Second-Generation Tryptamine Derivatives Potently Sensitize Colistin Resistant Bacteria to Colistin

Article abstract of DOI:10.1021/acsmedchemlett.9b00135

Antibiotic resistance has significantly increased since the beginning of the 21st century. Currently, the polymyxin colistin is typically viewed as the antibiotic of last resort for the treatment of multidrug resistant Gram-negative bacterial infections. However, increased colistin usage has resulted in colistin-resistant bacterial isolates becoming more common. The recent dissemination of plasmid-borne colistin resistance genes (mcr 1-8) into the human pathogen pool is further threatening to render colistin therapy ineffective. New methods to combat antibiotic resistant pathogens are needed. Herein, the utilization of a colistin-adjuvant combination that is effective against colistin-resistant bacteria is described. At 5 μM, the lead adjuvant, which is nontoxic to the bacteria alone, increases colistin efficacy 32-fold against bacteria containing the mcr-1 gene and effects a 1024-fold increase in colistin efficacy against bacteria harboring chromosomally encoded colistin resistance determinants; these combinations lower the colistin minimum inhibitory concentration (MIC) to or below clinical breakpoint levels (≤2 μg/mL).

Full text of DOI:10.1021/acsmedchemlett.9b00135

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Second-Generation Tryptamine Derivatives Potently Sensitize
Colistin Resistant Bacteria to Colistin
Bradley M. Minrovic,† Veronica B. Hubble,† William T. Barker,† Leigh A. Jania,‡ Roberta J. Melander,†
Beverly H. Koller,‡ and Christian Melander*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
‡Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
S
* Supporting Information
ABSTRACT: Antibiotic resistance has significantly increased since the beginning of
the 21st century. Currently, the polymyxin colistin is typically viewed as the antibiotic
of last resort for the treatment of multidrug resistant Gram-negative bacterial
infections. However, increased colistin usage has resulted in colistin-resistant bacterial
isolates becoming more common. The recent dissemination of plasmid-borne colistin
resistance genes (mcr 1−8) into the human pathogen pool is further threatening to
render colistin therapy ineffective. New methods to combat antibiotic resistant
pathogens are needed. Herein, the utilization of a colistin-adjuvant combination that
is effective against colistin-resistant bacteria is described. At 5 μM, the lead adjuvant,
which is nontoxic to the bacteria alone, increases colistin efficacy 32-fold against
bacteria containing the mcr-1 gene and effects a 1024-fold increase in colistin efficacy
against bacteria harboring chromosomally encoded colistin resistance determinants;
these combinations lower the colistin minimum inhibitory concentration (MIC) to or below clinical breakpoint levels (≤2 μg/
mL).
KEYWORDS: Antibiotic resistance, multidrug resistant bacteria, colistin, antibiotic adjuvant
he impact of antibiotics cannot be understated; they have
significantly contributed to the quality of life since the
The escalation of carbapenem resistance in Gram-negative
bacteria has resulted in the increased usage of the “last resort”
antibiotic colistin, a macrocylic cationic polypeptide with a
bactericidal mechanism that involves direct physical disruption
of the Gram-negative outer membrane.7 Colistin is, however,
nephrotoxic with ca. 25% of patients who are administered a
full regimen experiencing some degree of nephrotoxicity (this
does not include patients whose regimen was halted
prematurely due to toxicity).8 For this reason, physicians
have traditionally avoided the use of colistin; however, the rise
in resistance to other more well-tolerated antibiotic classes has
forced colistin usage as of late. Prior to 2016, all known clinical
resistance to colistin was mediated by chromosomally located
genes. This situation changed dramatically with a report in
2016 of a patient presenting with a colistin-resistant Escherichia
coli infection, in which the gene (mcr-1) encoding the colistin
resistance machinery was located on a plasmid,9 raising the
possibility that colistin resistance could become rapidly spread
through lateral gene transfer. Indeed, mcr-1 and its variant
genes (mcr-2−8) have now been reported globally.10 The mcr-
1 gene encodes a phosphoethanolamine transferase that
covalently modifies lipid A with phosphoethanolamine, which
serves to decrease the overall net negative charge of the
T
discovery of penicillin in the 1930s.1 Despite the subsequent
discovery of numerous antibiotic classes spanning multiple
modes of action, bacteria have acquired resistance to every
antibiotic in the clinician’s arsenal. Antibiotic resistance will
likely continue to become more common, as antibiotic usage
rose 65% from 2000 to 2016, and history has shown us that
resistance is more likely to be acquired when antibiotic
consumption is increased.2 Currently, the Centers for Disease
Control and Prevention estimates approximately two million
people present with an antibiotic resistant infection annually in
the U.S., and the death toll from these infections is estimated
to be at least 23,000.3 A majority of these fatal infections are
caused by the ESKAPE pathogens: the Gram-positive bacteria
Enterococcus faecium and Staphylococcus aureus, and the Gram-
negative bacteria Klebsiella pneumoniae, Acinetobacter bauman-
nii, Pseudomonas aeruginosa, and Enterobacter spp.
