Small molecules with structural similarities to siderophores as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis

Article abstract of DOI:10.1016/j.bmcl.2008.03.025

Drugs inhibiting the iron scarcity-induced, siderophore-mediated iron-scavenging systems of Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) may provide new therapeutic lines of defense. Compounds with structural similarities to siderophores were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions, which mimic the iron scarcity these pathogens encounter and must adapt to in the host, and under standard iron-rich conditions for comparison. New antimicrobials were identified, some of which warrant exploration as initial leads against potentially novel targets and small-molecule tools to assist in the elucidation of targets specific to iron-scarcity adapted Mtb and Yp.

Full text of DOI:10.1016/j.bmcl.2008.03.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2662–2668
Small molecules with structural similarities to siderophores as
novel antimicrobials against Mycobacterium tuberculosis
and Yersinia pestis
Karen L. Stirrett,a,ꢀ Julian A. Ferreras,a,ꢀ Venkatesan Jayaprakash,b,* Barij N. Sinha,b
Tao Renc and Luis E. N. Quadria,*
aDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA
bDepartment of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, India
cNew England Regional Center of Excellence, Harvard Medical School, Boston, MA 02115, USA
Received 7 February 2008; revised 3 March 2008; accepted 6 March 2008
Available online 18 March 2008
Abstract—Drugs inhibiting the iron scarcity-induced, siderophore-mediated iron-scavenging systems of Mycobacterium tuberculosis
(Mtb) and Yersinia pestis (Yp) may provide new therapeutic lines of defense. Compounds with structural similarities to siderophores
were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions, which mimic the iron scarcity
these pathogens encounter and must adapt to in the host, and under standard iron-rich conditions for comparison. New antimicro-
bials were identified, some of which warrant exploration as initial leads against potentially novel targets and small-molecule tools to
assist in the elucidation of targets specific to iron-scarcity adapted Mtb and Yp.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (Mtb), the etiologic agent of
tuberculosis and Yersinia pestis (Yp), the causative agent
of plague and a potential agent of biowarfare and bioter-
rorism, are pathogens with serious impacts on global
public health. Multidrug-resistant (MDR) tuberculosis
is an emerging pandemic and the surfacing of extensive
drug-resistant (XDR) tuberculosis poses a new global
threat.1,2 Plague is a re-emerging disease and the occur-
rence of MDR Yp strains and self-transferable Yp plas-
mids conferring antibiotic resistance raises concerns
about future plague control.3,4 These scenarios under-
score the need for expanding the anti-tuberculosis and
anti-plague drug repertoires. Anti-infective drugs against
in vivo conditionally essential targets may offer novel ther-
apeutic possibilities, help the fight against MDR/XDR
strains and the prevention of their selection and dissemi-
nation, and increase biodefense preparedness.5
Anti-infective drugs inhibiting the siderophore-medi-
ated, iron-scavenging systems of Mtb and Yp may pro-
vide lines of defense against tuberculosis and plague,
respectively. The Mtb siderophores (mycobactins and
carboxymycobactins) and the Yp siderophore (yersin-
iabactin) (Fig. 1) have high affinity for Fe3+
Keywords: Siderophore; Antimicrobial; Drug target; Mycobacterium
tuberculosis; Yersinia pestis; Diaryl-carbothioamide-pyrazolines.
*
Corresponding authors. Tel.: +91 651 2275843 (V.J.); tel.: +1 212 746
4497 (L.E.N.Q.); e-mail addresses: venkatesanj@bitmesra.ac.in;
leq2001@med.cornell.edu
ꢀ
Figure 1. Structures of M. tuberculosis and Y. pestis siderophores.
