Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens

Article abstract of DOI:10.1021/jm025502x

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogen

Full text of DOI:10.1021/jm025502x

© Copyright 2002 by the American Chemical Society
Volume 45, Number 10
May 9, 2002
Letters
not be susceptible to established mechanisms of bacte-
rial resistance.
Na n om ola r In h ibitor s of Sta ph ylococcu s
a u r eu s Meth ion yl tRNA Syn th eta se w ith
P oten t An tiba cter ia l Activity a ga in st
Gr a m -P ositive P a th ogen s
The aminoacyl tRNA synthetase family of enzymes
are responsible for charging tRNA molecules with their
cognate amino acid in an essential step of protein
synthesis.3,4 The clinical antibiotic mupirocin (pseudo-
monic acid), a recommended topical agent for the
prevention and eradication of MRSA outbreaks, is an
inhibitor of bacterial isoleucyl tRNA synthetase. The
other 18 aminoacyl tRNA synthetases present in Gram-
positive bacteria are unexploited as antibacterial tar-
gets, and selective inhibition of one of these enzymes
would provide an antibiotic acting by a novel mode of
action. We have undertaken high throughput screening
of all aminoacyl tRNA synthetases from S. aureus.5-7
Here we describe potent Gram-positive antibacterial
agents derived from a file compound screening hit
against methionyl tRNA synthetase (MRS).
Richard L. J arvest,*,† J ohn M. Berge,† Valerie Berry,‡
Helen F. Boyd,† Murray J . Brown,† J ohn S. Elder,†
Andrew K. Forrest,† Andrew P. Fosberry,†
Daniel R. Gentry,‡ Martin J . Hibbs,†
Deborah D. J aworski,‡ Peter J . O’Hanlon,†
Andrew J . Pope,† Stephen Rittenhouse,‡
Robert J . Sheppard,† Courtney Slater-Radosti,‡ and
Angela Worby†
GlaxoSmithKline, New Frontiers Science Park,
Third Avenue, Harlow, Essex CM19 5AW, U.K.,
and GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426
Scr een in g Lea d . High throughput screening of S.
aureus MRS8,9 afforded the hit 1 which had an IC50
value of 350 nM. The MRS inhibition was found to be
competitive with respect to methionine with a Ki of 100
nM, despite little formal similarity to the tightly bound
intermediate methionyl adenylate (2). In a standard
antibacterial assay against a range of pathogens, com-
pound 1 had no Gram-positive antibacterial activity up
to the highest concentration tested (64 µg/mL).10
Received J anuary 4, 2002
Ab st r a ct : Potent nanomolar inhibitors of Staphylococcus
aureus methionyl tRNA synthetase have been derived from a
file compound high throughput screening hit. Optimized
compounds show excellent antibacterial activity against sta-
phylococcal and enterococcal pathogens, including strains
resistant to clinical antibiotics. Compound 11 demonstrated
in vivo efficacy in an S. aureus rat abscess infection model.
In tr od u ction . The inexorable rise in the incidence
of bacterial resistance and the attendant healthcare
issues have been widely noted. Of particular concern
has been the rise of methicillin resistant Staphylococcus
aureus (MRSA), with MRSA strains now accounting for
over 40% of cases of S. aureus bacteraemia.1 An enor-
mous increase has also occurred in the incidence of
infections with vancomycin resistant enterococci (VRE),
many strains of which have become resistant to all
established classes of antibiotics.2 This scenario has
driven the search for new classes of antimicrobial agents
which act on novel bacterial targets and thus should
Syn th esis. The inhibitors reported here were syn-
thesized as shown in Scheme 1. The key intermediate
is the amine (5), prepared in two steps from the
quinolone (3). Reductive alkylation of 5 with 3,4-
dichlorobenzaldehyde afforded 9 (SB-299683). A similar
reaction under more forcing conditions with the ketones
7 and 8 gave the tetrahydroquinoline derivatives 10
(SB-362916) and 11 (SB-425076). Where necessary, the
* Corresponding author. Tel: +44 (0)1279 627629. Fax: +44 (0)-
1279 622260. E-mail: Richard.L.J arvest@GSK.com.
† GlaxoSmithKline, Essex.
‡ GlaxoSmithKline, Collegeville.
10.1021/jm025502x CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/09/2002

1960 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Letters
Sch em e 1a
Ta ble 1. In Vitro Minimum Inhibitory Concentration (MIC) of
MRS Inhibitors against Strains of S. aureus and E. faecalis
MIC (µg/mL)
amoxicillin mupirocin
9
10
11a
S. aureus Oxford
0.125
0.125
0.25
64
64
4
8
2
2
<0.06
0.25
0.25
S. aureus WCUH29 >64
E. faecalis 1
E. faecalis 7
0.125
0.5
0.5
<0.06 <0.06
<0.06 0.125
a
SB-425076 was tested as the dihydrochloride salt.
Ta ble 2. In Vitro Antibacterial Activity of Compounds 10 and
11 against Panels of Staphylococci and Enterococci, Where
MIC90 Is the Concentration Required To Inhibit 90% of the
Strains Tested13
MIC90 (µg/mL)
S. aureus S. epidermidis E. faecalis E. faecium
(n ) 31)
(n ) 10)
(n ) 10)
(n ) 10)
amoxicillin
10
11
>16
0.5
1
8-16
0.5
1
>16
0.03
0.25
>16
<0.016
0.06
a
Reagents: (a) H2N(CH2)3NH2/60 °C/48 h; (b) conc. HCl/reflux/
24 h; (c) 3,4-diCl-PhCHO/NaBH3CN/AcOH/MeOH; (d) propiolac-
tone/MeCN/reflux/3 h; (e) P2O5/polyphosphoric acid/100 °C/2 h; (f)
ICl/2 M HCl; (g) 7 or 8/NaBH3CN/AcOH/MeOH/reflux.
excellent antibacterial activity against these organisms.
Against the major respiratory tract pathogens, the
compounds showed good activity against some strains
of Streptococcus pneumoniae but no activity against the
Gram-negative organism Haemophilus influenzae.
cyclic ketone was prepared from a suitably substituted
aniline, for example 8 from 6.
En zym e In h ibition . Initial structure-activity analy-
sis of the MRS inhibition of close analogues of 1
indicated the quinolone moiety was important for MRS
inhibition but that there was significant potential for
variation at the left-hand side and linker parts of the
molecule. Substantially improved inhibition was ob-
tained by introducing a secondary amine in the linker
between the left-hand aryl ring and the quinolone
heterocycle; for example the dichlorobenzyl-amine com-
pound (9) had an IC50 of 16 nM against S. aureus MRS.
Compound 9 maintained competitive kinetics with
respect to methionine, with a Ki value of 5 nM.
Cyclization around the left-hand aryl ring led to the
tetrahydroquinolines 10 and 11. These compounds were
potent inhibitors of S. aureus MRS with IC50 values of
8 nM and 12 nM, respectively.9 The concentration of
enzyme in the assay is 3 nM, and as the IC50 values
are approaching the tight binding limit of the assay, the
true potency of the compounds is likely to be somewhat
greater than these figures suggest. To substantiate this,
the inhibitors were tested in a pyrophosphate exchange
assay using a large excess of substrates. This type of
exchange assay has successfully differentiated tight
binding inhibitors of other tRNA synthetases.11 In the
exchange assay, compounds 9, 10, and 11 had IC50
values of 500 nM, 1.0 nM, and 1.4 nM, respectively,
indicating that the tetrahydroquinolines are consider-
ably more potent than 9.
The inhibitors were all highly selective for the bacte-
rial MRS, showing no inhibition of rat liver MRS at
concentrations up to 1000 nM. Further details of the
structure-activity studies of the series will be published
elsewhere.
In vitr o a n tiba cter ia l a ctivity. Whole-cell antibac-
terial activity of compounds 9-11 was determined by
broth microdilution.12 In line with its increased enzyme
inhibition over 1, compound 9 shows significant anti-
bacterial activity against S. aureus and Enterococcus
faecalis (Table 1). The further enhancement in MRS
potency achieved by compounds 10 and 11 results in
Compounds 10 and 11 were tested against larger
collections of staphylococcal and enterococcal isolates
using the NCCLS recommended procedure for broth
microdilution.13 The organisms tested consisted of refer-
ence as well as clinical isolates. As shown in Table 2,
the MIC90 values against S. aureus, Staphylococcus
epidermidis, E. faecalis, and Enterococcus faecium were
all e1 µg/mL. Included in this evaluation were strains
resistant to many clinical antibiotics, including 18
MRSA, 9 mupirocin resistant S. aureus, 3 mupirocin
resistant S. epidermidis, and 2 vancomycin resistant S.
epidermidis isolates. The E. faecalis and E. faecium
collections each contained two VRE strains.
Time-kill viability studies were performed with com-
pound 10 at concentrations up to 100 µg/mL against S.
aureus Oxford and E. faecalis 1.14 Although growth was
inhibited, there was no significant reduction in bacterial
counts from the test inoculum (1 × 106 cfu/mL) for either
organism, up to 24 h. These results suggest that the
inhibitory effect of this compound is bacteriostatic,
consistent with what would be expected for a tRNA
synthetase inhibitor.
Mech a n ism of An tiba cter ia l Action . An expected
response to the treatment of S. aureus with a tRNA
synthetase inhibitor is an increase in the intracellular
pool of guanosine 3′-diphosphate, 5′-triphosphate (pp-
pGpp) due to the induction of the stringent response.15,16
Cultures of S. aureus were treated with varying con-
centrations of compound 9 for 30 min, and nucleotides
were extracted and then separated by HPLC.17,18 Com-
pound 9 at concentrations equal to MIC and 2 times
MIC led to an increase in intracellular pppGpp pools
between 9-fold and 10-fold relative to an untreated
control (Figure 1). Identical cultures treated with 4
times MIC of mupirocin exhibited an approximately 30-
fold increase in the intracellular pppGpp pool. Control
compounds chloramphenicol and ciprofloxacin failed to
elicit an increase in pppGpp pools leading to the

