5-Alkyloxytryptamines are membrane-targeting, broad-spectrum antibiotics

Article abstract of DOI:10.1016/j.bmcl.2016.10.004

Antibiotic adjuvant therapy represents an exciting opportunity to enhance the activity of clinical antibiotics by co-dosing with a secondary small molecule. Successful adjuvants decrease the concentration of antibiotics used to defeat bacteria, increase activity (in some cases introducing activity against organisms that are drug resistant), and reduce the frequency at which drug-resistant bacteria emerge. We report that 5-alkyloxytryptamines are a new class of broad-spectrum antibacterial agents with exciting activity as antibiotic adjuvants. We synthesized 5-alkyloxytryptamine analogs and found that an alkyl chain length of 6–12 carbons and a primary ammonium group are necessary for the antibacterial activity of the compounds, and an alkyl chain length of 6–10 carbons increased the membrane permeability of Gram-positive and Gram-negative bacteria. Although several of the most potent analogs also have activity against the membranes of human embryonic kidney cells, we demonstrate that below the minimum inhibitory concentration (MIC)—where mammalian cell toxicity is low—these compounds may be successfully used as adjuvants for chloramphenicol, tetracycline, ciprofloxacin, and rifampicin against clinical strains of Salmonella typhimurium, Acinetobacter baumannii and Staphylococcus aureus, reducing MIC values by as much as several logs.

Full text of DOI:10.1016/j.bmcl.2016.10.004

Accepted Manuscript
5-alkyloxytryptamines are membrane-targeting, broad-spectrum antibiotics
Katherine C. Faulkner, Katherine A. Hurley, Douglas B. Weibel
PII:
S0960-894X(16)31032-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.004
BMCL 24307
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2016
1 October 2016
4 October 2016
Please cite this article as: Faulkner, K.C., Hurley, K.A., Weibel, D.B., 5-alkyloxytryptamines are membrane-
targeting, broad-spectrum antibiotics, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.10.004
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5-alkyloxytryptamines are membrane-targeting, broad-spectrum antibiotics
Katherine C. Faulknera^, Katherine A. Hurleya^, Douglas B. Weibela,b,c
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a Department of Biochemistry, University of Wisconsin-Madison, 440 Henry Mall,
Madison, WI 53706 USA
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b Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue,
Madison, WI 53706 USA
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c Department of Biochemical Engineering, University of Wisconsin-Madison, 1550
Engineering Drive, Madison, WI 53706 USA
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^ equal contribution
*Author to whom correspondence should be addressed:
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Douglas B. Weibel
Departments of Biochemistry, Chemistry, and Biomedical Engineering
6424A Biochemical Sciences Building
440 Henry Mall
Madison WI 53706 USA
Phone: +1 (608) 890-1342
Fax: +1 (608) 265-0764
E-mail: weibel@biochem.wisc.edu
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Key Words: serotonin; tryptamine; antibacterial; bacterial membrane; antibiotic
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Abstract
Antibiotic adjuvant therapy represents an exciting opportunity to enhance the activity
of clinical antibiotics by co-dosing with a secondary small molecule. Successful
adjuvants decrease the concentration of antibiotics used to defeat bacteria, increase
activity (in some cases introducing activity against organisms that are drug resistant),
and reduce the frequency at which drug-resistant bacteria emerge. We report that 5-
alkyloxytryptamines are a new class of broad-spectrum antibacterial agents with
exciting activity as antibiotic adjuvants. We synthesized 5-alkyloxytryptamine analogs
and found that an alkyl chain length of 6-12 carbons and a primary ammonium group
are necessary for the antibacterial activity of the compounds, and an alkyl chain length
of 6-10 carbons increased the membrane permeability of gram-positive and gram-
negative bacteria. Although several of the most potent analogs also have activity against
the membranes of human embryonic kidney cells, we demonstrate that below the
minimum inhibitory concentration (MIC)—where mammalian cell toxicity is low—
these compounds may be successfully used as adjuvants for chloramphenicol,
tetracycline, ciprofloxacin, and rifampicin against clinical strains of Salmonella
typhimurium, Acinetobacter baumannii and Staphylococcus aureus, reducing MIC values by
as much as several logs.