These authors contributed equally to this work.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.025

K. L. Stirrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2662–2668
2663
(Kd < 10ꢀ25 M), their production is induced under iron
scarcity, and they are believed to be required for scav-
enging iron inside the host, where free iron is scarce
(10ꢀ25–10ꢀ15 M) and pathogens experience iron-limiting
conditions.6,7 The Mtb siderophore-deficient mutant is
impaired for growth in macrophages and iron-limiting
culture medium.8 The Mtb mutant lacking the IrtAB fer-
ri-siderophore uptake system is impaired for multiplica-
tion in macrophages, mouse lung, and iron-limiting
medium.9 Siderophore system-deficientYp strains are
avirulent in mice infected subcutaneously (a route imi-
tating the fleabite transmission of Yp) and unable to
multiply in iron-limiting medium.10,11 Siderophore sys-
tem-deficient mutants of enteropathogenic Yersinia
spp. are also attenuated in mice.12–14
We recently developed the first antibacterial targeting
siderophore biosynthesis, a salicyl-AMP biosynthetic
intermediate analog called salicyl-AMS (Fig. 2).15 Sali-
cyl-AMS is a potent inhibitor of salicylic acid adenyla-
tion domains involved in biosynthesis of salicylate-
derived siderophores, blocks production of Mtb and
Yp siderophores, and inhibits Mtb and Yp growth with
greater potency in iron-limiting media, where sidero-
phores are crucial for uptake of essential iron.15 More
recently, others have independently reported the activity
of salicyl-AMS.16 Continuing the line of our previous
work, we hypothesized that compounds with structural
features resembling Mtb and Yp siderophores may also
impair the siderophore system by, for example, inhibit-
ing biosynthetic enzymes or (ferri-)siderophore trans-
port systems, and halt bacterial growth under iron-
limiting conditions. To begin testing this hypothesis,
we synthesized a 32-member pilot library of 3,5-diaryl-
1-carbothioamide-pyrazoline derivatives (compounds
1–32, Fig. 3) with structural features resembling the
hydroxyphenyl-oxazoline/thiazoline containing half of
the siderophores and tested these compounds as Mtb
and Yp growth inhibitors in iron-limiting media, which
mimic the iron-scarcity condition that the pathogens
encounter in the host, and in standard iron-rich media
for comparison.17 We also assessed whether selected
compounds were bactericidal or bacteriostatic in iron-
limiting media.18 The ability of the compounds to inhi-
bit YbtE (the Yp salicylation enzyme that is the intended
target of salicyl-AMS15) in vitro was also examined.19
Lastly, cytotoxicity toward mammalian cells was evalu-
ated using a HeLa cell-based assay.20
Figure 3. Structures of compounds 1–32. In 6 and 7, 2-thiophenyl and
2-furyl groups replace, respectively, the R1–3-bearing phenyl group. In
32, H replaces the R5–6-bearing phenyl group.
pared by adding 60% aqueous solution of sodium
hydroxide or potassium hydroxide to the mixture of ke-
tone and aldehydes in methanol at 0 ꢁC and stirring the
reaction mixture for 4 h. Adjusting the pH of the reac-
tion mixture to 2 using 6 N hydrochloric acid precipi-
tated
hydroxy
chalcones.
20,40-Dihydroxy
acetophenone-derived chalcones required 2 days with
occasional stirring.22 Pyrazoline derivatives were ob-
tained by condensing 20-hydroxy chalcones with 80%
hydrazine hydrate in ethanol.23 Hydrazine hydrate was
used in excess and after 3 h of reflux, pyrazolines were
precipitated out upon cooling. 20,40-Dihydroxy chal-
cone-derived pyrazolines were extracted using chloro-
form from the concentrated reaction mixture. The final
products (1–31) were obtained by the reaction of pyraz-
oline derivatives with phenyl isothiocyanates.23 Most of
the thiocarboxamide derivatives precipitated out while
the reaction mixture was hot, few upon cooling, and
the rest upon concentration. Compound 32 was ob-
tained by the reaction of chalcone with thiosemicarba-
zide in alkaline medium.24 After 8 h of reflux, the
reaction mixture was diluted with cooled water and
acidified to precipitate it out. All the intermediates were
characterized by IR spectroscopic analysis and elemen-
tal analysis for CHNS. In the elemental analysis, the ob-
served values were within 0.4% of the calculated
values. Final compounds were characterized by 1H
NMR and FAB-MS.21
Compounds 1–32 were synthesized from 20-hydroxy
chalcone derivatives21 (Scheme 1). Hydroxy chalcones
were prepared through Claisen–Schmidt condensation.
20-Hydroxy acetophenone-derived chalcones were pre-
Figure 2. Reaction intermediate mimic 50-O-[N-(salicyl)-sulfamoyl]-
adenosine (salicyl-AMS).

