2
R. Rajamuthiah et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
showed that PSPC is comparable to vancomycin in prolonging sur-
vival of MRSA infected worms at a concentration as low as 0.78 g/
2
l
g/mL for vancomycin (Table 1). A time-kill assay at 10Â MIC
l
(40 g/mL) showed that PSPC is bactericidal, killing essentially
l
mL, though it is also toxic to nematodes at high concentrations.
PSPC does not cause hemolysis of erythrocytes but displays cyto-
toxicity to mammalian cell lines causing almost 69% mortality at
100% of MW2 cells during a 4-hour exposure period (Fig. 3). The
minimum bactericidal concentration of PSPC against MW2 is
16 lg/mL (data not shown), 4 times the MIC. These data show that
a concentration of 4
l
g/mL. These findings support the need for
PSPC functions directly to block the growth of MRSA MW2.
The ability of PSPC to function as a broad-spectrum antibiotic
was determined by measuring its MIC against the ESKAPE patho-
gens: Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa and Enterobacter spp., in addi-
tion to S. aureus. The six ESKAPE pathogens are the main causes of
nosocomial infections, which are often difficult to treat due to
antimicrobial resistance.11 Similar to S. aureus, the MIC of PSPC
further studies to improve the antibacterial potency and diminish
the toxicity of PSPC before in vivo testing in mammalian infection
models.
The C. elegans-MRSA liquid infection assay is a well-established
high throughput screening platform that allows simultaneous
assessment of both the toxicity and antibacterial efficacy of test
compounds in 384-well formats.5–7 The end point of the assay is
to measure the survival of MRSA-infected worms that have been
treated with test compounds. This liquid infection assay was used
to identify antibacterial hits from a library of 21,472 pre-selected
compounds provided by Boehringer-Ingelheim GmbH (BI). The
compounds were pin-transferred to 384-well plates at a final assay
against the Gram-positive species E. faecium was 8
but PSPC displayed higher MICs against the Gram-negative
bacteria K. pneumoniae (64 g/mL), A. baumannii (32 g/mL),
P. aeruginosa (>64 g/mL) and Enterobacter spp. (>64 g/mL).
lg/mL (Table 1),
l
l
l
l
Two analogs of PSPC were synthesized (see Supplementary
methods) and tested for antibacterial activity against the ESKAPE
pathogens (Table 1). The sulfide analog PSPC-1S (Fig. 1) was found
concentration of 20
l
g/mL. Each sample plate contained 16 wells
each of positive control (vancomycin at 10
l
g/mL in 1% DMSO)
and negative control (1% DMSO). The percent survival of infected
worms treated with library compounds was determined at the
end of the assay and a Z score was calculated for each well to iden-
tify hits. Of the 21,472 compounds tested, 318 (1.4% hit rate) pro-
tected worms from MRSA-mediated death. Among the hits, PSPC
(Fig. 1) was chosen for follow up studies because the compound
is a novel, low molecular weight scaffold that has not previously
been reported to have antibacterial activity.
to be inactive across all species in the panel (MICs > 64 lg/mL). In
contrast, the sulfoxide analog PSPC-6S (Fig. 1) showed identical
potency to PSPC against all species. These findings indicate that
the activity of PSPC is structure-dependent and requires the polar
functionality attached to the sulfur atom, which supports a tar-
get-based mechanism.
The peptidomimetic compound phenylalanine arginyl
b-naphthylamide (PAbN) is a broad spectrum pump inhibitor, that
is, effective against resistance/nodulation/division (RND) pumps in
various Gram-negative bacteria, restoring the antibiotic suscepti-
bility of these strains.12 We examined whether PAbN also pos-
sessed the ability to directly inhibit the growth of bacteria, by
testing the antimicrobial activity of PAbN against the Gram-nega-
tive ESKAPE pathogens in a broth microdilution assay. PAbN did
not show any growth inhibitory effects against these strains at
To confirm the activity of PSPC, the compound was resynthe-
sized (see Supporting information) and the C. elegans-MRSA liquid
infection assay was repeated using serial dilutions in the range of
0.78–100 lg/mL. The activity of the resynthesized PSPC was com-
pared to vancomycin over the same concentration range. At the
lowest concentration, both PSPC and vancomycin were effective
in prolonging survival of more than 90% of infected worms
(Fig. 2). However, at the higher concentrations of 50 and 100
lg/
the maximum tested concentration of 64 lg/mL (data not shown).
mL, worms treated with PSPC showed only 46% and 16% survival,
respectively, whereas vancomycin-treated worms showed >97%
survival at these concentrations. These findings suggest that
although PSPC ameliorates MRSA-associated lethality in worms
at low concentration, the compound is toxic to the worms at higher
concentrations. The concentration of DMSO in all sample wells did
not exceed 1% DMSO, the concentration of DMSO in the negative
control, and therefore, the toxicity of PSPC at higher concentrations
is not due to DMSO.
As we have previously described, compounds identified in
chemical screens that prevent a pathogen from killing C. elegans,
can inhibit the growth of the pathogen, block the virulence of the
pathogen, and/or enhance C. elegans immunity.8–10 To help distin-
guish these possibilities, we determined first whether PSPC could
directly affect the growth of the pathogen. We found that PSPC
does inhibit the growth of the MRSA strain MW2 in Müller-Hinton
Accordingly, we examined whether the activity of PSPC against
Gram-negative ESKAPE pathogens could be potentiated in the
presence of PAbN at a fixed concentration of 64 lg/mL. In the pres-
ence of PAbN, the MIC of PSPC against K. pneumoniae, A. baumannii,
P. aeruginosa and Enterobacter spp. decreased by more than 4-fold
(Table 2).
We tested the ability of polymyxin B, a lipopeptide antibiotic,
that is, effective against a variety of Gram-negative bacteria, to
function synergistically with PSPC against K. pneumoniae and A.
baumannii. A microplate checkerboard assay was used to test for
growth inhibition of the strains in the presence of PSPC adjusted
to final concentrations in the range of 1–64
lg/mL and polymyxin
B adjusted to final concentrations of 0.063–64
lg/mL. The mini-
mum concentrations at which the two compounds inhibited bacte-
rial growth, both alone and in combination, were determined in
order to calculate the Fractional Inhibitory Concentration (FIC)
index.13 The FIC indices of polymyxin B/PSPC against K. pneumoniae
and A. baumannii were found to be 0.141 and 0.375, respectively
(Table 3). FIC indices <0.5 indicate that polymyxin B exhibits syn-
ergistic activity with PSPC against both pathogens.
broth with an in vitro MIC of 4 lg/mL, compared to an MIC of
PSPC was found to be toxic to worms at high concentrations
(Fig. 2), suggesting the possibility of toxicity to other eukaryotic
systems. In vitro cytotoxicity of PSPC was evaluated against
HEK-293 cells. The cells were treated with serial dilutions of PSPC
over the concentration range 1–64 lg/mL and cell viability deter-
mined. The mitochondrial toxin rotenone, which interferes with
the electron transport chain in mitochondria, was included as a
positive control. At concentrations up to 2 lg/mL of PSPC, almost
90% of treated cells survived (Fig. 4A). Higher concentrations of
Figure 1. Chemical structures of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC)
and its sulfide (PSPC-1S) and sulfoxide (PSPC-6S) analogs.
PSPC caused a dose dependent increase in toxicity to HEK-293