Photoactivated 2,3-distyrylindoles kill multi-drug resistant bacteria

Article abstract of DOI:10.1016/j.bmcl.2018.04.001

Compounds based on the 2,3-distyrylindole scaffold were found to exhibit bactericidal properties upon irradiation with white light. At the concentration of 1 μM, the lead compound 1 completely (ca. 10⁹ CFU/mL) eradicated such Gram-positive orga

Full text of DOI:10.1016/j.bmcl.2018.04.001

Accepted Manuscript
Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria
Leslie Edwards, Danielle Turner, Cody Champion, Megha Khandelwal, Kailee
Zingler, Cassidy Stone, Ruwini D. Rajapaksha, Jing Yang, Mahinda I.
Ranasinghe, Alexander Kornienko, Liliya Frolova, Snezna Rogelj
PII:
S0960-894X(18)30300-7
https://doi.org/10.1016/j.bmcl.2018.04.001
BMCL 25748
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 October 2017
27 March 2018
2 April 2018
Please cite this article as: Edwards, L., Turner, D., Champion, C., Khandelwal, M., Zingler, K., Stone, C.,
Rajapaksha, R.D., Yang, J., Ranasinghe, M.I., Kornienko, A., Frolova, L., Rogelj, S., Photoactivated 2,3-
Distyrylindoles Kill Multi-Drug Resistant Bacteria, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.04.001
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Photoactivated 2,3-Distyrylindoles Kill Multi-Drug Resistant Bacteria
a,*
a,*
a
a
Leslie Edwards, Danielle Turner, Cody Champion, Megha Khandelwal, Kailee
a
b
b
c
b
Zingler, Cassidy Stone, Ruwini D. Rajapaksha, Jing Yang, Mahinda I. Ranasinghe,
b
b,**
a,**
Alexander Kornienko, Liliya Frolova, Snezna Rogelj
a
Department of Biology, New Mexico Tech, 801 Leroy Place, Socorro, NM 87801, USA
b
Department of Chemistry, New Mexico Tech, 801 Leroy Place, Socorro, NM 87801, USA
c
Department of Chemistry and Chemical Biology, The University of New Mexico, 1 University of
New Mexico, NM 87131, USA
*
**Equal contribution
Corresponding authors. Email: liliya.frolova@nmt.edu (L. Frolova); snezna.rogelj@nmt.edu (S.
Rogelj)
Graphical Abstract
Light
Cl
Mask
Cl
Killed zone
N
H
Bacteria-inoculated
agar plate
Abstract
Compounds based on the 2,3-distyrylindole scaffold were found to exhibit bactericidal properties
upon irradiation with white light. At the concentration of 1 µM, the lead compound 1 completely (ca.
9
1
0 CFU/mL) eradicated such Gram-positive organisms as S. aureus (MRSA, MSSA), E. faecalis
(
VRE), S. pyogenes and S. mutans when irradiated with white light for 2 minutes. At the
concentration of 5 µM and in the presence of polymyxin E at non-bactericidal 1.25 µg/mL
concentration, 1 also showed a 7-log to 9-log reductions in bacterial counts of such Gram-negative
organisms as multi-drug resistant (MDR) A. baumannii, MDR P. aeruginosa, E. coli and Klebsiella
pneumoniae (CRE: KPC and NDM-1), also when irradiated with white light for 2 minutes. The
structure-activity relationship studies revealed that unsubstituted at benzene rings 2,3-distyrylindole 2
was most potent and gave a 5-order of magnitude eradication of a MRSA strain at the concentration
of 30 nM upon irradiation with white light. Initial mechanistic experiments revealed the disruption of
bacterial cell membrane, but indicated that singlet oxygen production, which is commonly associated
with photodynamic therapy, may not play a role in the bactericidal effects of the 2,3-distyrylindoles.
Keywords
1

Multi-drug resistance; MRSA; VRE; CRE; light-activated; S. mutans
Since the discovery of penicillin, antibiotics have been used globally as life-saving drugs and
th
revolutionized medicine in the 20 century. However, their effectiveness and easy access have led to
1
2
3,4
their overuse in healthcare and agriculture leading to the build-up of antibiotics in the environment
5
6
7
and the emergence of resistance through mutations and horizontal gene transfer. Despite the
8
implementation of numerous social awareness campaigns with the goal of decreasing the
inappropriate use of antibiotics in human healthcare and animal husbandry, there is an urgent need
for novel or underexplored therapeutic approaches to fight bacterial infectious diseases.
As part of the efforts to address the growing global resistance to antimicrobial medicines, on
February 27, 2017 the World Health Organization (WHO) published a list of bacteria that pose the
9
greatest threat to human health and thus require new effective antibiotics. The list of bacteria was
prioritized based on the current level of resistance, number of deaths they cause, the frequency of
infection and the burden they place on health care systems. The list includes Gram-positive
organisms, such as vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus
aureus (MRSA), as well as Gram-negative strains, such as Acinetobacter baumannii, Pseudomonas
aeruginosa and Carbapenem-resistant Enterobacteriaceae (CRE). Indeed, for example, CRE can kill
up to half of the patients who develop bloodstream infections and has been referred to as “nightmare
1
0
bacteria.”
One emerging approach for the treatment of drug-resistant bacterial infections is photodynamic
11,12
inactivation (PDI). It involves the use of a harmless visible light combined with a light-sensitive
dye, referred to as a photosensitizer. When irradiated, the dye is promoted to an excited state and
passes the excitation energy to oxygen resulting in the formation of reactive oxygen species. The fact
that the ensuing cell death is spatially limited to the areas of where light is applied makes PDI a
highly selective treatment approach for localized disease. PDI was originally described in 1900 as an
observation that a microorganism Paramecia was killed by a combination of a photosensitizing dye
1
3
acridine and sunlight. Currently, however, the two main applications of PDI are for the treatment of
14
various types of cancer and age-related macular degeneration.
Starting in the 1990s, significant effort has been applied to develop PDI as an antimicrobial
11,12
therapy. An important advantage of PDI over the existing antibiotics is its ability to work equally
1
5
well against both resistant and sensitive strains. Moreover, PDI has been shown to be incapable of
1
6,17
inducing resistance in bacteria even after 20 cycles of partial killing with the following regrowth.
Other advantages of PDI over systemically administered antibiotics include: rapid killing of
microbial cells, while the systemic antibiotics’ action may take hours to days; rapid achievement of
sufficient concentrations at the site of burns or damaged tissue, whose compromised blood supply
may prevent the systemic antibiotic from reaching the infected sites; broad spectrum of action
1
8
resulting in PDI’s quick administration before the infectious agent is identified. Furthermore, it is
now possible to deliver the photosensitizing agent and light to almost any site in the body through the
1
9
application of endoscopes and narrow-diameter interstitially inserted needles and fiber optics.
Although PDI, involving the generation of singlet oxygen (also termed photodynamic therapy
PDT)), is the most well-studied approach for antimicrobial applications, recently, treatment
(
strategies utilizing light activation of antibacterial agents not relying on oxygen or reactive oxygen
species have been described. For example, the incorporation of an azobenzene photo-switchable
element into the structure of quinolone antibiotics resulted in photo-controlled antibacterial agents
2
0
active against E. coli. In another example, a photo-isomerizable diarylethene moiety was introduced
into the backbone of an antimicrobial peptide gramicidin S and the antibacterial activity of the
21
resulting peptidomimetic could be controlled by light. Although such approaches indeed allow for
spatial control of antibacterial treatment, they do not offer better efficacy at eradicating resistant
2

strains than the original antibiotics from which they are derived.
Previously, as part of our screening efforts, we described the synthesis of compound 1 (Figure 1)
22
possessing a novel 2,3-distyrylindole scaffold. We reported that this compound was devoid of
antibacterial, antifungal and anticancer activities in the assays we utilized in our screening program.
More recent testing of compound 1, however, led to a serendipitous discovery that its lack of
antimicrobial activity is dramatically reversed when it is irradiated with white light. Herein, we
describe these recent findings by showing that 1 and its structural analogues represent significant
promise as light-activated antimicrobial agents active against resistant Gram-positive and Gram-
negative strains, including those that pose the greatest risk to human health as identified by the WHO,
as discussed above.
Cl
Cl
N
H
1
Figure 1. Previously synthesized 2,3-distyrylindole 1.
Our initial experimentation involved the utilization of a multidrug resistant MRSA strain (ATCC
BAA-44, HA-MRSA), a clinical isolate from a hospital in Lisbon, Portugal. In addition to beta-
lactams, this strain is resistant to antibiotics of many other structural and mechanistic types, such as
23,24
8
erythromycin, tetracycline, ciprofloxacin, tobramycin.
Thus, the bacterial isolates (ca. 10
CFU/mL) were incubated with 1 at different concentrations and then irradiated (or left in the dark in
the control experiment) with white light from a LumaCare lamp for 2 min. The samples were diluted
o
1
0-fold, drip-streaked on TSA plates and incubated overnight at 37 C. The colony counts were then
performed using a dilution that produces a drip-streak with 30-300 colonies, as shown in Figure 2B.
The bar graph derived from the colony counts is shown in Figure 2A. It illustrates a strong
bactericidal effect of 1, activated with light. A 5-log reduction in bacterial count is observed at the
concentration as low as 0.1 µM and a complete killing is achieved at a concentration between 0.5-1
µM.
Figure 2. Compound 1 eradicates MRSA (ATCC BAA 44) in the presence of light. A: Graph,
showing a dose-dependent effect of 1 with and without a 2-minute irradiation with white light. B.
Drip-streak plates utilized in the experiment illustrating the CFU counts. The data are derived from a
single experiment performed in 3 replicates and serve as an example of 3 independent experiments
producing similar results. The stars represent no observable colonies.
3

As mentioned above, PDI is attractive because it allows for high spatial and temporal resolution.
To illustrate this, a bacterial patterning experiment was performed. An agar plate, containing MRSA
8
cells (ca. 10 CFU/mL) and pre-treated with 1 (1 µM), was prepared. A mask was then placed on top
of the agar plate and the inoculum was irradiated with white light for 2 min (Figure 3, right). The
o
plate was then incubated at 37 C overnight and photographed. As can be seen on the photograph
shown in Figure 3 (left), bacterial colonies were observed only where the area of the plate was
covered to prevent the irradiation. Biological patterning, including the utilization of microorganisms,
is an emerging area of research with potential applications in biosensors, drug discovery and
2
5
diagnostics; thus, our results illustrate another area of promise for the light-controlled antimicrobial
properties of 2,3-distyrylindoles.
Figure 3. Bacterial patterning with light. Left: MRSA (ATCC BAA-44) cells pre-treated with 1 (1
µM), spread over an agar plate and irradiated with light through a mask for 2 minutes, and then
o
incubated at 37 C. NMT: New Mexico Tech. Right: Schematic of the bacterial patterning
experiment.
To investigate whether the bactericidal effect of 1 was not limited to HA-MRSA (ATCC BAA-
44), we performed experiments with other Gram-positive bacterial strains. The panel included:
another multidrug-resistant MRSA strain (ATCC BAA1717, CA-MRSA); drug-sensitive
Staphylococcus aureus; vancomycin-resistant Enterococcus faecalis, known as VRE and representing
2
6
another bacterial genus on the WHO list as discussed above; Streptococcus pyogenes, a causative
agent of important human diseases, ranging from superficial skin infections to life-threatening
2
7
systemic infections; Streptococcus mutans, a species commonly found in human oral cavity and the
2
8
main contributor to tooth decay. The latter organism is of particular interest due to its resistance to
2
9
various classes of antibiotics and the convenience of light delivery into the oral cavity.
As can be seen in Figure 4, all these Gram-positive organisms are sensitive to the bactericidal
effects of 1. While MRSA (ATCC BAA1717), S. aureus, E. faecalis, and S. mutans are completely
eradicated at the concentration of 1 µM, S. pyogenes is particularly sensitive and its complete killing
is achieved at the concentration of 0.1 µM. Importantly, compound 1 has a significantly lower
4

toxicity in the dark (Figure 4). Indeed, a light-activated agent should possess low toxicity to the target
tissue until light is applied. To confirm that the lack of toxicity in the dark is also observed toward
mammalian cells, the effects of 1 and several clinically used antibiotics on the viability of MCF-7
3
0
breast adenocarcinoma cells were evaluated using the MTT colorimetric assay. It was found that 1,
with the IC of 47.0±2.5 µM, is not any more toxic toward mammalian cells than the clinically used
5
0
antibiotics, such as tetracycline (IC of 41.6±0.3 µM) or norfloxacin (IC of 13.8±2.1 µM).
5
0
50
Figure 4. Compound 1 is effective against a variety of drug resistant Gram-positive bacterial strains
in the presence of light. A. MRSA (ATCC BAA1717). B. S. aureus (ATCC 29213). C. E. faecalis
(
ATCC 51299). D. S. pyogenes (ATCC 8133). E. S. mutans (Ward’s 85W 2357). The data are
derived from a single experiment performed in 3 replicates and serve as an example of 3 independent
experiments producing similar results. The stars represent no observable colonies.
5

The application of the above procedure to Gram-negative bacteria did not lead to the desired
killing. Although initially disappointing, this observation is consistent with the literature precedent
and explained by the fact that Gram-negative bacteria are typically characterized by an impermeable
outer cell membrane that protects the cell from antibiotics, dyes, and detergents. Thus, the outer
membrane of Gram-negative bacteria plays an important role in resistance to many antibiotics that
are highly effective against Gram-positive bacteria, e.g. macrolides, novobiocin, rifamycin,
lincomycin, clindamycin and fusidic acid. Accordingly, for a light-activated therapeutic agent to
work, it needs to permeate the outer membrane to get in the cytoplasm. This is believed to be the
3
1
reason for the higher occurrence of Gram-negative infections in hospital environments.
A number of approaches to overcome the above obstacle and make a PDI agent active against
3
2
Gram-negative bacteria have been reported in the literature. This can be achieved, for example, by
taking advantage of the outer membrane’s intrinsic negative charge due to teichoic acid residues and
adding a positive charge to the PDI agent, or coupling it with a positively charged moiety, such as
3
2
poly-L-lysine, polyethyleneimine and polymyxin nonapetptides. These agents are believed to work
by disorganizing the outer cell membrane in Gram-negative bacteria and making it penetrable to
3
2
hydrophobic agents. For example, a combination treatment with polymyxin B (PMB),
33
deuteroporphyrin and light resulted in killing of E. coli and P. aeruginosa.
Thus, a combination including both 1 and polymyxin E (PME) was evaluated against a panel of
Gram-negative organisms. This panel included: multidrug resistant Acinetobacter baumannii, a strain
that is becoming increasingly important causing hospital-acquired infections and responsive to only a
3
4
few antibiotics; Pseudomonas aeruginosa, also a causative agent of hospital acquired infections,
3
5
such as ventilator-associated pneumonia and various sepsis syndromes; Klebsiella pneumoniae, the
most significant member of the Enterobacteriaceae, mostly affecting people with weakened immune
system and, as mentioned in the Introduction, is associated with mortality rate of 50%, even with
36
37
antibiotic therapy; Escherichia coli, whose serotypes can cause serious food poisoning.
As can be seen in Figure 5, treatments involving 1 (5 µM) or PME (1.25 − 2 µg/mL) by
themselves had little or no effect either in the dark or with light. However, when 1 and PME were
combined at the same concentrations as in the single agent treatments, pronounced effects were
observed with irradiation. Indeed, P. aeruginosa and Klebsiella were completely eradicated (Figure
5B and D), while ca. 7-log reductions in bacterial counts were observed in the case of A. baumannii
and E. coli (Figure 5A and C). At present, the reason for the dark toxicity of this combination
treatment toward E. coli and Klebsiella (Figure 5C and D) is not completely understood and may
reside in potentiation of the PME’s action by hydrophobic 1 through the disruption of cells
membranes.
6

Figure 5. Compound 1 is effective against a variety of drug resistant Gram-negative bacterial strains
in the presence of PME and light. A. A. baumannii (ATCC BAA 1797). B. P. aeruginosa (ATCC
BAA-2114). C. E. coli (ATCC 29522) D. Klebsiella pneumoniae, CRE (ATCC BAA-2146). The data
are derived from a single experiment performed in 3 replicates and serve as an example of 3
independent experiments producing similar results. The stars represent no observable colonies.
To ascertain whether the bactericidal properties are associated with the novel 2,3-distyrylindole
scaffold, rather than specifically with compound 1, a group of analogues with differing substitution
patterns were synthesized (Figure 6). The synthesis is based on our previously discovered process to
2
2
access compound 1 by the oxidative Heck reaction of p-chlorostyrene with indole. Thus, analogues
with alternative positioning of chlorine substituent on the benzene ring 5 and 6 were prepared in
acceptable yields (Figure 6). Further, unsubstituted compound 2 and analogues with a different para-
substituent 3 and 4 were obtained in acceptable yields as well. Overall, the discovered reaction
appears to be useful for the preparation of a large number diverse analogues for the optimization of
their antibacterial properties. Importantly, these antibacterial agents are prepared in one step from
indole and various substituted styrenes. The spectra of the compounds are included in the supporting
information. In a few instances the compounds retained small amounts of solvents even after a
prolonged drying. To confirm the structures of the synthesized distyrylindoles, compound 2 was
1
13
treated with Pd/C under reflux in xylenes to generate known carbazole 7. The H and C NMR data
38
were fully consistent with those in the published report.
7

Figure 6. a) Synthesis of compounds of 2,3-distyrylindoles 1–6 and b) structure confirmation through
the synthesis of known carbazole 7.
Evaluation of the synthesized compounds against our original MRSA (ATCC BAA44) strain
revealed the initial structure-activity relationship (SAR) in this series of compounds. Specifically, the
replacement of chlorine with fluorine at the para-position in compounds 3 and 4 is detrimental for
activity and complete killing was not achieved even at a concentration as high as 5 µM (Figure 7B
and C, compare with 1 in Figure 2). Moving chlorine to the meta-position, as in compound 6,
similarly reduces potency considerably (Figure 7E). Chlorine at the ortho-position, as in 5, appears to
retain activity better, but it is still worse than that of compound 1 (Figure 7D). In contrast, analogue
2, with unsubstituted benzene rings, is the most potent so far. The eradication of bacteria by 5 orders
of magnitude is observed at concentrations as low as 30 nM (Figure 7A).
8