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K. L. Stirrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2662–2668
Scheme 1. Synthesis of compounds 1–32. Conditions: (a) (i) R2, R3, R4 AC6H2ACHO, aq. NaOH (60%), stirring at rt 4–48 h, (ii) ice cold HCl (6N),
pH adjusted to 2; (b) NH2NH2ÆH2O (80%) excess, C2H5OH, reflux 3–6 h; (c) R5, R6 AC6H3ANCS, C2H5OH or CH3OH, reflux 15–30 min.; (d) (i)
NH2NHC(S)NH2, NaOH excess, CH3OH, reflux 8h, (ii) ice cold HCl (3N), pH adjusted between 2–4; (e) thiophene-2-carboxaldehyde or
furfuraldehyde followed by step (b) and (c).
Testing against Mtb revealed that 15 compounds had
IC50s and MICs (3–500 lM range; Table 1) within the
concentration series tested in the iron-limiting medium,
GAST-D. Nine of these compounds also had determin-
able IC50s and MICs (4–500 lM range) in the iron-rich
medium, GAST-D-Fe. Interestingly, 10, 12, 16, 25, 26,
and 32 were P3-fold more potent against Mtb cultured
under iron scarcity (Table 1). Compound 32, with 16-
fold higher potency in GAST-D, stood out in this group.
This compound, along with 13 and 16, was the only bac-
tericidal compound (>99% inoculum killing) among
those examined for mode of action against Mtb (10,
12, and 16, tested at 2· MICGAST-D; 13 and 32, tested
at 5· MICGAST-D). These bactericidal compounds had
CD50s against eukaryotic cells in the 21–398 lM range
and were among those with the lowest cytotoxicity in
the library, in which compounds had 10- to 3980-fold
lower cytotoxicity than the reference cytotoxic com-
pound cycloheximide25 (CD50 = 0.1 lM; Table 3). Com-
pound 13, with the highest activity against Mtb
(IC50 = 3–4 lM, MIC = 12–13 lM; Table 1) and the sec-
ond best selectivity index relative to Mtb (SIMtb = 14;
Table 3), and 16 were 420- and 210-fold less cytotoxic
than cycloheximide, respectively, but 5- and 9-fold more
cytotoxic, respectively, than the reference anti-tubercu-
losis drug rifampicin used in our assay. Rifampicin
had a CD50 of 190 lM (1900-fold lower cytotoxicity
than cycloheximide) and the same antimicrobial potency
Table 1. Antimicrobial activity against M. tuberculosis
a
b
Compound
IC50 (lM)
Ratio
MIC90 (lM)
Ratio
Mode of action
GAST-D-Fe
GAST-D
GAST-D-Fe
GAST-D
1–3, 5, 9, 14, 15, 17–20, 22, 31
4
P500
>250
500
72
P500
>250
250
73
nd
nd
2
P500
>250
>500
208
P500
>250
>500
250
83
nd
nd
nd
1
nd
nd
nd
nd
nd
BS
nd
BS
BC
BC
nd
nd
nd
nd
nd
nd
nd
nd
nd
BC
nd
6
7
1
8
65
29
28
2
100
1
10
11
12
13
16
21
23
24
25
26
27
28
29
30
32
RIF
167
42
6
>250
>500
>250
12
63
>4
>5
>4
1
28
27
2
104
63
208
4
8
3
29
1
13
63
83
96
3
417
7
101
167
80
1
>500
>250
250
417
>250
125
167
125
125
>250
125
125
21
>1
nd
2
>250
156
250
333
55
>2
2
63
84
4
>500
>500
125
>3
>4
1
4
51
1
167
105
42
125
83
1
>250
250
167
nd
2
1
24
2
1
125
nd
8
nd
16
nd
333
0.008
16
1
0.008
All values were rounded to the nearest non-fractional number. RIF, rifampicin; nd, not determined; BS, bacteriostatic; BC, bactericidal.
a IC50s were calculated from sigmoidal curves fitted to triplicate sets of dose–response data.
b MIC90s are means of triplicates.

K. L. Stirrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2662–2668
2665
in GAST-D and GAST-D-Fe (MIC = 8 nM). Notably,
32, with one of the strongest antitubercular activity (Ta-
ble 1), was 2-fold and 3980-fold less cytotoxic than the
rifampicin and cycloheximide references, respectively,
and had the best selectivity index relative to Mtb
(SIMtb = 50; Table 3).
