Yield)
O
DMF (75 % Yield)
OTIPS
OPMB
OTIPS
PMBO
O
HO
10
11
12
13
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OH
N3
N3
14
N3
N3
PMBO
O
O
PMBO
Br
12
N3
Br
N3
N3
N3
TBAF, THF
(84 % Yield)
O
O
N3
O
N3
PMBO
PMBO
O
O
Br
OH
OPMB
NaH, DMF,
(85 % Yield)
O
O
PMBO
PMBO
12
OPMB
N3
N3
15
16
O
N
O
N3
O
F
OMe
HO
N
CAN,
N3
N3
O
O
CH3CN/H2O(4:1)
HN
N3
O
HO
O
O
Br
(74 % Yield)
HO
12
OH
O
N3
K2CO3, DMF, (55 % Yield)
(i) LiOH, THF
17
O
N3
O
OMe
HO
F
N3
N3
N
O
N3
O
N
HO
O
(ii) PMe3, NaOH(0.1 M),
THF, (63 % Yield)
O
O
N
HO
OH
12
N3
O
O
NH2
O
OH
HO
F
18
NH2
HO
N
H2N
O
NH2
N
HO
O
O
O
N
O
OH
12
NH2
3
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Antibacterial properties, toxicity and in vivo data. The hybrids 1ꢀ3 were tested for
antibacterial activity by determining the minimal inhibitory concentration (MIC) against select
Gramꢀnegative and Gramꢀpositive bacteria (Table 1). Among hybrids 1ꢀ3, hybrid 1 showed
consistently the highest antibacterial activity and displayed good activity (MIC = 1 ꢁg/mL)
against Staphylococcus aureus and methicillinꢀresistant Staphylococcus aureus (MRSA). Of
special consideration is the potent activity of hybrid 1 against three P. aeruginosa strains (MIC =
4ꢀ8 ꢁg/mL) including two gentamicinꢀresistant P. aeruginosa strains. Interestingly, hybrid 1
(MW = 1217.5) displays comparable antipseudomonal activity against these three P. aeruginosa
strains than ciprofloxacin (MW = 367.8) when the changes in molecular weight are factored in.
The promising antipseudomonal properties of hybrid 1 provided the rational to study the
antibacterial activity of hybrid 1 against a select panel of MDR and XDR P. aeruginosa isolates
obtained from multiple Canadian hospitals which are resistant to tetracyclines (doxycycline),
cephalosporins (cefepime), carbapenems (meropenem and imipenem), fluoroquinolones
(ciprofloxacin and moxifloxacin) and aminoglycosides (tobramycin and gentamicin) but remain
susceptible to colistin. We also included two PDR P. aeruginosa strains (#91433 and #101243)
resistant to all known antipseudomonal agents including colistin (Table 2). Our results
demonstrate that hybrid 1 possesses potent activity (MIC = 1ꢀ8 ꢁg/mL) against a panel of 8
MDR, XDR and PDR P. aeruginosa isolates. In comparison, hybrids 2 and 3 in which the linker
is attached to a different position in tobramycin are > 8ꢀtimes less active than hybrid 1 indicating
that the positioning of the tether in tobramycin is crucial for potent antibacterial activity. Similar
MIC ranges (2ꢀ8 ꢁg/mL) of hybrid 1 were also seen against a panel of 12 fluoroquinoloneꢀ
resistant (ciprofloxacinꢀresistant and moxifloxacinꢀresistant) but tobramycinꢀsusceptible MDR P.
aeruginosa isolates from multiple Canadian intensive care units indicating potent activity against
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19
20
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25
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Table 1. Antibacterial activity of hybrids 1ꢀ3, tobramycin (TOB), moxifloxacin (MOX) and
ciprofloxacin (CIP) against Gramꢀpositive and Gramꢀnegative pathogens.
