Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin

Article abstract of DOI:10.1016/j.bmcl.2018.01.022

We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus (MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloenzymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs. Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance. Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant bacteria to mupirocin.

Full text of DOI:10.1016/j.bmcl.2018.01.022

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel inhibitors of Staphylococcus aureus RnpA that synergize with
mupirocin
Nicole Lounsbury a,1, Tess Eidem b, Jennifer Colquhoun b, George Mateo a, Magid Abou-Gharbia a,
Paul M. Dunman b, , Wayne E. Childers a,
⇑
⇑
a Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA, United States
b University of Rochester School of Medicine and Dentistry, 601 Elmwood Street, Box 672, Rochester, NY, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus
(MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of
the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloen-
zymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an
inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant
strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing
the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs.
Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but
two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic
used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance.
Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant
bacteria to mupirocin.
Received 12 December 2017
Revised 10 January 2018
Accepted 14 January 2018
Available online xxxx
Keywords:
Antibiotic
Mupirocin
RnpA
RNase P
Synergy
Ó 2018 Elsevier Ltd. All rights reserved.
Staphylococcus aureus infections are often associated with high
rates of morbidity and mortality.1 First described in 1961,2,3 the
emergence of methicillin-resistant strains of S. aureus (collectively
referred to as MRSA) has become a significant health issue. On a
positive note, a recent study by the U.S. Center for Disease Control
found that the prevalence of community-acquired and hospital-
acquired MRSA in the U.S. has declined somewhat since 2005
due, in part, to enhanced awareness and more careful healthcare
practices.4 However, there has been an increase in the number of
multidrug resistant strains of S. aureus that respond poorly to treat-
ment by multiple classes of antibiotics, including the current stan-
dard of care for MRSA, vancomycin. Taken together with the
observation that antibiotic drug discovery research has declined
significantly since the 1990s, it has become obvious that there is
a real need for the development of new classes of antibiotics.5
Today’s antibiotics act through a number of drug targets to
exert their antimicrobial effects.6 Resistant strains of S. aureus have
acquired mechanisms that reduce the effectiveness of many of the
known classes of antimicrobial agents. As a result, recent antibiotic
drug candidates that act through familiar pathways have not been
able to differentiate themselves from existing agents and have not
received regulatory approval. What is needed is new classes of
antibacterial drugs.
We recently disclosed our discovery of RnpA as a novel drug
discovery target for S. aureus, including MRSA.7 S. aureus RnpA is
an essential protein that is thought to perform two required cellu-
lar processes. As a component of the RNA degradosome, it medi-
ates mRNA degradation, providing substrates for new RNA
synthesis. Together with rnpB, it forms RNase P holoenzymes,
which are required for tRNA maturation. Thus, small molecule
RnpA inhibitors have the potential to pose as dual threat antimi-
crobial agents, acting as multi-target ligands that eliminate the
organism’s ability to catalyze both mRNA decay and tRNA matura-
tion. This, coupled with the facts that the enzyme is expressed in
disease-associated S. aureus (including MRSA), is highly conserved
across other emerging and re-emerging pathogens, and has no sig-
nificant similarity to any human protein, make RnpA an attractive
antibacterial drug target.
⇑
Corresponding authors.
E-mail addresses: paul.dunman@urmc.rochester.edu (P.M. Dunman), wayne.
No direct inhibitors of RnpA have been reported in the litera-
ture, although four classes of small molecule RNase P inhibitors
have been described (Fig. 1).8 Puromycin mimics the 30-terminal
childers@temple.edu (W.E. Childers).
1
Current address: Larkin University School of Pharmacy, 18301 N. Miami Avenue,
Suite 1, Miami, FL, United States.
https://doi.org/10.1016/j.bmcl.2018.01.022
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lounsbury N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.022

2
N. Lounsbury et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
cause significant toxicity. In the present report we describe our
efforts to identify suitable bioisosteric replacements for the furan
group.