25, along with 6 and 28, had the best selectivity indexes
relative to Yp (SIYp; Table 3). Twenty-two compounds
evaluated for mode of action against Yp were bacterio-
static when tested at up to the maximum multiple of
the MICPMH-D permitted by solubility, which ranged
from 1–38 · MICPMH-D (Table 2).
Most compounds were more active against Yp than
against Mtb, which has a thick, waxy cell envelope that
makes penetration of many drugs difficult. Twenty-five
compounds had considerable activity against Yp in the
iron-limiting medium, PMH-D (IC50s = 0.01–17 lM
range; Table 2). Notably, 17 compounds had both
IC50s and MICs that were at least >3-fold higher in
PMH–D–Fe (iron-rich medium) than in PMH-D (Table
2). In this group were 18, 20, and 23–26, each of which
had >30-fold more potent IC50s and MICs against Yp
cultured under iron scarcity. These compounds were
130- to 630-fold less cytotoxic than cycloheximide, but
have over 250- to 1250-fold greater cytotoxicity than
the reference anti-plague drug streptomycin. Streptomy-
cin had a CD50 >500 lM (>5000-fold less cytotoxic than
cycloheximide) and the same antimicrobial activity in
PMH-D and PMH-D–Fe (MIC = 0.2 lM). Compound
Compounds 1–32 were not specifically designed to inhi-
bit a particular enzyme in the siderophore biosynthetic
pathways. However, the compounds were tested as
inhibitors of YbtE, which is the intended target of sali-
cyl-AMS (Fig. 2).15 None of the compounds was as po-
tent as the bona fide inhibitor salicyl-AMS (Table 4).
The compounds had IC50s in the 0.2- to >12.5-lM range
and were 29- to >1786-fold less potent than salicyl-AMS
(IC50s = 0.007 lM). Moreover, no clear structural–
activity relationships emerged from these data. Interest-
ingly, however, the three library compounds (29–31)
lacking the hydroxyl ortho to the 5-membered ring as
seen in the siderophores were among the four com-
pounds with drastically increased IC50s (>12.5 lM; Ta-
ble 4) compared with salicyl-AMS. No meaningful
correlation trend between the IC50s in the YbtE assay
and the IC50s in the Yp growth assay was observed.
Table 2. Antimicrobial activity against Y. pestis
a
a
Compound
IC50 (lM)
Ratio
MIC90 (lM)
Ratio
Mode of action
PMH-D-Fe
PMH-D
PMH-D-Fe
PMH-D
1
2
>9
>5
>9
>5
0.5
0.4
0.7
0.01
1
nd
nd
>9
>5
>9
>5
1
nd
nd
nd
nd
BS
nd
nd
nd
BS
BS
BS
BS
BS
BS
nd
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
nd
nd
nd
nd
nd
3
>5
>5
>10
>13
>27
>900
>9
>5
>5
>5
nd
4
>5
>19
>9
2
5
>19
>9
>19
>9
nd
nd
6
7
>9
>9
>5
8
>19
>5
0.7
2
>27
>3
>19
>5
2
>10
>1
9
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
STR
>2
1
>2
>2
3
>0.7
>4
>19
>9
2
>10
>11
nd
>19
>9
5
0.8
>19
2
2
>5
nd
>19
>19
>19
>19
>9
>19
>19
>19
>19
>9
>19
6
>10
>27
>6
>3
0.7
3
2
>10
>4
5
0.9
2
>10
>75
>9
3
>3
>150
>9
>150
>9
5
>30
>2
1
6
>150
>19
>19
>150
>150
>150
>150
>38
>9
0.6
3
>250
>6
>150
>19
>19
>150
>150
>150
>150
>38
>9
2
>75
>2
9
1
>19
>150
>214
>375
>375
>2
2
>10
>75
>50
>30
>75
nd
1
2
0.7
0.4
0.6
17
0.2
>75
>19
>38
>38
nd
3
5
2
>38
1
>45
nd
>9
nd
>75
>19
>38
>38
nd
>75
>19
>38
>38
0.2
>75
>19
>38
>38
0.2
nd
nd
nd
nd
nd
nd
nd
1
All values <1 and values >1 were rounded to one significant digit and to the nearest non-fractional number, respectively.