7
8
9
MIC (ꢁg/mL)
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
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38
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42
43
44
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49
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53
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59
60
Test organism
1
1
2
4
3
16
16
8
TOB
≤0.25
≤0.25
≤0.25
1
MOX
≤0.25
≤0.25
≤0.25
64
CIP
≤0.25
≤0.25
≤0.25
128
1
S.aureus ATCC29213
MRSA ATCC 33592
1
8
MSSE CANWARDꢀ2008 81388
MRSE CANꢀICU 61589 (CAZ >32)
E. faecalis ATCC 29212
2
2
16
4
8
8
32
16
32
4
32
16
64
16
64
64
128
64
32
8
≤0.25
2
E. faecium ATCC 27270
16
1
8
8
S. pneumoniae ATCC 49619
E. coli ATCC 25922
2
≤0.25
≤0.25
0.5
1
0.5
4
0.5
8
≤0.25
≤0.25
≤0.25
256
1
E. coli CANꢀICU 61714 (GENꢀR)
E. coli CANꢀICU 63074 (AMK 32)
E. coli1 CANWARDꢀ2011 97615
P. aeruginosa ATCC 27853
64
32
128
16
128
4
8
1
128
4
128
0.5
16
32
4
P. aeruginosa CANꢀICU 62308 (GENꢀ
R)
8
16
2
P. aeruginosa2 CANWARDꢀ2011 96846
S. maltophilia CANꢀICU 62584
A. baumannii CANꢀICU 63169
K.pneumoniae ATCC 13883
4
32
16
1
>128
64
256
16
4
4
32
>128 >128 >512
128
4
>128
16
32
1
2
≤0.25
≤0.25
≤0.25
1(GENꢀR, TOBꢀR, CIPꢀR) aac(3')iia (GENꢀR, TOBꢀR) CIP = Ciprofloxacin TOB =
Tobramycin GEN = Gentamicin AMK = Amikacin TOB, CIP MIC values have been reported in
Ref. 17.
2
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diverse P. aeruginosa phenotypes (Table 3). It is interesting to note that hybrid 1 performed
poorly compared to moxifloxacin and tobramycin in wild type P. aeruginosa PAO1 but at the
same time was much more potent in the clinical isolates when compared to moxifloxacin and
tobramycin. This suggests that the resistance mechanisms which deactivate moxifloxacin and
tobramycin in P. aeruginosa do not apply to hybrid 1.
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14
15
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17
18
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60
Table 2. Antibacterial activity MIC (ꢁg/mL) of hybrids 1ꢀ3 and select classes of antibiotics
against MDR, XDR and PDR P. aeruginosa isolates.
Antibiotic (MW)
Clinical PA DOX
CPM
480.6
MER
383.5
DOR
420.5
CIP
MOX GEN
TOB
467.5
COL
1
2
3
isolatea
444.4
331.3
401.4
128
128
512
64
477.6
>32
>0.5
>32
>32
>32
>32
32
1155.4 1217.5 1217.5 1217.5
100036
>32
>32
>32
32
4
8
4
1
5
0.5
>32
32
8
64
32
256
64
32
32
4
64
<0.5
>64
32
2
ND
ND
>32
>32
32
ND
ND
>32
32
8
8
2
1
4
4
8
8
4
101885
1
259ꢀ96918
260ꢀ97103
262ꢀ101856
264ꢀ104354
91433
>64
16
32
32
16
64
ND
>32
16
32
>32
8
1
1
>32
>32
32
128
64
>64
>64
8
2
1
>32
>32
ND
ND
>32
16
8
32
16
4
ND
ND
>32
101243
8
16
ND
16
ND
2
8
>32
ND
>64
0.5
1024
1
PAO1
ND
<0.2
1
a)PA = Pseudomonas aeruginosa; DOX = Doxycycline (tetracycline); CPM = Cefepime
(cephalosporin); MER = Meropenem (penem); DOR = Doripenem (penem); CIP =
Ciprofloxacin (fluoroquinolone); MOX = moxifloxacin (fluoroquinolone); GEN = Gentamicin
(aminoglycoside); TOB = Tobramycin (aminoglycoside); COL = colistin; ND = not determined.