Figure 7. Eradication of MRSA (ATCC BAA44) by analogues of 1. The data are derived from a
single experiment performed in 3 replicates and serve as an example of 3 independent experiments
producing similar results. The stars represent no observable colonies.
The initial mode of action studies, aimed at understanding how light activation of 2,3-
distyrylindoles results in the bactericidal effect, were initiated. Electron microscopy of MRSA cells
treated with both 1 and light revealed a morphology involving an apparent cell membrane disruption
9

and leakage of the intracellular contents. This morphology was not observed in untreated cells or
cells treated only with 1 or only with light (Figure 8). To confirm this rationalization of the SEM
imaging, a propidium iodide (PI) staining assay was performed. PI is a positively charged fluorescent
intercalating agent that can cross the damaged cell membrane and bind to nucleic acids. As can be
seen in Figure 9, cells labeled with red fluorescence due to PI are observed only when they were
treated with 1 and light together. This red fluorescence is similar to the positive control phenylarsine
oxide (PAO), a general cytotoxic agent. In contrast, cells treated with 1 only exhibit green
fluorescence due to 1.
39
Cell membrane disruption is a characteristic outcome of photodynamic therapy, which is based
on the generation of singlet oxygen by the irradiated sensitizer, as discussed in the Introduction.
Thus, the ability of 1 to generate singlet oxygen was investigated (Figure 10). The reference samples
Rubpy and Rose bengal showed singlet oxygen emission at 1270 and 1285 nm, respectively. In
contrast, 1 showed a concentration dependent emission at 1210 nm. It is thus unlikely that the
bactericidal effect of 1 is due to the production of singlet oxygen. Although matrix effects can shift
the singlet oxygen emission by a few nanometers, this significant spectral shift cannot be explained
by matrix effects.
Figure 8. SEM images of MRSA (ATCC BAA44) cells treated with 1.
10

Figure 9. Permeability of MRSA (ATCC BAA44) cell membrane probed by PI. Green fluorescence
is due to 1. Red fluorescence is indicative of the disruption of cell membrane.
3 6 2
Figure 10. Lack of singlet oxygen generation by 1. Positive controls [Ru(bpy) ](PF ) and Rose
bengal show emission at 1270-1285 nm likely due to singlet oxygen. 1 shows emission at 1210 nm.
Although matrix effects can shift the singlet oxygen emission by a few nanometers, the significant
spectral shift by 1 cannot be due matrix effects.
As the post-antibiotic era is in sight, the possibility of infectious diseases becoming a major cause
11

of lethality can no longer be ignored. Fatal infections are not confined to tropical regions, or even
contained in hospitals, they are appearing more and more frequently in the community. Multi-drug
resistant Gram-positive organisms, such as vancomycin-resistant enterococci (VRE) and methicillin-
resistant Staphylococcus aureus (MRSA), as well as Gram-negative strains, such as Acinetobacter
baumannii, Pseudomonas aeruginosa and Carbapenem-resistant Enterobacteriaceae (CRE) are
becoming increasingly less and less susceptible to the currently available antibiotics. From this
perspective, research described herein is significant. The combination of 2,3-distyrylindoles and light
appears to overcome bacterial resistance regardless of the type of bacteria or the mechanism of
resistance. Importantly, these compounds are readily prepared via a one-step synthesis, wherein three
fragments, indole and two styrene units, are assembled in a convergent manner. Due to its simplicity
and functional group tolerance, the synthesis should be amenable to the preparation of a large number
of analogues with the desired substitution patterns in either indole or the styrene moieties, enabling
the optimization of these compounds during preclinical and possible clinical development. The next
step in this research is the elucidation of the MOA. Our preliminary data appear to rule out the singlet
oxygen generation as the mechanism responsible for the bactericidal properties of these compounds
and this leads us to the exciting process of hypothesis-driven MOA investigations. These studies will
be reported in due course.
Acknowledgments
This project was supported by grants from the National Institute of General Medical Sciences
(
P20GM103451), SR, LF and ALP acknowledge their NMT Presidential Research Support.
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-
-
-
2,3-Distyrylindoles exhibit bactericidal properties upon irradiation with light.
The compounds eradicate Gram-positive organisms when irradiated for 2 minutes.
In the presence of polymyxin E, the compounds eradicate Gram-negative organisms.
14