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The juxtaposition between the slow pace of antibiotic discovery and the accelerated rate
of antibacterial resistance is a current concern in the medical and research community.1–
3 Of particular concern are gram-negative bacteria, which use multiple mechanisms to
evade antibiotics, including the diffusion barrier presented by their bilayer membranes,
pumps for drug efflux, and enzymes that modify and deactivate drugs.4–7 Antibiotic
adjuvant therapy is an antibacterial development strategy gaining attention in
combating these three major hurdles, improving the efficacy of existing antibiotics, and
suppressing the emergence of drug resistance.8–13
In antibiotic adjuvant therapy, two or more compounds—one of which is
traditionally an antibiotic—are combined to potentiate function and improve activity.
There are several successful examples of combination therapy, including Augmentin™:
the clinically successful combination of a beta-lactam (e.g., amoxicillin) with clavulanic
acid.14 Several recent adjuvant approaches draw on a combination of an antibiotic with
a mechanism for inhibiting resistance mechanisms,15 inhibiting efflux pumps,16,17
dispersing biofilms,18 increasing reactive oxygen species production19 and exploiting
bacterial metabolism.20,21
Another strategy for adjuvant therapy pairs an antibiotic with a molecule that
improves the transport of the compound into bacterial cells to increase the
concentration and bioavailability of an antibiotic. For example, the membrane-
permeabilizing properties of colistin sensitize multidrug-resistant Acinetobacter
baumannii to vancomycin.22 Phenylpropanoids are a family of natural products that
damage membranes and create synergistic interactions with various antibiotics that use
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different mechanisms of action against gram-negative and gram-positive bacteria.23,24
The discovery of new families of small molecules that target bacterial membranes and
increase the transport of antibiotics into cells may advance this emerging area of
antibiotic chemotherapy.
We discovered 5-nonyloxytryptamine (5-NOT, 1) in a high throughput screen of
small molecules that cause bacterial cells to form anucleate daughter cells (i.e., cells
lacking chromosomes) (Figure 1A).25 1 belongs to a family of serotonin-like compounds
that bind 5-hydroxytryptamine (serotonin) receptors (5-HT), and was previously
synthesized to test its selectivity in binding the human 5-HT1D receptor.26,27 Multiple
variants of human 5-HT1 receptors exist and elicit different neurological responses.28 5-
HT1D causes endothelium dependent relaxation in arteries and previous research
suggests that 5-HT1D receptor agonists inhibit neurotransmitter release and relax cranial
arteries.29 Sumatriptan selectively binds to the 5-HT1D receptor and is an effective
strategy for treating migraines.27,30 A structure-activity relationship study of
sumatriptan and other serotonin-like compounds led to the discovery of 1, which binds
to 5-HT1D receptors with 300-fold selectivity over non-specific serotonin 5-HT1
receptors.27
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Figure 1. A) The chemical structure of 5-nonyloxytryptamine (1), an O-alkylated
derivative of serotonin. A list of analogs of 1 synthesized separated by structural
modifications: B) modification of alkyl chain, C) modification of the substituent at 3-
position and the amine, and D) substitution of the indole ring by a phenyl ring and
modification of the length of the ethylene chain.
We performed minimum inhibitory concentration (MIC) assays of 1 against E.
coli BW25113 (6.0 µg/mL), B. subtilis 168 (0.76 µg/mL), and E. coli BW25113 ∆tolC (1.5
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µg/mL) (Table 1). These experiments made it possible for us to determine its potency
against a model gram-negative and gram-positive bacterium, and a bacterium in which
the AcrAB-TolC drug efflux pump system was partially disabled to potentially increase
in the intracellular concentration of the compound (compared to the parent E. coli
BW25113 strain). We also measured the antibacterial activity of 1 against various BSL-2
bacterial strains; 1 was effective against all strains tested with MIC values ranging from
3.0-24 µg/mL (Table 1). We determined the minimum bactericidal concentration (MBC)
of 1 against BSL-1 and BSL-2 strains to establish whether it was bacteriostatic or
bactericidal (Table 1).31 The ratio of MBC to MIC for 1 indicated that it was bactericidal
against: Shigella boydii, Klebsiella pneumonia, Acinetobacter baumannii, Edwardsiella tarda,
Morganella morganii, Staphylococcus aureus, and Streptococcus pyogenes and bacteriostatic
against: Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio cholera, and Enterobacter
aerogenes (Table 1).
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MBC
(µg/mL)
MIC
(µg/mL)
Bacterial Strain
MBC/MIC
Mode
E. coli BW25113
12
6.0
1.5
0.76
24
2
4
1
4
Bactericidal
Bacteriostatic
Bactericidal
Bacteriostatic
E. coli BW25113
∆tolC
6
B. subtilis 168
0.76
96
Pseudomonas
aeruginosa
Salmonella
typhimurium
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24
6.0
6.0
6.0
4
4
1
Bacteriostatic
Bacteriostatic
Bactericidal
Vibrio cholerae
Shigella boydii
6.0
6

Klebsiella pneumoniae
Enterobacter aerogenes
6.0
>24
6.0
12
6.0
6.0
6.0
6.0
12
1
>4
1
Bactericidal
Bacteriostatic
Bactericidal
Bactericidal
Bactericidal
Bactericidal
Bactericidal
Acinetobacter
baumannii
Edwardsiella tarda
Morganella morganii
Staphylococcus aureus
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24
2
6.0
24
3.0
24
2
Streptococcus
pyogenes
1
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Table 1. Minimum inhibitory concentration (MIC) and minimum bacterial
concentration (MBC) values of 1 against E. coli BW25113, E. coli BW25113 ∆tolC, B.
subtilis 168, and BSL-2 bacterial strains.
We initiated our structure-activity relationship (SAR) study by testing the MIC
activity of commercially available molecules with structures related to serotonin:
tryptamine (2), serotonin (3), 5-methoxytryptamine (4), 2-methyl-5-hydroxytryptamine
(5), and 6-methoxytryptamine (6) (Figure S1). We determined the MIC values of 2-6
against E. coli BW25113, E. coli BW25113 ∆tolC, and B. subtilis 168 and found they were
all >56 µg/mL, consistent with the importance of the long alkyl group of 1 on its
antibacterial properties (Table 2). We continued our SAR study by synthesizing new
analogs of 1 in which we varied the alkyl group.
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MIC values against bacterial strains (µg/mL)
E. coli
BW25113
E. coli
BW25113
B. subtilis
Compound
Number
S. aureus
A. baumannii
cLogP
168
∆tolC
1
2
3
4.65
1.13
0.79
6.0
>51
1.5
>51
0.76
>51
0.6
>51
6.0
>51
>56.4
>56.4
>56.4
>56.4
>56.4
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1.06
1.19
1.06
6.41
2.38
3.29
5.11
6.01
6.92
7.38
4.63
5.18
5.22
6.55
4.61
4.61
4.18
4.5
>60.8
>60.8
>60.8
>130
>74.3
42
>60.8
>60.8
>60.8
>130
>74.3
42
>60.8
>60.8
>60.8
>130
>74.3
42
>60.8
>60.8
>60.8
>130
>74.3
42
>60.8
>60.8
>60.8
>130
>74.3
42
5
6
8
17
18
19
20
21
22
23
24
25
26
27
28
29
30
6.3
3.2
1.6
3.2
6.3
26
6.9
1.7
3.4
28
>119
>124
13
>119
>124
3.2
15
3.4
>119
>124
13
>124
1.6
>124
6.3
14
1.8
1.8
3.5
7.0
>83
52
>83
6.5
>83
52
>83
6.5
>83
52
5.3
2.6
0.66
0.66
4.7
5.3
5.3
5.3
1.3
2.6
5.3
9.4
4.7
9.4
9.4
>40
10
40
>80
>80
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123
Table 2. Estimated cLogP and measured MIC (µg/mL) values of compounds 1-6, 8, 17-
30 against E. coli BW25113, E. coli BW25113 ∆tolC, B. subtilis 168, S. aureus, and A.
baumannii.
To create 5-alkyloxytryptamine analogs, we used a three-step synthesis:
protection of the primary amine of serotonin, alkylation of the hydroxyl group, and
deprotection of the primary amine (Figure 2).27 We synthesized 1 by converting
serotonin to N-Boc-protected serotonin (7) in 84%. We alkylated 7 with 1-bromononane
to yield the alkylated product 8 (30%) and removed the Boc protecting group to provide
1 in 85% yield. We used a similar synthesis procedure to prepare analogs 17-24 (Figure
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1B). Serotonin was protected with di-tert-butyl dicarbonate, alkylated with different
bromoalkanes to yield Boc-protected intermediates 8-16, and deprotected under acidic
conditions to provide final products 17-24 (Figure 2).
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Figure 2. The general synthetic strategy for the synthesis of 1 and its analogs: Boc
protection of the amine, alkylation of the hydroxyl group, and acidic deprotection to
produce the HCl salt of the free amine. Percent yields included in this figure are for the
synthesis of 1.
We next turned our attention to modification of the substituent at the 3-position.
We synthesized the N-Boc-protected 5-nonyloxytryptamine analog 8 by stopping at the
O-alkylation intermediate (Figure 1C); the yield of 8 was 47% over two steps. We
synthesized 25 by O-alkylation of 1H-indol-5-ol with 1-bromononane in 52% yield. 1
was trimethylated with methyl iodide in ethanol to produce analog 26 in yield of 27%
(Figure 1C). We synthesized analogs 27-30 by starting with a different scaffold and
using the same reaction conditions for 1 (Figure 1D). We produced 27 in 82% yield from
3-(2-aminoethyl)phenol. Analog 28 was synthesized from 4-(2-aminoethyl)phenol in
82% yield. We synthesized 29 from 3-(aminomethyl)phenol in 65% yield. Finally, we
synthesized 30 from 4-aminophenol in 42% yield.
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We used the library of 5-NOT analogs to make correlations between the structure
of 1 and its antibacterial activity. First, we tested how altering the length of the alkyl
chain of 1 affected antibacterial activity (Figure 1B). The MIC value for analogs with
butyl and hexyl saturated alkyl chains (17, 18) displayed a decrease in antibacterial
activity against E. coli BW25113. Butane analog 17 had an MIC value >74 µg/mL against
a panel of BSL-1 and BSL-2 bacterial strains (Table 2 and Table S1). The MIC value of
the hexyl analog 18 was 42 µg/mL against E. coli BW25113, E. coli BW25113 ∆tolC, and B.
subtilis 168 and ≥42 µg/mL against a panel of BSL-2 bacterial strains (Table 2 and Table
S1). 1, 17, and 18 have cLogP values of 4.65, 2.38, and 3.29, respectively. Compound 19
has a 10 carbon-chain, MIC values that are comparable to 1 against E. coli BW25113
(MIC, 6.3 µg/mL), E. coli BW25113 ∆tolC (MIC, 3.2 µg/mL), and B. subtilis 168 (MIC, 1.6
µg/mL) (Table 2), and a cLogP value of 5.11, which is close to the cLogP value of 1
(Table 2). Increasing the length of the saturated alkyl groups (20 has a 12 carbon-chain,
21 has a 14 carbon-chain, and 22 has a 15 carbon-chain) decreased the antibacterial
activity of compounds (Figure1B). Of the analogs with longer saturated chains, 20 had
the highest antibacterial activity with MIC values of 26, 6.9, and 1.7 µg/mL against E.
coli BW25113, E. coli BW25113 ∆tolC, and B. subtilis 168. (Table 2). 21 had antibacterial
activity only against B. subtilis 168 (MIC, 15 µg/mL) and S. aureus (MIC, 3.4 µg/mL),
and 22 did not display antibacterial properties (Table 2 and Table S1). cLogP values of
compounds 20-22 increased with chain length: cLogP values were 6.01 (20), 6.92 (21),
and 7.39 (22) (Table 2). These results suggest that the most active analogs have an
optimal alkyl length of 6-12 carbon atoms and a cLogP value of ~5.