All control antibiotic MIC values have earlier been reported in Ref. 17.
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tobramycin in the clinical isolates are ineffective on hybrid 1. We also assessed whether the
potent antipseudomonal properties of hybrid 1 can be extended to a panel of MDR E. coli and
MDR A. baumanii strains. These studies indicate that hybrid 1 did not display potent activity
against E. coli (MIC ≥ 64 ꢁg/mL) and A. baumannii (8ꢀ64 ꢁg/mL) clinical isolates but in contrast
retained potent activity against wild type E. coli strains (MIC < 2 ꢁg/mL Table 1 and Supporting
Table S1). This likely reflects differences in pathogenꢀspecific resistance mechanisms. It is likely
that hybrid 1 primarily affects intrinsic resistance in P. aeruginosa as it relates to outer
membrane permeability barrier and efflux which is less pronounced in E. coli and A. baumannii.
After demonstrating that hybrid 1 possesses potent and selective antipseudomonal activity
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11
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Table 3. Antibacterial activity of hybrids 1 against tobramycin susceptible P. aeruginosa
clinical isolates.
MIC (ꢁg/mL)
P. aeruginosa
1
CPM CEFT MER MOX IMI CIP GEN TOB COL
Clinical isolate
86052
2
16
64
64
32
16
16
16
>32
>32
>32
>32
>32
>32
32
8
>32
>32
32
16
8
>16
16
32 >16
16 >16
4
4
1
1
1
1
86053
4
4
4
4
4
2
86067
16
>32
4
>32
8
>64
2
0.5
1
86079
>16
>16
16
16 >16
86141
16
8
8
4
2
8
1
1
92014
4
1
1
108590
8
16
32
16
4
1
79199
80621
8
8
32
8
32
16
16
>16
8
4
8
2
4
2
2
1
2
>32
32
16
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83182
84745
85322
8
8
8
16
16
16
>32
>32
>32
16
16
16
16
16
8
16
16
32
4
2
2
4
8
4
1
1
1
1
2
1
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1: Hybrid 1; CPM: Cefepime; CEFT: Ceftazidime; MER: Meropenem; MOX:Moxifloxacin;
IMI: Imipenem; CIP: Ciprofloxacin; GEN: Gentamicin; TOB: Tobramycin; COL: Colistin.
against diverse MDR and XDR P. aeruginosa pathogens, we then studied the killing kinetics of
P. aeruginosa PAO1 and observed complete eradication of P. aeruginosa PAO1 at 2x MIC
concentration over a 24 hour time period (Figure. 2A). Efficacy of hybrid 1 was demonstrated in
Figure 2. Effects of hybrid 1 on bacterial killing and in vivo efficacy. (A) Timeꢀkill curves
showing the effect of varying concentrations of hybrid 1 and MIC concentration of colistin (Col)
on the viability of P. aeruginosa PAO1 cells grown in MH broth. No colonyꢀforming units
(CFU) were found with 1 at 2 × MIC (blue) after 24 hours. Experiment was performed three
times independently and each data point is an average of three determinations ± SEM. (B)
Enhanced dose dependent efficacy of hybrid 1 in comparison with tobramycin (TOB) and
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moxifloxacin (MOX) in XDR P. aeruginosa (#104354) over a period of 24 h was demonstrated
in Galleria mellonella in vivo infection model. Larvae infected with #104354 (106 CFU/mL)
resulted in 73% killing after 24h. In contrast, single dosage monotherapy (50 mg kg) of
moxifloxacin, or tobramycin or hybrid 1 resulted in 27%, 20% or 100% survival of the larvae,
respectively, after 24 h. No killing was observed due to physical trauma when larvae were
injected with equal volumes of PBS. Two independent experiments were conducted for each
dosage of antibiotic/hybrid, where each expermient involved 15 worms (n = 30). Significant
differences between 0 and 24 h are indicated by * (p value ≤ 0.05).