The general method of Li et al. was used to synthesize most of
the required thiahydrazide analogs (Scheme 1).19 Acid chlorides
(3) were sequentially treated in acetonitrile with potassium thio-
cyanate followed by 2-(4-isopropylphenoxy)acetohydrazide (4) to
give the desired products (5) in 15–80% yield.20 Final products that
did not precipitate from the reaction were obtained by reversed-
phase chromatography. In our hands, the use of potassium thio-
cyanate and acetonitrile provided better yields than the original
conditions described by Li et al. (ammonium thiocyanate, CH2Cl2/
PEG400), although the final products tended to precipitate out of
the reaction mixture more often when the original conditions were
used.
The synthetic sequence shown in Scheme 1 worked well when
the acid chloride component (3) was either an aryl acid chloride
(e.g., 2-furoyl chloride) or a cinnamoyl chloride (e.g., 2-(2-fur-
oyl)-acryloyl chloride). However, when benzylic hydrogens were
present in the acid chloride component no reaction with thio-
cyanate was realized. The final product in these reactions resulted
from direct combination between the acid chloride (3) and the
acylhydrazide (4). An alternate synthetic sequence was devised
to prepare the desired homologated thiahydrazide analogs
(Scheme 2).21 2-(4-Isopropylphenoxy)acetyl chloride (6) was trea-
ted with semicarbathiazide to give the resulting carbothioamide
(7), which was subsequently reacted with acid chloride (3) to
provide the desired final product (8) in low yield.
The compounds were tested for their ability to inhibit recombi-
nant in vitro S. aureus RnpA-mediated mRNA degradation activity
(Table 1; ꢀ97% protein purity), as previously described.7,22 Like-
wise, in vitro inhibitory effects on tRNA maturation activity were
measured using RNase P reconstituted with equimolar amounts
of S. aureus RnpA and rnpB, as previously described.22 Antibacterial
susceptibility testing was performed using S. aureus strain UAMS-
1, a well-characterized methacillin-sensitive osteomyelitis clinical
Fig. 1. Known RNase P inhibitors.
CCA sequence of tRNA and serves as a competitive inhibitor of
tRNA precursor cleavage by RNase P.9 Retinoids such as acitretin
inhibit the action of both human10 and D. discoideum11 RNase P,
although the exact mechanism of inhibition is not known. Amino-
glycosides, including neomycin B and kanamycin A and B (struc-
tures not shown), have been shown to inhibit RNase P in vitro
activity by interfering with the binding of active site magnesium
ions that are essential for activity.12 Most recently, a series of ben-
zamide-based RNase P inhibitors, exemplified by MES 10609, was
disclosed in a patent awarded to Message Pharmaceuticals.13 All
of these various series displayed potencies that were in the low
micromolar to low millimolar range.
A high throughput screen of over 29,000 small molecules iden-
tified a number of compounds that inhibited mRNA turnover by
recombinant S. aureus RnpA, including RNPA1000 (1) and
RNPA2000 (2) (Fig. 2).7 Compounds 1 and 2 demonstrated moder-
ate antibacterial activity in vitro against the predominant methi-
cillin-susceptible and MRSA lineages circulating throughout the
U.S., were effective against S. aureus biofilm associated cells, and
showed selectivity for RnpA over a number of other ribonucleases
evaluated, including E. coli RNase H, RNase A, RNase I and S. aureus
RNase J1. In vivo, 1 reduced S. aureus pathogenesis in a murine
acute lethal model of infection. However, the presence of a reactive
Michael acceptor group in compound 1 drove us to select com-
pound 2 for initial hit-to-lead activities. Key tasks for this scaffold
included eliminating the thiourea14–16 hydrazide17 and furan moi-
eties,18 which can be metabolized to give reactive species that
Scheme 1. Synthesis of thiahydrazide inhibitors of RnpA. Reagents and conditions:
(a) Potassium thiocyanate, CN3CN, rt; (b) add 2-(4-isopropyl-phenoxy)acetohy-
drazide (4), stir at room temperature.