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After alkane modification, we synthesized and tested two analogs with similar
chain length containing various substituents: 23 has an 8 carbon branched chain bearing
two methyl groups, while 24 has a 7 carbon-tail modified with a terminal phenyl group
(Figure1B); 23 and 24 have cLogP values of 4.63 and 5.18, respectively. 23 and 24 had
comparable MIC values of 13-14 µg/mL against E. coli BW25113 (Table 2) and displayed
MIC values of 3.2-25 µg/mL against a panel of BSL-2 strains, with the exception of P.
aeruginosa (25 and 56 µg/mL, respectively) and M. morganii (>101 and >112 µg/mL,
respectively) (Table S1).
We next investigated the function of the primary amine on the ethylene chain
located at the 3-position of the indole ring to determine whether the ammonium group
was important for the antibacterial properties of these compounds. We synthesized
analogs with a protected primary amine (8), lacking the ethylene amine chain (25), with
a quaternary ammonium (26) to determine the role of the amine on the antibacterial
activity of compounds. Trimethylating the primary amine created quaternary
ammonium analog 26 (Figure 1C). Protecting the amine (8) and removing it completely
(25) eliminated the antibacterial properties (MIC, >83 µg/mL) against E. coli BW25113, E.
coli BW25113 ∆tolC, and B. subtilis 168 cells (Table 2). Trimethylamine analog (26) had
decreased antibacterial activity compared to 1: MIC values of 52 µg/mL (E. coli
BW25113), 6.5 µg/mL (E. coli BW25113 ∆tolC), and 52 µg/mL (B. subtilis 168) (Table 2).
These results confirm our hypothesis that the amine is important for the antibacterial
activity of these compounds and that the small size and presence of acidic protons on
the ammonium group are contributing factors to the antibacterial activity.
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We next investigated the impact of the indole ring of 1 on its activity. We
replaced the indole ring with benzene (27) to alter the structure, yet maintain the same
spacing between the ethylene amine group and hydrophobic tail in 1 (Figure 1D); the
ethylene amine at the 3-position and O-alkyl chain at the 5-position on the indole ring is
structurally similar to the 1 and 3-positions on the benzene ring. The MIC value of
analog 27 was 5.3 µg/mL against E. coli BW25113, 2.6 µg/mL against E. coli
BW25113∆tolC, 0.66 µg/mL against B. subtilis 168, and matched the MIC value of 1
against S. boydii, K. pneumonia, A. baumannii, E. tarda, M. morganii, P. aeruginosa, S.
typhimurium, V. cholera, and E. aerogenes (Table 2 and Table S2). Since 1 and 27 exhibited
similar antibacterial activity, we tested the position of the ethylene amine relative to the
hydrophobic tail on the benzene scaffold. Instead of a 1,3 configuration of the alkyl
group and ethylene amine groups, we synthesized a 1,4 configuration (28) (Figure 1D).
The MIC values of 28 against E. coli BW25113 (5.3 µg/mL), E. coli BW25113 ∆tolC (1.3
µg/mL), B. subtilis 168 (0.66 µg/mL) were approximately the same as for 27 (Table 2)
and identical to 27 and 1 against S. boydii, K. pneumonia, A. baumannii, S. typhimurium,
and V. cholera. The MIC values of 28 increased to >84 µg/mL against P. aeruginosa, E.
tarda, and M. morganii and 10 µg/mL against E. aerogenes (Table S2). Both 27 and 28
have cLogP values of 4.61, which are similar to the cLogP value of 1 (Table 2). These
studies suggest that the 1,3 orientation of the alkyl tail and the ethylene amine groups
around the scaffold structure is not required for antibacterial activity.
Finally, we tested the contribution of the ethylene chain connecting the primary
amine at the 3-position of the benzene scaffold to the antibacterial activity of this family
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of compounds. We synthesized two analogs that maintain the 1,3 configuration:
methylene amine analog 29 and amino analog 30 (Figure 1D). The MIC values of 29 and
30 were against E. coli BW25113 (9.4 and >40 µg/mL, respectively), E. coli BW25113
∆tolC (4.7 and 10 µg/mL, respectively), and B. subtilis 168 (4.7 and 40 µg/mL,
respectively) suggest that a distance of ~3-5 Å between the primary amine and the
aromatic ring is ideal for activity (Table 2).
These results suggest that the hydrophobic tail and the charged, primary amine
head of 1 are essential for its antibacterial activity, making it plausible that these
compounds mimic the general structural features of lipids and lead to bacterial cell
death through altering the physical properties of membranes.32 We investigated the
effect of 1 on membrane permeability using the membrane impermeable DNA probe
propidium iodide (PI) to label DNA in cells in which the membrane is compromised
(Figure 3A).33,34 We treated E. coli BW25113 cells with 1 at 6.0 µg/mL (1×MIC) or 12
µg/mL (2×MIC), 50% v/v ethanol (positive control), and an equivalent volume of
DMSO (solvent control) and used flow cytometry to measure the fluorescence intensity
of individual cells in each of the treatment populations in the presence and absence of
PI (background fluorescence control). Ethanol-treated cells and cells treated with 1 at
both concentrations gave a significantly higher fluorescent signal than the population
treated with DMSO and untreated cells, suggesting 1 increases the membrane
permeability of E. coli cells.
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Figure 3. A) A plot depicting the fluorescence intensity of propidium iodine (PI) after
treating E. coli BW25113 cells with DMSO, 50% ethanol, 1×MIC of 1 (6.0 µg/mL), and
2×MIC of 1 (12.0 µg/mL), labeling using PI, and measurements using flow cytometry.
Each condition includes a sample size of n>7,000 cells. The three treatment conditions
(50% ethanol, 1×MIC, and 2×MIC) are statistically significant compared to DMSO
treatment (**p<0.001). A Q-test was preformed to define and remove outliers. B) The
MIC and percent of membrane permeability after treatment of E. coli BW25113 cells
with compounds 1 and 17-22 are presented on the same axes. The
fluorescence/absorbance ratio was normalized to the fluorescence/absorbance ratio of
the 50% ethanol treatment control.
We also explored the effect of alkyl chain length on the membrane permeability
of E. coli BW25113 cells treated with 1xMIC of 1 (6.0 µg/mL), 18 (42 µg/mL), 19 (6.3
µg/mL), and 20 (26 µg/mL) and the highest concentration of 17 (74 µg/mL), 21 (119
µg/mL), and 22 (124 µg/mL) that we tested in the SAR study (as these compounds did
not display significant antibiotic activity). We treated cells with compound for 15 min,
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added PI, and measured the absorbance and fluorescence intensity of both labeled and
unlabeled cells (the latter a background fluorescence control) using a microplate reader
(Figure 3B). When normalized to the 50% ethanol positive control, we observed 100%
membrane permeability when cells were treated with 1, 34% after treatment with 18,
83% after treatment with 19 and 10% after treatment with 20. Compounds 17, 21, and 22
displayed very little membrane permeability—3%, 11%, and 13%, respectively, which is
consistent with their lack of biological activity in MIC assays. An alkyl chain length of 6-
10 carbons maximizes bacterial cell membrane permeability and minimizes the MIC,
suggesting that biological activity of these compounds is related to membrane
permeability (Figure 3B).
To rule out the activity of 1 also arising from binding to a protein target in cells,
we performed a spontaneous resistant mutant screen. We isolated stable E. coli mutants
that were resistant to 10×MIC of 1, isolated genomic DNA of two mutants, deep
sequenced them, and assembled their genomes. We identified loci for mutations that
mapped to four genes; pykA, otsA, thrS, ydeA (Table S3). Using a multiple copy
suppression assay, we demonstrated that overexpressing each protein did not reduce
the MIC of E. coli MG1655 cells to 1 (Table S3). These results suggest that this compound
does not have a specific protein target in bacteria and that its activity arises from its
interaction with the membrane.