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Galleria mellonella infection model. Galleria mellonella larvae is an established in vivo model to
study the efficacy of antimicrobial therapy.30,31 In pilot studies, we determined that hybrid 1
causes < 2.5% hemolysis of human erythrocytes at 1000 ꢁg/mL (Supporting Figure. S1) and
shows low cytotoxicity CC50>> 30 ꢁM against cancer cell lines (Supporting Figure. S2). We
assessed the tolerability of hybrid 1 in Galleria mellonella and did not see any toxic effects up to
the maximal dose of 600 mg/kg over a period of 96 hours (Supporting Figure. S3). Efficacy
studies were performed by infecting the larvae with a dose (106 CFUs) of XDR P. aeruginosa
strain #104354 (resistant to all classes of antipseudomonal agents except colistin) followed by
injection of hybrid 1 or tobramycin or moxifloxacin at 2 h post infection. Therapy with a single
dose of moxifloxacin (50 mg/kg) or tobramycin (50 mg/kg) or no drug resulted in 27% or 20%
or 27% survival of the larvae at 24 h, respectively indicating that both antibiotics provide
insufficient protection. In contrast, single dose therapy (50 mg/kg) of hybrid 1 resulted in 100%
survival after 24 h (Figure. 2B). We also observed enhanced longꢀterm survival effects upon
treatment with hybrid 1. For instance, treatment of the infected larvae with a single dose (75
mg/kg) of moxifloxacin, or tobramycin or hybrid 1 resulted in 7%, 0% or 20% survival after a 96
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hour period. Survival rate of the larvae upon therapy with hybrid 1 was doseꢀdependent
5
6
(Supporting Figure. S4).
7
8
9
Mode of action studies. To understand the protective function of hybrid 1, we studied the
membrane interactions of 1 with P. aeruginosa PAO1. Initially, we demonstrated that hybrid 1
permeabilizes the outer membrane of P. aeruginosa in a doseꢀdependent manner using the NPN
(1ꢀNꢀphenylnapthylamine) assay (Figure. 3A).32 In addition, we also assessed whether the
combination of hybrid 1 with other classes of antibiotics were additive or synergistic by using
the fractional inhibitory concentration (FIC) index as a measure of the interaction between two
antibacterial agents.33 FIC indices of 1ꢀ2, ≤ 0.5, and ≥ 4 indicate no interaction, synergy and
antagonism, respectively.34 We observed strong synergy of hybrid 1 with outer membrane
impermeable agents interacting with intracellular targets including novobiocin (FIC index 0.12),
rifampicin
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Figure 3. (A) Effects of hybrid 1 on the outer membrane. Concentrationꢀdependent
permeabilization of the outer membrane by tobramycinꢀmoxifloxacin hybrid 1 is indicated by the
accumulation of 1ꢀNꢀphenylnaphthylamine (NPN) in P. aeruginosa PAO1 cells. 50 ꢁg/mL
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(medium plum), 32 ꢁg/mL (cyan), 16 ꢁg/mL (gold), or 8 ꢁg/mL (pale lavender) of hybrid 1 were
used, along with 5 ꢁg/mL colistin (Col) (pale violet red) as positive control. Hybrid 1 was added
at 120 seconds. Experiment was performed in triplicate and each data point is an average of three
determinations ± SEM. (B) Synergistic FIC index of hybrid 1 in combination with various
classes of antibiotics against PAO1.
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(FIC index 0.15), vancomycin (FIC index = 0.26) and erythromycin (FIC index = 0.26)
indicating that hybrid 1 enhances cellular uptake of these agents into P. aeruginosa. Moreover,
we also observed strong synergy with other classes of antibiotics including chloramphenicol
(FIC index 0.15), minocycline (FIC index 0.15), moxifloxacin (FIC index = 0.18), trimethoprim
(FIC index = 0.25), ceftazidime (FIC index = 0.37) and colistin (FIC index = 0.37) but not with
meropenem (FIC index = 2), tobramycin (FIC index > 2) and gentamicin (FIC = 5) against P.
aeruginosa PAO1 (Figure 3B and Supporting Table S2). Importantly, hybrid 1 strongly
Table 4A. Effect of fixed concentration of hybrid 1 (≤ 2 ꢁg/mL; ≤ 0.25 x MIC) on MIC of
ciprofloxacin (CIP) or moxifloxacin (MOX)
MIC (ꢁg/mL)
Clinical PA isolate
CIP
Absolute MIC1
MOX
Absolute MIC2
PA259ꢀ96918
PA260ꢀ97103
256
16
<1
1
512
32
<1
1
PA262ꢀ101856
PA264ꢀ104354
91433#
16
16
8
1
64
32
8
2
1
1
0.25
0.06
0.12
0.06
101243#
2
8
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1 MIC of ciprofloxacin when < 0.25 x MIC (≤ 2 ꢁg/mL) of 1 was used, 2 MIC of moxifloxacin
when < 0.25 x MIC (≤ 2 ꢁg/mL) of 1 was used, PDR strain resistant to all antiꢀpseudomonal
classes including colistin.
3
4
5
6
#
7
8
9
10
11
12
13
14
15
16
17
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synergizes with the fluoroquinolones ciprofloxacin and moxifloxacin against a panel of MDR
and XDR P. aeruginosa isolates. For instance, ciprofloxacinꢀsusceptible (MIC = 1 ꢁg/mL) CLSI
break points were reached for 6/6 ciprofloxacinꢀresistant, MDR, XDR or PDR P.
aeruginosaisolates at 0.25 x MIC of hybrid 1 (≤ 2 ꢁg/mL). In comparison, the same susceptible
breakpoints were reached for moxifloxacin in 5/6 moxifloxacinꢀresistant, MDR, XDR or PDR
isolates indicating that hybrid 1 strongly synergizes with both clinically used fluoroquinolone
antibiotics (Table 4A). At the same time the presence of ciprofloxacin or moxifloxacin at or
below their respective 0.25 x MIC (≤ 4 ꢁg mL) synergizes hybrid 1 to lower its MIC < 1 ꢁg/mL
for the 6 tested MDR, XDR and PDR P. aeruginosa isolates (Table 4B).
Table 4B. Effect of fixed concentration of ciprofloxacin (≤ 4 ꢁg/mL; ≤ 0.25 x MIC) or
moxifloxacin (≤ 4 ꢁg/mL; 0.25 x MIC) on MIC of hybrid 1
MIC (ꢁg/mL)
Clinical PA isolate
Absolute MIC1
Absolute MIC2
1
PA259ꢀ96918
PA260ꢀ97103
2
1
< 0.5
<1
< 0.25
0.25
PA262ꢀ101856
PA264ꢀ104354
91433#
4
4
8
8
< 0.5
< 0.5
< 1
<1
< 1
< 1
< 1
101243#
< 1
1: Hybrid 1; 1 MIC of 1 when ≤ 0.25 x MIC (≤ 4 ꢁg/mL) of ciprofloxacin was used, 2 MIC of 1
when ≤ 0.25 x MIC (≤ 4 ꢁg/mL) of moxifloxcin was used, # PDR strain resistant to all antiꢀ
pseudomonal classes including colistin.
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Next, we assessed the ability of hybrid 1, moxifloxacin and tobramycin to depolarize the
cytoplasmic membrane in P. aeruginosa PAO1 using the membrane potentialꢀsensitive dye bisꢀ
(1,3ꢀdibutybarbituric acid) trimethine oxonol.35 Our results demonstrate that the tobramycinꢀ
moxifloxacin hybrid 1 induces doseꢀdependent depolarization of the cytoplasmic membrane in a
comparable manner to colistin (Figure. 4A and 4B). In contrast, moxifloxacin was not able to
induce membrane depolarization while tobramycin showed weak doseꢀdependent cytoplasmic
membrane depolarization (Figure 4C and 4D).