STR, streptomycin; nd, not determined; BS, bacteriostatic.
a IC50s were calculated from sigmoidal curves fitted to triplicate sets of dose–response data.

2666
K. L. Stirrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2662–2668
Table 3. Cytotoxicity and selectivity assessment
Table 4. Inhibition of YbtE by compounds 1–32
a
Compound
CD50
(lM)
SIMtb
(CD50/IC50GAST-D
SIYp
(CD50/IC50PMH-D)
Compound
IC50 (lM)
IC50 1–32 / IC50 SAMS
)
1
0.5
2
71
1
2
3
<0.006
<0.01
<0.01
<0.02
<0.01
0.01
0.04
0.1
<0.3
<1
10
15
7
2
286
100
429
143
286
43
5
3
0.7
3
3
5
4
4
6
5
1
5
5
6
2
6
3
300
3
7
0.3
0.6
0.8
0.2
1
7
3
8
86
8
3
4
9
114
29
9
15
1
<0.03
0.04
0.6
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
SAMS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
RIF
STR
CHX
1
143
71
17
11
42
1
9
0.5
0.4
0.3
7
0.4
14
14
<2
0.5
6
57
43
0.002
<0.008
0.7
1000
100
57
4
0.7
0.4
0.2
0.8
0.5
1
21
3
7
0.007
<0.03
0.02
<0.05
0.1
3
29
13
11
25
15
16
63
23
62
30
26
31
248
19
29
398
190
>500
0.1
7
114
71
11
42
5
143
71
0.5
1
<0.03
0.4
0.3
16
63
33
155
50
2
143
86
0.6
>12.5
0.7
0.2
1
1786
100
29
1.0
0.4
0.5
0.2
143
1786
1786
1786
71
155
<3
<1
<0.8
<10
nd
nd
nd
>12.5
>12.5
>12.5
0.5
0.007
3
0.8
0.06
50
1
nd
nd
All values <1 and values >1 were rounded to one significant digit and
to the nearest non-fractional number, respectively. The maximum
compound concentration tested in the assay was 12.5 lM. SAMS,
salicyl-AMS.
nd
SIMtb and SIYp, selectivity index relative to activity against M. tuber-
culosis and Y. pestis, respectively. CD50 and SI values <1 and values >1
were rounded to one significant digit and to the nearest non-fractional
number, respectively. RIF, rifampicin; STR, streptomycin; CHX,
cycloheximide; nd, not determined.
targets.5 Some of these antimicrobials may impair sider-
ophore system functioning as discussed above, a prop-
erty that would result in bacteriostatic activity
conditional to environmental iron scarcity (e.g., as seen
with 25 against Yp). Others may inhibit functions condi-
tionally essential to the iron scarcity-associated physiol-
ogy, which is expected to be adopted by the pathogens in
the iron-limiting environments of the host, and could
have bactericidal activity (e.g., as seen with 32 against
Mtb). Compounds with antimicrobial activity that was
independent from the iron content of the media were
also identified (e.g., 13 against Mtb). These antimicrobi-
als may target essential bacterial functions required
under both low and high iron conditions.
a CD50s were calculated from sigmoidal curves fitted to triplicate sets of
dose–response data.
Considering that the compounds active against Yp had
IC50s in the high nanomolar–low micromolar range in
the cellular assay under the iron-scarcity condition (Ta-
ble 2) and that the activity of these compounds is likely
to be drastically reduced in the cellular assay (due to fac-
tors such as penetration/efflux, intracellular stability,
and off-target binding) compared with the enzymatic as-
say, it is unlikely that YbtE inhibition plays a major role
in the antimicrobial activity of these compounds.
In sum, 30 compounds of our pilot library had detect-
able antimicrobial activity. To our knowledge, these
are novel scaffolds not previously shown to have this
property. In line with our aforementioned hypothesis,
several compounds had higher potency under iron-limit-
ing conditions. Under this condition bacteria depend on
siderophores for iron scavenging and engage an adap-
tive response to tailor their physiology to iron scarcity,
thus exposing novel potential in vivo conditional
This study provides proof-of-principle for the effective-
ness of screening compound libraries in iron-limiting
conditions to identify antimicrobials that may selectively
target iron scarcity-adapted bacteria. Our screening ap-
proach allowed us to identify compounds with antimi-
crobial activity that is conditional to iron scarcity and
would not have been revealed in conventional screens,
which are performed under iron-rich conditions. Some
of the identified antimicrobials warrant exploration as

K. L. Stirrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2662–2668
2667
15. Ferreras, J. A.; Ryu, J. S.; Di Lello, F.; Tan, D. S.; Quadri,
L. E. Nat. Chem. Biol. 2005, 1, 29.
initial leads against potential in vivo conditionally essen-
tial targets and small-molecule tools to assist in the elu-
cidation of targets and pathways critical to iron-scarcity
adaptation in Mtb and Yp. Studies are underway to elu-
cidate the molecular mechanisms of action of selected li-
brary compounds, a process that may lead to the
discovery of novel mechanisms of antimicrobial activity
and drug target candidates.
16. (a) Miethke, M.; Bisseret, P.; Beckering, C. L.; Vignard,
D.; Eustache, J.; Marahiel, M. A. FEBS J. 2006, 273, 409;
(b) Somu, R. V.; Boshoff, H.; Qiao, C.; Bennett, E. M.;
Barry, C. E., III; Aldrich, C. C. J. Med. Chem. 2006, 49,
31; (c) Vannada, J.; Bennett, E. M.; Wilson, D. J.; Boshoff,
H. I.; Barry, C. E., III; Aldrich, C. C. Org. Lett. 2006, 8,
4707.
17. Antimicrobial activity was tested in dose–response exper-
iments using standard, 96-well plate-based, twofold-mic-
rodilution assays as reported previously.15 Mtb (strain
H37Rv)26 was grown in iron-limiting GAST-D medium
and GAST-D supplemented with 100 lM FeCl3(GAST-
D-Fe).15 Yp (strain KIM6-2082.1+)27 was grown in iron-
limiting PMH-D medium and PMH-D with 100 lM
FeCl3(PMH-D-Fe).15 This Yp strain is avirulent due to
the lack of the Lcr virulence-conferring plasmid and
excluded from the Select Agent Program (http://
www.cdc.gov/od/sap/sap/exclusion.htm). Cultures were
started with ꢁ5 · 104 colony-forming units (CFU)/mL.
Growth was assessed as optical density after incubation at
37 ꢁC (Mtb: 15 days, stationary condition; Yp: 26 h,
200 rpm) using a Spectra Max Plus plate reader (Molec-
ular Dynamics). Compounds were evaluated at up to the
highest concentration permitted by their solubility, with a
500 lM upper testing limit, and added as DMSO solu-
tions. DMSO was kept at 0.5% in treated and DMSO-
control cultures. IC50s were calculated from sigmoidal
curves fitted to triplicate dose–response data using Kale-
idaGraph (Synergy Software). MIC90s were calculated as
the lowest concentration tested that inhibited growth by
P90% relative to DMSO controls.
Acknowledgments
This work was supported in part by NIH Grants
AI063384 and AI056293-01, and the New England
Regional Center of Excellence in Biodefense and
Emerging Infectious Disease. The Department of
Microbiology and Immunology acknowledges support
from the W.R. Hearst Foundation. L.Q. is a Niar-
chos Scholar. We are grateful to the Sophisticated
Analytical Instrument Facility (CDRI, Lucknow, In-
dia) for providing spectral data, to Dr. D.S. Tan’s
laboratory (Memorial Sloan-Kettering Cancer Center)
for providing salicyl-AMS for this study, and to R.
Moy, G. Sadhanandan and Dr. W. He (Cornell)
for assistance with antimicrobial testing and helpful
discussions.
Supplementary data
18. After cultures were treated with compounds and incubated
as above, the mode of action was evaluated by enumer-
ating CFU/mL after plating serial dilutions of triplicate
Yp and Mtb cultures on TBA plates (Difco) and Middle-
brook 7H11 supplemented as reported,28 respectively.
Plates were incubated at 37 ꢁC for 4 weeks for Mtb and at
30 ꢁC for 3 days forYp before colony counting.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.03.025.
References and notes
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PLUS plates (PerkinElmer) as reported15 and described in
the Supplementary material. Briefly, YbtE-catalyzed
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bound aryl carrier protein domain of Yp protein HMWP2
in the absence or presence of test compounds at a range of
concentrations was quantified using a plate counter and
IC50s were derived from the dose–response data.
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