The arduous nature of developing effective therapies to treat
infections caused by multidrug-resistant (MDR) Gram-
negative bacteria has been well documented4 and is hampered
by the presence of both intrinsic and acquired resistance
mechanisms that can render bacteria resistant to nearly, if not
all, antibiotics on the market.5 Indeed, in 2017, a woman in the
United States succumbed to septic shock that stemmed from
an infection caused by a strain of K. pneumoniae that was
resistant to 26 antibiotics, including all aminoglycosides and
polymyxins tested.6
Received: March 26, 2019
Accepted: April 12, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00135
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
membrane and greatly impacts the ability of colistin to disrupt
the bacterial membrane due to decreased electrostatic
interactions.11
Table 1. Potentiation of Colistin (MICs μg/mL) with Pilot
and Brominated Library Adjuvants Tested at 50 μM
K. pneumoniae
A. baumannii
As the antibiotic pipeline for the treatment of MDR Gram-
negative infections is sparse, the development of novel
approaches to combat these bacteria are warranted. One
such method is the antibiotic-adjuvant approach, in which a
conventional antibiotic is codosed with a nontoxic small
molecule that increases the antibiotic’s efficacy.12,13 In a
previous study from our lab, tryptamine derivative 1a (Figure
1) exhibited a 32-fold and a 2-fold reduction in the colistin
E. coli YD626
parent +mcr-1
2210291
17978
compound
parent
+mcr-1 parent +mcr-1
1
8
4
4
4
2
4
4
4
2
1
16
8
4
16
2
4
4
8
1
2
0.25
1
0.25
1
0.5
1
16
4
4
8
2
4
4
8
1
1a
1b
1c
1d
6a
6b
6c
6d
0.0625
0.25
0.0625
0.5
0.125
0.5
0.125
0.5
0.25
0.5
0.25
0.5
0.25
0.5
0.0625
0.25
0.5
0.5
groups attached to a phenyl ring with either trifluoromethyl
groups or various halogens can augment activity in the context
of antibiofilm activity and colistin potentiation (2-AI 3 and
meridianin D analog 4, Figure 1).16 To explore the effects of
these structural modifications to tryptamine in the context of
colistin potentiation, seven additional compounds were readily
synthesized by acylating the appropriate tryptamine derivative
(Scheme 1) and were then assayed for activity.
Figure 1. Active adjuvants that reduce the MIC of colistin against
various MDR Gram-negative bacteria.
Scheme 1. General Synthetic Route for Pilot Library
minimum inhibitory concentration (MIC) against Francisella
philomiragia and F. novicida, respectively, when tested at 50
μM.14 Additional indole and 2-aminoimidazole (2-AI)
derivatives (2, 3, and 4, Figure 1) have also demonstrated
the ability to potentiate colistin activity against several MDR
Gram-negative bacterial strains.15−17 Building upon these
initial studies, we report the synthesis and screening of 32
novel tryptamine derivatives for their ability to enhance colistin
efficacy against a range of MDR Gram-negative bacteria.
During these studies, we identified seven compounds that, at
30 μM or less, increased colistin efficacy, reducing the MIC
below the susceptible breakpoints defined by the Clinical and
Laboratory Standards Institute (2 μg/mL for the bacteria
tested).18 The lead compound from this series, at 5 μM,
elicited a 1024-fold reduction of the colistin MIC against a
chromosomally resistant K. pneumoniae strain, reducing the
MIC from 512 to 0.5 μg/mL. Herein, we describe the
structure−activity relationship (SAR) study that led to the
discovery of these novel small molecule adjuvants that increase
colistin efficacy in vitro.