The membrane permeability activity of 1, 18, 19, 20, and 27 led us to explore
these compounds as antibiotic adjuvants to facilitate antibiotic transport into bacterial
cells, decrease the concentration of therapeutic antibiotics (and thereby reduce the
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susceptibility for bacteria developing drug resistance), and increase the susceptibility of
bacteria to the drugs. We used chloramphenicol, tetracycline, ciprofloxacin and
rifampicin as model clinical antibiotics that hit a range of drug targets. We treated E. coli
BW25113 cells with a combination of each of these antibiotics and 1, 18, 19, 20, or 27 at a
concentration of either 0.25× or 0.5×MIC. Analogs 18, 19, and 20 at 0.5×MIC
significantly changed the MIC of certain antibiotics against E. coli BW25113, while 1 had
no effect on the MIC of all four antibiotics (Table 3). Co-dosing 1, 18, 19, or 27 at
0.25×MIC did not have a significant effect on the MIC of the four antibiotics. 18 and 19
(at 0.5×MIC) reduced the MIC of rifampicin by ≥8-fold. Co-dosing 20 (at 0.5×MIC)
reduced the MIC of chloramphenicol and ciprofloxacin by 16-fold, the MIC of
tetracycline by 8-fold, and the MIC of rifampicin by 32-fold against E. coli BW25113. 10
was also active at 0.25×MIC and reduced the MIC of chloramphenicol by 4-fold and
rifampicin by 8-fold (Table 3). From these data we selected 18, 19, rifampicin, and
tetracycline to test additional drug combinations against S. typhimurium, A. baumannii,
P. aeruginosa, K. pneumoniae, S. aureus.
Analog
Concentration (µg/mL)
1
1
18
18
19
19
20
20
27
27
No
Analog
Antibiotic
3.0 1.5
21 10.5 3.2 1.6
14
6.9 2.6 1.3
Chloramphenicol
(µg/mL)
8
2
8
2
8
2
4
1
4
2
2
4
1
0.5
2
1
4
1
8
2
Tetracycline
(µg/mL)
0.25
0.25
Ciprofloxacin
(ng/mL)
Rifampicin
13
8
13
8
13
8
13
1
13
4
13
1
13
8
0.8
13
1
25
2
25
4
0.25
16

(µg/mL)
289
290
291
292
293
Table 3. The MIC (µg/mL) of 4 antibiotics against E. coli BW25113 supplemented with 1,
18, 19, 20, and 27. Gray shading highlights co-treatment combinations tested against
BSL-2 bacterial strains, which form the basis for Table 4.
Table 4 displays MIC data for rifampicin dosed with 18 (21 µg/mL) and
tetracycline dosed with 19 (3.2 µg/mL; with the exception of S. aureus, for which we
used 1.6 µg/mL) against all five bacterial strains. The activity of 18 and 19 as potential
adjuvants was most notable against S. typhimurium, A. baumannii, and S. aureus. The
MIC of rifampicin against S. typhimurium and A. baumannii was 8 µg/mL and 2 µg/mL,
respectively. Co-dosing rifampicin with 18 (21 µg/mL) decreased the MICs to 0.5
µg/mL and 0.016 µg/mL, respectively. The MIC of tetracycline was 1 µg/mL for both S.
typhimurium and A. baumannii; adding 19 (3.2 µg/mL) decreased the MIC to 0.016
µg/mL and 0.25 µg/mL, respectively. The only combination effective against S. aureus
was 19 (1.6 µg/mL) and tetracycline, which reduced the MIC of tetracycline by over
100-fold to 0.001 µg/mL (Table 4, Figure 4).
294
295
296
297
298
299
300
301
302
303
Rifampicin
No Analog
Rifampicin Tetracycline Tetracycline
Antibiotic (µg/mL)
Analog/[Analog] (µg/mL)
18/21
No Analog
19/3.2
Salmonella typhimurium
Acinetobacter baumannii
Pseudomonas aeruginosa
Klebsiella pneumoniae
Staphylococcus aureus
8
2
16
16
0.004
0.5
0.016
16
1
1
32
0.016
0.25
16
8
2
2
0.004
0.125
0.001*
304
305
306
307
308
309
310
311
* We used a concentration of 19 (1.6 µg/mL) with tetracycline when treating S. aureus to
keep to maintain 0.5×MIC of the hydroxytryptamine analog.
Table 4. The MIC (µg/mL) of rifampicin and tetracycline against BSL-2 strains:
Salmonella typhimurium, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella
pneumoniae, and Staphylococcus aureus supplemented with sub-MIC concentrations of 18
and 19.
17

Absorbance (λ
)
600
0
0.62
0.0003
0.0005
0.001
0.002
0.004
0.008
0.016
0.031
0.063
0.13
0.25
[19] (µg mL-1)
312
313
314
315
316
317
Figure 4. A plot depicting a checkerboard assay with varying concentrations of
tetracycline and 19 against S. aureus. The MIC of tetracycline and 19 against S. aureus is
0.13 µg/mL and 3.2 µg/mL, respectively.
To explore 5-NOT analogs as antibiotic adjuvants from a therapeutic perspective,
we investigated the cytotoxicity of several analogs against human embryonic kidney
(HEK) cells. We measured HEK cell viability 24 and 48 h after treatment with 18, 19, 20,
and 27 at the concentrations used in the co-dosing experiments described above (Table
S4). We found that 19 (0.25×MIC, 1.6 µg/mL) and 27 (0.25×MIC, 1.3 µg/mL) reduce
HEK cell viability to 29.6% and 60.5% after 24 h of treatment. At this concentration, 19
reduced the susceptibility of S. aureus to tetracycline from 0.125 µg/mL to 0.001 µg/mL
(Table 4, Figure 4). These data suggest that 19 at sub-MIC concentrations can potentiate
the activity of clinical antibiotics, however further structural modifications will be
needed to reduce HEK cell cytotoxicity.
318
319
320
321
322
323
324
325
326
18

327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
Although 1 was selected in a high throughput screen designed to detect
anucleate cells after compound treatment, cells treated with this compound and the
DNA-intercalating dye 4’,6-diamidino-2-phenylindole (DAPI) became fluorescent,
indicating that cells contained a nucleoid. To test the hypothesis that molecules that
disrupt the cell envelope are false positives in the anucleate assay, we tested three β-
lactams (meropenem, aztreonam, and cefotaxime) that inhibit the biosynthesis of
peptidoglycan35,36 and an antimicrobial peptide (cecropin A) that disrupts the bacterial
membrane.37 We used rifampicin as a positive control and chloramphenicol as a
negative control.25 We treated E. coli SH3210 cells with each compound and the β-
galactosidase substrate, DDAOG and measured the fluorescence signal indicating
production of the cleaved fluorescent byproduct. Meropenem, aztreonam, cefotaxime,
cecropin A, and 1 produced a significantly higher β-gal signal than the negative control,
chloramphenicol and the DMSO solvent control (Figure S2). These results confirm our
reasoning that compounds that interact with the membrane or inhibit cell wall
biosynthesis are false positive hits in the anucleate cell assay.
In conclusion, we report the discovery and characterization of 5-
alkyloxytryptamine analogs as a new family of broad-spectrum antibiotics. Members of
this family of compounds were described previously as human 5-HT1D receptor
agonists. We describe a series of compounds in this family that target membranes and
display MIC values as low as 0.6-5 µg/mL against a range of pathogenic bacteria. At
sub-MIC concentrations—toward the end of the spectrum where toxicity against HEK
cells is minimized—the compounds are effective adjuvants that potentiate the activity
19

349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
of clinical antibiotics. For example, co-dosing S. aureus cells with 19 reduced the MIC of
tetracycline from 0.125 µg/mL to 0.001 µg/mL. Exploring other co-dosing formulations
and 5-alkyloxytryptamine analogs may increase potency while reducing toxicity.
Acknowledgements
This research was supported by grants from the National Institutes of Health
(1DP2OD008735) and the Human Frontiers Science Program (RGY0076/2013). K.
Faulkner acknowledges a fellowship from the Biochemistry Department of UW
Madison provided by the Genentech Corporation and K. Hurley acknowledges a
fellowship from the American Foundation for Pharmaceutical Education (AFPE). We
acknowledge Soren Rozema and his synthetic efforts toward new analogs during the
revision process. We also acknowledge the flow cytometry instrumentation facility
supported by University of Wisconsin Carbone Cancer Center Cancer Center Support
Grant P30 CA014520.
Supplementary data
Supplementary data (figures, tables, synthetic procedures, compound characterization
data and biological methods) associated with this article can be found in the online
version.
20

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374
375
376
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381
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385
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23

*Graphical Abstract (for review)
positively charged
primary amine
CH3
Hypothesized compound insertion
in the phospholipid bilayer
n = 4-8
NH3
Cl
O
N
H
indole or benzene ring
Structural elements necessary for
membrane permeability activity