Scheme 2. Synthesis of homologated thiahydrazides. Reagents and conditions: (a)
Semicarbathiazide, CN3CN, rt; (b) R-CH2-C(O)Cl (3), THF, 60 °C.
Fig. 2. Structures of RNPA1000 and RNPA2000.
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N. Lounsbury et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Table 1
RnpA inhibitory activity, MIC and cytotoxicity of compounds 2, 5a–5bb and 8.
Entry R Group
RnpAa
RNase Pb
MIC, UAMS-1
g/mLc
HEPG2
g/mLd
Entry R Group
RnpAa
RNase Pb
MIC, UAMS-1
g/mLc
HEPG2
l
g/mLd
IC50
,
l
M
IC50
,
lM
l
l
IC50
,
l
M
IC50
,
lM
l
2
275
140
16
16
64
64
32
>128
5n
100
32
64
>256
>128
128
5a
5b
5c
5d
250
250
150
25
16
5o
5p
5q
5r
60
1
32
>256
>256
>128
>250
50
25
100
0.5
128
>256
16
>500
>500
395
512
>500
50
50
>256
5e
5f
>500
>250
64
16
64
16
5s
5t
40
90
50
32
64
>128
>256
25
240
5g
100
1
32
>128
5u
50
50
32
32
5h
5i
>500
>500
160
150
64
>256
>512
5v
50
50
>256
256
>512
256
128
5w
>500
100
5j
20
30
150
105
32
32
>128
>128
5x
5y
250
<50
< 50
<50
16
64
32
5k
256
5l
30
40
32
>128
5z
150
90
128
512
5m
8
40
25
50
25
64
64
5aa
5bb
30
26
>256
64
256
512
25
16
32
>500
100
350
175
Mupirocine
>0.25f
>0.25f
0.125
>512 g
1
RNPA1000e
32
a
b
c
d
e
f
Inhibition of mRNA degradation by recombinant S. aureus RnpA.
Inhibtion of tRNA cleavage by a 1:1 mixture of S. aureus RnpA and RnpB.
Minimum inhibitory concentration toward S. aureus strain UAMS-1.
Lowest concentration causing cytotoxicity in HepG2 cells.
Data taken from Eidem et al.22
Highest concentration tested (2X MIC for S. aureus strain UAMS-1).
g
Rabbit corneal endothelial cells.26
isolate,23 according to Clinical and Laboratory standards guidelines
(CLSI)24 in 96-well format (Table 1). Minimum inhibitory concen-
trations (MICs) were defined as the lowest concentrations of test
compounds at which there was no visible bacterial growth. Overt
cytotoxicity was assessed in human HepG2 hepatocytes using the
MTT method.25
to undergo oxidative metabolism in the 5-position to give reactive
electrophilic species that can cause toxicity.18 The presence of a
heteroatom in this ring appeared to be necessary since substitution
of the furan by a phenyl ring (5bb) essentially abolished the RnpA
mRNA degradation inhibitory activity and greatly reduced RNase P
inhibitory potency. Substitution of the furan with a thiophene
group (compound 5b, Table 1) resulted in a compound with
similar inhibitory potency against both mRNA degradation and
The goal of this initial SAR campaign was to identify suitable
replacements for the furan moiety of compound 2, which is known
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4
N. Lounsbury et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 2
FICIs of selected compounds.
RNase P activity as that seen for 2. Having the heteroatom in the 2-
position appeared to be favorable for furans, thiophenes and
pyridines (compounds 5a–5i). While structural information on
the binding site of these molecules is not available, the pocket har-
boring the furan seems to be fairly lipophilic since oxazole (5e) and
isoxazole (5d) were not tolerated, especially when measuring
RnpA-mediated mRNA degradation activity. That lipophilic
pocket also seems to be somewhat longer than originally hypoth-
esized from the activity of compound 2, as suggested by the
enhanced potency of derivatives possessing longer lipophilic
groups in place of furan. For example, analogs possessing lipophilic
substitutions on the 5-position of the furan moiety (5k–5m) as
well as benzofuran, benzothiophene and indole (5o–5s) all demon-
strated superior activity against the enzyme’s mRNA degradation
and RNase P functions compared to 2. Homologation also enhanced
potency, as evidenced by the results seen with compounds 5n, 5aa
and 8.