We first evaluated compound 1a for its ability to potentiate
colistin against three representative Gram-negative strains that
contain the mcr-1 plasmid as well as their parent strains (Table
1).19 This compound showed modest activity and, at 50 μM,
reduced the colistin MICs between two- and 16-fold, with the
greatest activity being a 16-fold MIC reduction observed
against the parent E. coli strain. In a parallel SAR study on
compound 2, we noted that replacement of the halogenated
pyrrole with diverse halogenated heterocycles abrogated
colistin potentiation activity against several of the same
bacterial strains.15 Therefore, it was interesting that compound
1a exhibited activity against these six Gram-negative strains.
We next elected to investigate whether substitution of the
alkyl tail was tolerated as well as bromination of the indole
ring. Previous studies in our group have shown that
bromination of the indole ring as well as substituting alkyl
a
Compound was previously synthesized.14
Initially, we measured the MIC of each adjuvant against
three engineered bacterial strains harboring the mcr-1 gene,
along with the corresponding parent strains.19 All adjuvants
were essentially nontoxic themselves, exhibiting MICs > 200
μM. We then screened each nonbrominated tryptamine
derivative (1b−d) at 50 μM (≤25% of their MIC so that
potential toxic effects of the adjuvants toward the bacteria
would be minimal) and observed that every adjuvant showed
at least modest activity against all strains tested (Table 1).
Compound 1d displayed the greatest activity across all mcr-1
containing strains, lowering the colistin MIC to 2 μg/mL in all
cases (between 4- and 8-fold reductions), which is the
susceptibility breakpoint (2 μg/mL), and demonstrated that
replacement of the alkyl group is tolerated. The brominated
indolic derivatives, compounds 6a−d, exhibited similar activity
to their nonbrominated analogues (potentiation was within 2-
fold against all six strains) (Table 1), with most exhibiting 2-
fold reductions in activity. However, compound 6d did exhibit
a 2-fold increase in activity against two of the three mcr-1
containing strains when compared to its nonbrominated
counterpart 1d.
With negligible traction gained from brominating the indole
ring or replacing the phenalkyl tail, we next turned our
B
DOI: 10.1021/acsmedchemlett.9b00135
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
attention to the amide linker. In the context of colistin
adjuvants based upon the 2-AI scaffold, it has been noted that
replacing the amide linker with a urea (i.e., compound 3,
Figure 1) increased colistin potentiation activity substan-
tially.17 Therefore, we elected to test whether this structural
modification translated between scaffolds, and we set out to
make both the brominated and nonbrominated tryptamine
derivatives containing a urea linker.
Compounds were synthesized via one of two methods
depending on the commercial availability of the starting
materials. If the appropriate isocyanate was available, it was
coupled directly with tryptamine or 5-bromotryptamine
(Scheme 2A); otherwise, in a one-pot procedure, the
Table 2. Potentiation of Colistin (MIC μg/mL) with Urea-
containing Adjuvants Tested at 50 μM
K. pneumoniae
2210291
A. baumannii
E. coli YD626
17978
compound
parent
+mcr-1 parent +mcr-1
parent
+mcr-1
1
1
8
8
2
1
1
1
8
2
1
1
16
16
1
1
0.5
1
16
2
2
0.5
0.5
2
2
16
16
1
0.5
0.5
1
16
2
2
1
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
0.25
0.0313
0.125
0.125
1
0.25
0.0313
0.25
0.25
0.5
0.25
0.25
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.125
0.5
0.5
2
0.5
0.25
0.5
0.5
4
0.5
1
Scheme 2. Synthesis of Urea-containing Indole Adjuvants
0.5
Figure 2. Second-generation urea-containing adjuvants synthesized
for SAR analysis.
MICs were >200 μM, and all were therefore tested at 50 μM in
combination with colistin. With the exception of compound
11c, all compound 11 derivatives exhibited a decrease in
activity. Compound 11c rivaled its tryptamine derivative 8c in
terms of activity. As for derivatives 12a−e and 13a−e, direct
attachment of the urea to the indole ring at the 5 or 6 position
abrogated activity, showing ≤2-fold reduction in the MIC of
colistin (Table 3).