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 1961
F igu r e 1. Quantitation of intracellular pppGpp pools in S. aureus following 30 min treatment with compound 9 (MIC ) 4 µg/
mL) and mupirocin (MIC ) 0.125 µg/mL). Levels are presented as pmol/A260 unit of extract. Levels were determined from extracts
run in triplicate which varied by less than 5%.
Ta ble 3. In Vivo Efficacy of Compound 11 against S. aureus
conclusion that 9 causes an amino acid starvation likely
WCUH29 in a Groin Abscess Infection in Rats
due to inhibition of MRS activity.
dose
bacterial counts
(log10 cfu)
The mode of action of compound 9 was further
confirmed by analysis of resistant mutants. S. aureus
RN4220 mutants resistant to 9 were obtained by both
chemical mutagenesis and serial passaging in the
presence of drug. Nitrosoguanidine-derived mutants
exhibited >4-fold increase in MIC to 9 following two
passages in the presence of drug. Nucleotide sequence
analysis of the MRS gene of these mutants revealed a
change at nucleotide position 699, leading to an isoleu-
cine to phenylalanine amino acid alteration. Spontane-
ous mutants isolated following one or four passages in
the presence of drug both revealed a nucleotide alter-
ation distinct from the chemically induced mutants, a
G to A conversion at nucleotide 148, resulting in a
glycine to serine amino acid alteration. Both classes of
mutants were subsequently shown to be resistant to
compound 10. The mutant metS genes were cloned into
a S. aureus/Escherichia coli shuttle vector, and the
resulting plasmids conferred >4-fold resistance to 9
confirming that the resistance was due to the alterations
contained in the mutant alleles.
compound
(mg/kg)
nontreated controls
erythromycin
11
7.0 ( 0.6
3.7 ( 1.2a
3.9 ( 0.8a
50
21
a
Significant difference compared to nontreated controls, p <
0.01.
classes of antibiotics. Compound 11 was also efficacious
against a S. aureus rat abscess infection in vivo. These
compounds represent a mechanistically novel class of
potential antibiotics against Gram-positive organisms.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and characterization data for compounds shown in
Scheme 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
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in rats. Animals (specific pathogen free Sprague-
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inoculated with approximately 6.0 log10 cfu S. aureus
WCUH29 in 0.5 mL of semisolid agar via subcutaneous
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good antibacterial activity against staphylococci and
enterococci, including strains with resistance to known

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J M025502X