For the indole group, substitution on the nitrogen (5t) was
detrimental while substitution in the 5- and 6-positions was toler-
ated (5u–5y). Structural changes did not always affect the two
activities similarly (i.e., changes that affected mRNA degradation
activity did not always impose a similar effect on tRNA processing).
The reason for this is not clear at present, although it may indicate
that the molecules elicit their effects on mRNA turnover and tRNA
processing by binding to two distinct sites on the protein.
Antimicrobial efficacy could not be accurately assessed for
many of these more potent analogs due to the presence of overt
cytotoxicity as demonstrated in human HepG2 cells (Table 1). Only
the analogs possessing unsubstituted 2-furanyl groups (5a, 8)
demonstrated antimicrobial potency similar to that seen with 2
Compound
Conc.
l
g/mL
Mupirocin
l
g/mL
FICI*
2
4
8
4
2
8
1
8
0.0625
0.0625
0.03125
0.0625
0.03125
0.0625
0.0625
0.49
0.5
0.5
0.3125
0.25
0.56
0.5
5g
5k
5l
5o
5r
5s
*
FICI = Fractional Inhibitory Concentration Index. MIC for mupirocin alone =
0.125 g/mL.
l
microtiter plate were inoculated with 1 ꢂ 105 CFU mlꢃ1 of S. aureus
UAMS-1. Each row contained increasing concentrations of the test
compound in 2-fold increments (0, 0.5, 1, 2, 4, 8, 16, 32
Each column contained increasing increments of mupirocin (0,
0.03125, 0.0625, 0.125 g/mL). The plates were incubated for 18
h at 37 °C and growth was measured. The FICIs were determined
using the formula: FICI = (MIC of test compound in combination/
MIC of test compound alone) + (MIC of mupirocin in combina-
tion/MIC of mupirocin alone). A potential synergistic combination
was defined as showing an FICI ꢁ 0.5.31 Results are summarized
in Table 2 and detailed data are provided in the Supplementary
Data Section.
Many of the compounds tested showed an additive effect (FICI
between 0.5 and 1.0) with mupirocin but compounds 5l and 5o
were shown to be synergistic based on the recommended guideli-
nes,31 exhibiting FICI values of <0.5 at multiple sub-MIC dose com-
binations. The superior synergy seen with these compounds
compared to 2 and the other analogs tested correlated most closely
with their ability to more potently inhibit both RnpA-associated
activities. This hypothesis is supported by two observations. First,
previously reported data shows that compound 1, which inhibits
RnpA-mediated mRNA degradation but does not prevent RNase
P-associated tRNA maturation, does not synergize with
mupirocin.22 Secondly, compounds like 5g and 5r, which showed
lg/mL).
l
(MIC = 16
However, in the other compounds tested, a trend toward greater
antibacterial potency against S. aureus UAMS-1 (MIC = 32 g/mL)
lg/mL) that could not be explained by overt cytotoxicity.
l
was seen with the compounds that demonstrated good activity
against both RnpA and RNase P functions. Compounds that were
potent at only one of these targets (e.g., 5i, 5w) provided relatively
weaker antibacterial activity. Another factor that might be influ-
encing the antimicrobial activity of these analogs is lipophilicity.