We next performed a dose−response study on the seven lead
compounds in combination with colistin against the three
strains that contain the mcr-1 plasmid (Table S1). The
concentration of the adjuvant was sequentially lowered to
determine the point at which the colistin MIC exceeded the
breakpoint for clinical colistin susceptibility (2 μg/mL). All
adjuvants maintained similar activity to the original 50 μM
dose when tested at 30 μM, and adjuvants 8b, 8c, 8e, 9d, and
9e successfully lowered the colistin MIC to the susceptibility
breakpoint at 15 μM against all three strains. Compound 9d
was the second most potent adjuvant and lowered the colistin
MIC to 1 μg/mL against all mcr-1 strains at 15 μM, while
adjuvant 9e displayed significant potentiation activity down to
5 μM, where it reduced the MIC of colistin from 8, 16, and 16
to 1, 2, and 1 μg/mL against mcr-1 plasmid containing E. coli,
K. pneumoniae, and A. baumannii, respectively.
isocyanate was formed in situ from the corresponding aniline
and triphosgene, and then immediately coupled with trypt-
amine or 5-bromotryptamine (Scheme 2B). These methods
afforded products 8a−e and 9a−e.
As observed with other tryptamine derivatives, compounds
8a−e and 9a−e were nontoxic to bacteria and displayed
standalone MICs > 200 μM against the six bacterial strains.
When tested at 50 μM in combination with colistin, most
compounds showed increased colistin potentiation when
compared to their amide counterparts (Table 2). Compounds
8b, 8c, 8d, 8e, 9d, and 9e resensitized all three mcr-1 strains
toward colistin, dropping the MIC to ≤2 μg/mL, demonstrat-
ing that the urea linkage generally is more active for colistin
potentiation than the amide.
Since the replacement of the amide with a urea moiety
provided analogs with increased colistin potentiation activity,
we next explored the effect of linker length between the indole
and urea and the position at which the urea was attached to the
indole ring. Three additional sets of compounds were
synthesized following the routes outlined in Scheme 2, but
with the urea placed either one methylene group closer to the
indole ring (11a−e, Figure 2) or directly attached to the indole
ring, at either the 5 or 6 position (12/13a−e, Figure 2). As
with all previously synthesized analogs, adjuvant standalone
To further investigate the lead compounds, we selected
compounds 8b, 9d, and 9e to test against chromosomally
colistin resistant K. pneumoniae and A. baumannii strains, which
are typically significantly more resistant to colistin than isolates
with resistance encoded by the mcr-1 gene. All three
compounds were highly active at 20 μM, eliciting between
64 and ≥2048-fold reductions in the MIC of colistin (Table
4). Compound 9e was again the most active compound and
C
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ACS Medicinal Chemistry Letters
Letter
compounds affected <2.5% cell lysis. Lastly, the potential for
eukaryotic cell toxicity was explored using a mouse mammary
gland tumor cell line, 4T1 (ATCC). Five analogs (8b, 8c, 8d,
9d, and 9e), in addition to an active compound from one of
our previous reports (14,17 Figure S1), were each tested in
monotherapy and in combination with colistin (1 μg/mL).
After 18 h of incubation with 4T1 cells, the culture medium
was aspirated, and cells were treated with a 0.5 mg/mL
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) in culture media to afford a colorimetric
assay for cell viability (Figure S2). The concentration at which
each compound resulted in 50% cell viability was recorded as
that compound’s CT50 value (Table 5). From these data, we
Table 3. Potentiation of Colistin (MIC μg/mL) with
Second-Generation Urea-containing Adjuvants Tested at 50
μM
K. pneumoniae
A. baumannii
E. coli YD626
parent +mcr-1
2210291
17978
compound
parent
+mcr-1
parent
+mcr-1
-
1
1
8
8
4
1
4
8
8
8
4
8
8
8
8
4
8
8
1
16
16
16
1
16
8
16
16
8
16
16
16
16
16
16
16
2
2
1
16
16
8
1
16
8
16
16
8
16
8
8
16
8
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
0.5
0.5
0.125
0.5
0.5
0.5
1
0.5
1
1
0.25
0.0313
0.5
0.25
0.5
1
0.5
0.5
0.5
0.5
1
0.125
2
1
1
1
1
2
1
2
2
1
2
2
Table 5. CT50 (μM) of Analogs in 4T1 Cells Dosed with and
without 1 μg/mL Colistin
compound alone
compound + colistin
1
1
1
1
8b
8c
8d
9d
9e
14
86.2
60.5
117.3
125.5
94.1
64.2
61.8
104.8
84.6
64.9
15.6
0.5
1
1
16
16
1
23.2
resensitized all four chromosomally colistin resistant strains to
or below the CLSI breakpoint (≤2 μg/mL) when tested at a
concentration of 20 μM, and resensitized three out of the four
strains to breakpoint colistin levels at a concentration of 7.5
μM. Against K. pneumoniae B9 and A. baumannii 4106,
compound 9e returned over a 2000-fold reduction of the
colistin MIC at 10 μM. Overall, these compounds, especially
adjuvant 9e, displayed potent in vitro colistin potentiation
activity against chromosomally resistant bacterial strains.