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Figure 4. Cytoplasmic membrane depolarization assay ascertained by DiSC3 fluorescence in P.
aeruginosa PAO1. Comparative membrane depolarization induced by varying concentrations of
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(A) hybrid 1 (MIC = 4 ꢁg/mL), (B) colistin (MIC = 1 ꢁg/mL), (C) moxifloxacin (MIC = 1
ꢁg/mL) and (D) tobramycin (MIC = 0.5 ꢁg/mL). Each data point represents ± SEM from three
independent samples.
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These results indicate that the cytoplasmic membrane destabilizing effects of tobramycin can be
enhanced by linking it to moxifloxacin. Moreover, flagellumꢀdependent swimming motility of P.
aeruginosa PAO1 was strongly reduced in a concentrationꢀdependent manner at sub MIC
concentration of hybrid 1 (Figure. 5, and Supporting Figure. S5). As flagellar function requires
an intact proton motive force (PMF),36 our results suggest that hybrid 1 dissipates the PMF.
Moreover, as the PMF drives the function of RNDꢀbased efflux pumps in P. aeruginosa,8 the
observed synergistic effects of sub lethal hybrid 1 with efflux susceptible antibiotics such as
fluoroquinolones likely reflect reduced efflux. The effects of hybrid 1 on the PMF are consistent
with the observed antagonistic effect of hybrid 1 with the aminoglycoside gentamicin and
additive effects with tobramycin in P. aeruginosa PAO1 as aminoglycosides require the
electrical component (∆Ψ) of the intact PMF for cellular uptake (Supporting Table S2).37
Figure 5. Motility of P. aeruginosa PAO1.Motile phenotype of cells in swim plates when
inoculated to an optical density (600 nm) of 1.0. (A) Swim plates with no drug. (B) Plate
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containing 1/16 × MIC hybrid 1 (0.25 ꢁg/mL). Swim plates were prepared using 0.3% agar,
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dried and solidified for 60 min under laminar flow. Images were taken after 20 h incubation at 37
°C.
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This suggests that hybrid 1 dissipates the electrical component (∆Ψ) of the proton motive force at
sub lethal concentration. The effect of hybrid 1 on the PMF may suggest that the hybrid alters the
function of RND efflux pumps. We therefore determined the MIC of hybrid 1 and moxifloxacin
in two effluxꢀdeficient strains. We tested a MexABꢀOprM deletion strain (PAO200), as well as
an effluxꢀsensitive strain (PAO750)38,39 that lacks five different clinicallyꢀrelevant RND pumps
(MexABꢀOprM, MexCDꢀOprJ, MexEFꢀ OprN, MexJK, and MexXY) and the outer membrane
protein OpmH for potentiation of antibiotic activity by hybrid 1. As expected the antibacterial
activity of moxifloxacin is greatly affected by the absence of RND pumps leading to a 4ꢀ and
128ꢀfold reduction of the MIC in the effluxꢀdeficient in PAO200
Table 5. Antibacterial activity of moxifloxacin and hybrid 1 in efflux pump deletion strains
derived from P. aeruginosa PAO1
MIC (µg/mL)
P. aeruginosa strains
moxifloxacin
hybrid 1
PAO1
1
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4
PAO2001
PAO7502
0.25
0.0078
0.5
2
1PAO1 strain deficient in MexABꢀOprM, PAO1 strain deficient in (MexABꢀOprM, MexCDꢀ
OprJ, MexEFꢀOprN, MexJK and MexXY) and the outer membrane protein OpmH.
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and PAO750 strains (Table 5). In contrast, the MIC of hybrid 1 was not affected by the absence
of the MexABꢀOprM pump and showed only an 8ꢀfold reduction in the PAO750 strain indicating
that hybrid 1 strongly resists efflux by P. aeruginosa RND pumps.
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Figure 6.(A) Differential interference contrast microscopic (DIC) image of P. aeruginosa PAO1
cells. Fluorescence microscopic image of (B) untreated P. aeruginosa PAO1 cells labeled with
propidium iodide (PI) with no fluorescence indicating intact cell membrane and (C) Cells treated
with 2 × MIC (8 ꢁg/mL) of hybrid 1 for 3h at 37°C that gives bright red fluorescence due to PI
uptake indicating membrane permeabilizing effect of hybrid 1. Scale bar represents 10 ꢁm.
Figure 7 Transmission electron microscopic images of (A) untreated P. aeruginosa PAO1 cells
and (B) cells treated with 2 × MIC (8 ꢁg/mL) of hybrid 1 for 3h at 37 °C. Arrow in panel B
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indicates loss of characteristic cell morphology, membrane blebbing and disintegration of the
cell membrane. Scales of the images are 100 nm.
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To assess the effects of hybrid 1 on both outerꢀ and innerꢀ membrane permeability barrier we
used the membrane impermeable fluorescent dye PI which stains nucleic acids.40,41 Cells grown
in the presence of growthꢀinhibitory amounts of hybrid 1 (8 ꢁg/ml) could be uniformly stained
with intracellular dye while cells in the absence of hybrid 1 did not show fluorescence indicating
that hybrid 1 enables internalization of PI (Figure. 6).
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Figure 8. Protein translation activity, gyrase A activity and emergence of resistance. (A)
Inhibition of in vitro protein translation in E. coli S30 extract by tobramycin, moxifloxacin,
hybrid 1 and 2. Protein translation assays were performed as described in methods section. The
percentage luminescence was calculated compared to no antibiotic control (assumed to be
100%). Each data point represents an average luciferase activity of the triplicates with ±
percentage error. The IC50 values (ꢁM) for tobramycin (0.0062 ± 0.0002), hybrid 1 (4.7 ± 0.21),
and hybrid 2 (0.13 ± 0.003) were obtained. (B) Inhibition of P. aeruginosa DNA gyrase A
activity in the presence of moxifloxacin, hybrid 1, 2 or 3. Each data point represents an average
value of band (supercoiled) intensities from two independent sets of reactions (see Appendix in
the supporting information). The percentage of supercoiled DNA was measured compared to the
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noꢀantibiotic control (assumed to be 100%). IC50 values (ꢁM) for moxifloxacin (2.6 ± 0.02),
hybrid 1 (54.7 ± 2.9), 2 (2.1 ± 0.08) and 3 (2.6 ± 0.14) were calculated. (C) Emergence of
resistance studies in P. aeruginosa after 25 serial passages in the presence of tobramycin,
moxifloxacin and hybrid 1: Col = colistin, TOB = tobramycin, MOX = moxifloxacin.
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We also examined the cellular morphology of P. aeruginosa PAO1 treated with 8 ꢁg/ml (2 x
MIC) hybrid 1 in MuellerꢀHinton (MH) broth for a period of 4h by transmission electron
microscopy.42 The obtained images indicate significant outer membrane damage and blebbing
(Figure. 7) that can cause membrane rupture, cytosolic leakage and cell death (Supporting
Figure. S6).
Finally, we assessed whether the hybrids retain their original modes of action. In a protein
translation assay hybrids 1 and 2 showed a >750ꢀfold and >20ꢀfold reduction in activity
respectively, when compared to tobramycin (Figure. 8A). Similarly, hybrid 1 was a 20ꢀfold less
potent inhibitor of gyrase A when compared to moxifloxacin while hybrids 2 and 3 displayed
equipotency than moxifloxacin (Figure. 8B). These studies indicate that the predominant,
original antibacterial effects of tobramycin and moxifloxacin are greatly reduced in hybrid 1.
Next we studied the potential of hybrid 1 to select for resistance. We used a procedure of
selective pressure in which P. aeruginosa PAO1 was exposed to sub inhibitory (0.5 x MIC)
concentrations of moxifloxacin, tobramycin and hybrid 1 during 24 successive subcultures
(Figure. 8C).43 As can be seen from the data, the relative MIC values of tobramycin,
moxifloxacin and hybrid 1 increased by 512ꢀ, 16ꢀ and 2ꢀfold respectively against PAO1
indicating that hybrid 1 strongly delays resistance development when compared to moxifloxacin
and tobramycin over the 25 day period of this experiment. The delayed resistance of hybrid 1 is
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