Almost all of the structural changes that resulted in improved
RnpA inhibitory activity resulted in increased lipophilicity com-
pared to compound 2 (Table S1). In general, effective antibiotics
that penetrate bacterial cell walls and act intracellularly tend to
be more hydrophilic compared to other drug classes and often pos-
sess a logP ‘‘sweet spot” that is characteristic of the structural
class.27 It is possible that the increased inhibitory activity against
the target, as seen in many of our improved molecules is not being
realized in the antibacterial assay due to reduced cellular
penetration.
potent RNase
P inhibitory activity (IC50 = 1 mM and 0.5 mM,
respectively) but weak RnpA mRNA degradation inhibitory
activity, also failed to show definitive synergistic effects (Table 2
and Checkerboard Synergy Charts, pS17 supplementary data).
Taken together, these data suggest that both RnpA and RNase
P inhibition must be present for these analogs to synergize
with mupirocin.
In conclusion, SAR within the RNPA2000 series of molecules
to find suitable replacements for the undesirable furanyl moiety
identified
a number of bioisosteric heterocyclic groups. In
particular, the 2-benzofuranyl and 2-indolyl groups provided
molecules with good RnpA inhibitory activity and potent RNase
P inhibitory activity. Most of the analogs tested demonstrated
somewhat less potent antibacterial activity and higher overt
cytotoxicity than the prototype molecule 2 but two analogs, 5l
and 5o, were shown to be synergistic with mupirocin against a
contemporary S. aureus clinical isolate and may find use as
combination agents in mupirocin decolonization. Studies to
identify suitable replacements for the acylhydrazide and thiourea
moieties in the scaffold are currently underway and will be
reported at a later date.
Antibiotics that function within the same biochemical pathway
often display synergy. A classic example is the synergy seen
between sulfonamides and trimethoprim which both function
within the folic acid biosynthesis pathway.28 Compound 2 was pre-
viously shown to synergize with mupirocin, an inhibitor of isoleu-
cyl-tRNA synthetase used topically for the treatment of impetigo
and S. aureus decolonization. However, the effectiveness of this
approach has come into question due to the emergence of bacterial
resistance to mupirocin.29 Since a synergistic approach represents
a potential means of re-sensitizing resistant bacteria to mupirocin
we explored the potential synergy of the present series with
mupirocin.
Compounds that displayed RnpA inhibitory activity, reasonable
Acknowledgements
antimicrobial activity (MIC ꢁ 32
lg/mL) and a high therapeutic
window over general cytotoxicity were examined for their ability
to synergize with mupirocin as previously described22 by deter-
mining their fractional inhibitory concentration30 index (FICI)
using standard checkerboard assays.31 Individual wells of a 96-well
This work was supported, in part, by University of Rochester
Technology Development Award and the National Institute of
Allergy and Infectious Diseases (NIAID) grant number AI103507
(PMD).
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N. Lounsbury et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
19. Li Z, Wang X, Da Y. Synthesis of 2-(5-(2-chlorophenyl)-2-furoylamido)-5-
aryloxymethyl-1,3,4-thiadiazoles under microwave irradiaton. Synth Commun.
2001;31:1829.
A. Supplementary data
Supplementary data (synthetic procedures and spectroscopic
characterization, logP calculation method, logP values for com-
pounds 2, 5a–5bb, 8, biological procedures, detailed checkerboard
synergy charts) associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmcl.2018.01.022.
20. The synthesis of (2) exemplifies a typical procedure: A mixture of 2-furoyl
chloride (1 eq) and potassium thiocyanate (1.7 eq) in dry acetonitrile was
stirred at room temperature for 1 hour. To the reaction was then added
2-(4-isopropylphenoxy)-acetohydrazide (0.97 eq) (5) and the resulting mixture
was stirred for an additional 1 hour at room temperature. In the case of (2), the
desired product precipitated as a white solid, which was collected by vacuum
filtration, washed with acetonitrile and dried under vacuum to obtain a 76%
yield of the desired product. In cases where products do not precipitate, the
reaction mixtures are concentrated on a rotary evaporator and the crude
reaction mixtures are purified on a Gilson 281 automated HPLC system using a
Phenomenex 5u Gemini C-18 column and a gradient of acetonitrile in water
with 0.1% trifluoroacetic acid.
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Please cite this article in press as: Lounsbury N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.022