Additional assays were performed with lead compound 9e to
confirm activity and examine toxicity. To rule out activity due
to nonspecific compound aggregation effects, the resensitiza-
tion of A. baumannii 17978+mcr‑1 to colistin was performed in
the presence of 0.01% Tween 80.20 Compound 9e displayed
equipotent colistin sensitization activity in the absence and
presence of Tween 80 when tested at 30 μM. Next, lead
adjuvant 9e and inactive compound 9a were tested for toxicity
against red blood cells at a concentration of 200 μM, and both
first note that the respective CT50 value of each compound was
not significantly altered by the presence of colistin in culture
media. Second, each compound was considerably less toxic
than 14, a compound previously shown to potentiate colistin.17
Finally, we sought to determine if our antibiotic−adjuvant
combination would translate into an in vivo model. We chose a
Galleria mellonella model of A. baumannii infection to test our
antibiotic−adjuvant combination as previous studies at the
Walter Reed Army Institute of Research (WRAIR) have shown
results from this model to be predictive of outcomes in murine
models of infection.21,22 Since the use of A. baumannii 4106 in
a G. mellonella model has not, to the best of our knowledge,
been previously reported, we first performed a dose escalation
study to determine the minimum lethal dose of bacteria in the
worms. Much to our surprise, this strain was notably virulent,
and 6 × 105 colony forming units (CFUs) brought about 100%
Table 4. Dose Response of Lead Compounds 8b, 8d, and 9e with Colistin (MIC μg/mL) against Chromosomally Colistin
Resistant Strains
K. pneumoniae B9
K. pneumoniae C3
A. baumannii 3941
A. baumannii 4106
-compound
8v
concentration (μM)
512
512
512
1024
20
15
10
7.5
5
≤0.25
0.5
1
4
32
2
4
8
8
8
16
4
4
8
9d
9e
20
15
10
7.5
5
20
15
10
7.5
5
≤0.25
0.5
1
2
8
≤0.25
≤0.25
≤0.25
≤0.25
0.5
1
2
4
2
4
8
2
2
4
0.5
0.5
1
2
4
2
4
4
≤0.5
≤0.5
1
1
4
16
D
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ACS Medicinal Chemistry Letters
Letter
death of moth larva by day 4, which is similar to the highly
virulent A. baumannii 5075 strain.21,22 Next, we sought to find
a positive control for this model; however, this strain was
essentially resistant to all antibiotic classes, with the antibiotic
tigecycline demonstrating the most potent in vitro MIC of 8
μg/mL. With this antibiotic as our only positive control option,
we determined that a dose of 100 mg/kg provided minimal
survival after 4 days (20% survival at day 4, Figure 3).
AUTHOR INFORMATION
Corresponding Author
*E-mail: cmelande@nd.edu.
ORCID
Christian Melander: 0000-0001-8271-4696
Author Contributions
All authors have given approval to the final version of the
■
manuscript.
Funding
The authors would like to thank the National Institutes of
Health (GM055769 and AI136904) for funding.
Notes
The authors declare the following competing financial
interest(s): Dr. Melander is co-founder and board of directors
member of Agile Sciences, a biotechnology company seeking
to commercialize antibiotic adjuvants.
ACKNOWLEDGMENTS
■
The authors would like to thank Robert Ernst (University of
Maryland, Baltimore) and Yohei Doi (University of
Pittsburgh) for mcr-1 containing bacterial strains.
Figure 3. Kaplan−Meier curve of worms inoculated with 6 × 105
colony forming units of A. baumannii 4106.
ABBREVIATIONS
MDR, multidrug-resistant; MIC, minimum inhibitory concen-
tration; SAR, structure−activity relationship
■
Treatment of infected worms with either 50 mg/kg colistin or
compound 9e provided only 7% and 0% survival, respectively,
after 4 days. On the contrary, when 50 mg/kg colistin was
paired with 50 mg/kg compound 9e, the combination
increased worm survival to 47% after 4 days, considerably
exceeding that of the tigecycline control.
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In conclusion, we report novel tryptamine derivatives that
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00135.
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Chemistry experimental; biology experimental; Galleria
mellonella day-by-day results; compound NMRs (PDF)
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DOI: 10.1021/acsmedchemlett.9b00135
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
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DOI: 10.1021/acsmedchemlett.9b00135
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX