Discovery and optimization of antibacterial AccC inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.057

The biotin carboxylase (AccC) is part of the multi-component bacterial acetyl coenzyme-A carboxylase (ACCase) and is essential for pathogen survival. We describe herein the affinity optimization of an initial hit to give 2-(2-chlorobenzylamino)-1-(cyclohe

Full text of DOI:10.1016/j.bmcl.2009.10.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6507–6514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of antibacterial AccC inhibitors
a
a,
Cliff C. Cheng a, , Gerald W. Shipps Jr. , Zhiwei Yang , Binyuan Sun a, Noriyuki Kawahata a,à, Kyle A. Soucy a,
*
Aileen Soriano b, Peter Orth b, Li Xiao b, Paul Mann b, Todd Black b
a Schering-Plough Research Institute, 320 Bent Street, Cambridge, MA 02141, United States
b Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The biotin carboxylase (AccC) is part of the multi-component bacterial acetyl coenzyme-A carboxylase
(ACCase) and is essential for pathogen survival. We describe herein the affinity optimization of an initial
hit to give 2-(2-chlorobenzylamino)-1-(cyclohexylmethyl)-1H-benzo[d]imidazole-5-carboxamide (1),
which was identified using our proprietary Automated Ligand Identification System (ALIS).1 The X-ray
co-crystal structure of 1 was solved and revealed several key interactions and opportunities for further
optimization in the ATP site of AccC. Structure Based Drug Design (SBDD) and parallel synthetic
approaches resulted in a novel series of AccC inhibitors, exemplified by (R)-2-(2-chlorobenzylamino)-
Received 20 August 2009
Revised 12 October 2009
Accepted 13 October 2009
Keywords:
Biotin carboxylase
AccC
Acetyl coenzyme-A carboxylase
ACCase
1-(2,3-dihydro-1H-inden-1-yl)-1H-imidazo[4,5-b]pyridine-5-carboxamide (40). This compound is
a
potent and selective inhibitor of bacterial AccC with an IC50 of 20 nM and a MIC of 0.8 lg/mL against a
sensitized strain of Escherichia coli (HS294 E. coli).
Ó 2009 Elsevier Ltd. All rights reserved.
BCCP
Biotin carboxyl carrier protein
Structure Based Drug Design (SBDD)
ALIS: Automated Ligand Identification
System
AS-MS: Affinity-Selection Mass
Spectrometry
HS294 E. coli
MIC
The emergence of antibiotic resistance continues to be a major
health problem.2 The development of broad spectrum antibiotics
directed against novel targets is an important strategy to meet
the urgent need for new antimicrobial agents. In this regard, the
enzyme components of bacterial acetyl coenzyme-A carboxylase
(ACCase) are excellent targets for antimicrobial drug discovery.3
ACCase catalyzes the first committed step in fatty acid biosynthesis
and is essential for bacterial viability.4,5 It is a multi-component
system comprised of two enzymes (AccC and AccAD) and the bio-
tin carrier protein (BCCP or AccB).6 The ACCase-catalyzed reaction
can be divided into two half-reactions (Fig. 1). The first half-reac-
tion, the ATP-dependent carboxylation of biotin, is catalyzed by
AccC (biotin carboxylase) and uses bicarbonate as the carbon diox-
ide source. In the second half-reaction, catalyzed by AccA/D (carb-
oxyltransferase), the carboxy group is transferred from biotin to
acetyl-CoA to form the final product, malonyl-CoA. The biotin
cofactor is attached to a lysine residue on BCCP and cycles between
the two half-reactions.
At the amino acid level the ACCase components are highly con-
served across all bacterial genera making ACCase an ideal broad
spectrum target. The pyrrolidine dione antibiotics, for example,
have broad spectrum antibacterial activity due to inhibition of
AccA/D.7 Although ACCase activity is also found in eukaryotic cells,
the three functionalities required for catalysis are located in a sin-
gle protein (Type I ACCase) which is distinct from the individual
proteins employed by bacteria (Type II ACCase). These differences
in active site organization suggest the potential to find specific
inhibitors of the bacterial ACCase.
Using a proprietary AS-MS platform (the Automated ligand
Identification System, ALIS) to screen a soluble version of the AccC
protein we identified a series of benzimidazoles as AccC ligands
from a mixture-based combinatorial library8 and these preliminary
hits were subsequently confirmed as low micromolar enzyme
inhibitors.11 Here, we describe the rapid hit-to-lead optimization
of this series using mixture-based optimization and computer-
aided drug design.
* Corresponding author.
E-mail address: cccheng@alum.mit.edu (C.C. Cheng).
Present address: Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008,
United States.
One of the initial hits, 2-(2-chlorobenzylamino)-1-(cyclo-
hexylmethyl)-1H-benzo[d]imidazole-5-carboxamide (1) (Fig. 2)
à
Present address: Aileron Therapeutics, Inc., 840 Memorial Dr., Cambridge, MA
02139, United States.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.057

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C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
-
Pi
ATP + HCO3
ADP +
Biotin Carboxylase (AccC)
O
S
Fatty Acid
Synthesis
O
S
HN
NH
-CO2
N-
HN
Cell Wall
Biosynthesis
BCCP
BCCP
Biotin Carboxyl Carrier Protein
(BCCP)
Carboxyltransferase (AccA/D)
Malonyl-CoA
Acetyl-CoA
Figure 1. The conversion of acetyl-CoA to malonyl-CoA takes place in two steps. Biotin carboxylase catalyzes the first reaction by transferring CO2 in an ATP-dependent
fashion from bicarbonate to the biotin moiety of BCCP (or AccB), resulting in carboxybiotinoyl-BCCP. The CO2 group is subsequently transferred from the carboxybiotin
moiety to acetyl-CoA by the carboxyltransferase subunits, resulting in malonyl-CoA formation.
screening nor did it show activity in the biochemical assay. The
NH of o-chlorobenzylamine was within H-bond distance from
Glu288 and the polarized o-Cl was well positioned towards
Tyr199. It was also observed that the o-chlorophenyl of the eastern
region fit well inside the protein hydrophobic pocket. However,
initial X-ray crystallography revealed that there might be nonpro-
ductive interactions between the cyclohexyl group and the protein
surface at the northern region; additionally, two polar residues,
His209 and Glu276, are located in this region. Based on the X-ray
crystal structure of 1, we initiated a hit optimization effort using
structure-based drug design (SBDD).
Figure 2. Initial benzimidazole carboxamide-based AccC ATP-site inhibitors.
The X-ray crystal structure and the initial SAR suggested that
the interactions of the primary amide with protein residues were
critical; focusing our chemistry efforts on the northern part of
was amendable to X-ray co-crystallography and its structure with
the molecule to incorporate hydrophobic and polar substituents.
the AccC protein was rapidly solved. Figure 3 illustrates the key
To access the northern part of the molecule quickly and efficiently,
compound–protein interactions.
our strategy was to leverage the established chemistry from the
Several hydrogen bonding interactions with AccC protein resi-
parent combinatorial library to create focused, SAR informing sub-
libraries9 and then screen these sets using the ALIS platform.1
dues were noticed, particularly the primary amide of 1 H-bonded
with Glu201 and Lys202 and this observation explained some of
Guided by the X-ray derived structural information approximately
the preliminary negative SAR. For example, the methylated com-
300 mass-encoded, mixture-based compounds were synthesized
on solid-phase using split/pool techniques. The syntheses of the
mixture libraries are outlined in Scheme 1. 4-Fluoro-3-nitrobenzoic
pound 2 (Fig. 2) was significantly less potent than initial hit and
the carboxylic acid analog 3 (Fig. 2) was not detected during ALIS
His209
Glu276
O
H
N
O
N
O
hydrophobic
Glu288
O
H
N
N
N
O
hydrophobic
N
H
H
Cl
H2
N
O
HN
O
H
H
O
O
Lys202
Lys159
Glu201
ADA motif
Figure 3. X-ray crystal structure of 1 bound to AccC ATP-site. PDB code 3JZF.
Tyr199

C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
6509
Scheme 1. Optimization of the northern part of the series using solid-phase synthesis. Reagents and condition: (a) (COCl)2, DMF (cat.) then Rink resin, DCM, DIEA, 4 °C; (b)
R1NH2, DIEA; (c) SnCl2; (d) DIC, 2-chlorobenzyl isothiocyanate; (e) TFA, H2O.
Scheme 2. Synthesis of the eastern analogs. Reagents and condition: (a) (COCl)2, DMF (cat.) then Rink resin, DCM, DIEA, 4 °C; (b) cyclohexylmethanamine, DIEA; (c) SnCl2; (d)
DIC, R2NCS; (e) TFA, H2O.
acid was converted into acid chloride followed by amide forma-
tion with Rink resin. Amine building blocks (R1NH2) were reacted
in the subsequent SNAr reaction to install the diversification point.
The nitro group was reduced to the amine (7) by SnCl2 and then
reacted with 2-chlorobenzyl isothiocyanate to give the benzimi-
dazoles. The mixture-based combinatorial libraries were cleaved
from the resin by TFA, and the reaction outcomes verified by
LC–MS, revealing that a minimum 85% of desired products were
observed in amounts sufficient for potential detection as ligands
to AccC.
pocket. Compounds (9 to 14) with larger hydrophobic R1 have
shown better hydrophobic interactions and the aromatic group of
compounds (9 to 14) might make additional interactions with
the side chain of adjacent His209. It was suggested that the protein
residues might be flexible at this region. Introducing H-bond donor
hydroxyl group in compounds (15 to 18) provided good improve-
ment in potency due to the polar interactions with protein residues
of the northern binding pocket. Moreover, the second X-ray crystal
structure of compound (16) was also resolved (data not shown)
and provided clearer optimization guidance.
ALIS screening revealed 16 compounds to be of potential inter-
est and these compounds were synthesized separately, purified to
>90% by preparative LC–MS and confirmed in the enzyme inhibi-
tion assay. The results are summarized in Table 1.
Informed by compound 1 and the new ligands we continued the
optimization process by investigating the northern part (R1) of
benzimidazole (Table 1). From the early X-ray crystal structure of
1 it was observed that the cyclohexyl occupied the hydrophobic
We further explored the possible opportunity to improve po-
tency by designing compounds to enhance the interaction with
Glu288 and Tyr199 residues at the eastern region. The facile, par-
allel synthesis chemistry is outlined in Scheme 2 and the results
summarized in Table 2.
Modifications of the eastern part of the molecules resulted in
significant loss of activity as we observed in the crystal structure.
It was concluded that o-chlorobenzyl group is an optimal component

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C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
Table 1
Table 2
Optimization of the northern part of benzimidazole
Eastern part of benzimidazole modifications
R1
N
NH
H2N
N
N
N
NH
R2
O
H2N
CI
Structure of R1
ACCase, IC50
(lM)
a,b
O
Compound
Compound
Structure of R2
ACCase, IC50
15.8
(lM)
a,b
1
5
19
9
0.7
20
21
16.9
143
F
Br
10
14.7
11
12
1.3
9.5
22
23
144
28
NH
F3C
13
14
15
8.9
2.1
0.4
24
25
139
241
O
Cl
OH
OH
Cl
16
0.5
HO
26
27
40
63
17
18
0.7
2
OH
OH
Cl
Cl
a
Values are means of duplicate experiments on two separate weightings.
[ATP] = 100 lM.
b
28
29
171
of this series to fit into the eastern pocket. Therefore, it is not
surprising that analogs (19 to 29) are less active than compound
(1) (Table 2).
41
a
Values are means of duplicate experiments on two separate weightings.
[ATP] = 100 lM.
b
Computer modeling results suggested the 4-position C–H of
benzimidazole carboxamide could be replaced with a nitrogen
atom (Fig. 4). The repulsive interaction of C–H at that position
would be turned into an H-bond to interact with charged
Lys159. Therefore compound (35) was considered to validate
this idea and the synthesis is outlined in Scheme 3. We started
from intermediate (30) which was subsequently methylated
with TMS-diazomethane to give phenol ether (31). Oxidation
of 31 with selenium dioxide gave a carboxylic acid followed
by methylation to afford methyl ester (32). The SNAr reaction
to displace the methoxy group of 32 with a primary amine
(Table 3) followed by reduction gave compound (34), which
was treated with isothiocyanates to give the cyclized aza-benz-
compared to compounds (9), (17) and (18). The results are
listed in Table 3.
From X-ray crystallography and modeling studies (Fig. 3), we
observed that it was possible to access the empty pocket of the
northern region to gain potency by modification of the 7-position
benzimidazole carboxamide. Designed compounds (65 to 79 in Ta-
ble 4) were synthesized according to Scheme 4. Chloro-dinitroben-
zoic acid (50) was the general starting material for the final
compounds (65 to 75). Methylation of 50 followed by SNAr dis-
placement with primary amines provided 52. The key transforma-
tion was the regioselective hydrogenation of 52 with 10% Pd/C
to give 53 after column chromatography. After benzimidazole
imidazole. The methyl ester was converted into
a primary
amide by aminolysis to give compound (35). As suggested by
computer modeling, compounds (36), (48) and (49) with a N
at the 4-position gained 10–20-fold in biochemical activity

C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
6511
formation the remaining nitro group of 54 was reduced to NH2 by
Zn powder in the presence of NH4Cl to give 55. A Sandmeyer
reaction of 55, followed by acid hydrolysis and amidation gave
compounds 65 and 71. Sulfones analogs (73 to 75) were synthesized
by diazotization of 55.
Representative compounds in the optimized chemical series
were tested for cell-based activity using a proprietary sensitive
strain of Escherichia coli (E. coli HS294) in which the major drug ef-
flux pumps have been deleted and cell permeability is enhanced by
a reduction in LPS synthesis (Table 5). Improvement in cell-based
activity were shown by these compounds relative to the initial
To synthesize sulfonamide analogs of this series at R7 we
started from 4-chloro-3-nitro-5-sulfamoylbenzoic acid (62), which
was converted to 63 using a standard protocol (Scheme 4). A Mits-
unobu reaction was employed for further substitutions of the sul-
fonamides followed by acid hydrolysis and amidation to achieve
compounds 76 to 79.
hit 1 (MICꢀ25
lg/mL), in line with improvements in enzyme inhi-
bition potency.
The antibacterial mechanism-of-action of these compounds
was tested against E. coli HS294 strain specifically sensitized
for AccC inhibitors. The targeted sensitization was achieved by
lowering the pool of functional ACCase complex in the cell via
overproduction of apo-BCCP. Overproduction of apo-BCCP down-
regulates transcription of the accB/accC operon and may also
form an inactive complex between AccC protein and apo-BCCP.12
Therefore, decrease in the MIC for the strain overproducing
apo-BCCP (E. coli HS294/accB) relative to E. coli HS294 control
strain indicates inhibition of AccC as the mode for antibacterial
activity. As shown in Table 6, compounds (40), (69) and (73)
gave a 4- to greater than 12-fold decrease in MIC upon overpro-
duction of apo-BCCP, indicating on-target antibacterial activity
while there is no change in the MIC for the antibiotic trimetho-
prim which does not inhibit ACCase. Furthermore, selective
inhibition of fatty acid biosynthesis in E. coli HS294 by these
Compounds (65 to 79) were tested in the enzyme inhibition as-
say and the results are summarized in Table 4. Electron withdraw-
ing groups at the R7 position of 59 have significant effects on
potency. Bulky R7 groups of 59 may also provide rigidity and con-
sequently limit the free rotation of R1 substituents, as illustrated in
Figure 5. The rigidity stabilizes the hydrogen bond network be-
tween R1 hydroxyl group and the organized water, which also
hydrogen-bonds to Glu276 and Glu288 side chains. The interac-
tions of the primary amide of benzimidazole carboxamide with
Glu201 and Lys202 were enhanced due to electron withdrawing
groups at R7 lowering electron density of the benzimidazole ring.
The coordinates have been deposited in the Protein Data Bank10
with code 3JZI.
Figure 4. (A) Computer-aided modeling predicted possible binding interactions between aza-benzimidazole carboxamide and charged Lys159. (B) Structure of 36 bound to
the AccC active site by molecular docking.
Scheme 3. Synthesis of compound 35. Reagents and conditions: (a) TMS–CHN2, MeOH; (b) SeO2, dioxane, 90 °C overnight; (c) TMS–CHN2, MeOH; (d) R1NH2, i-Pr2NEt, t-
BuOH, 160 °C, 15 min; (e) H2/Pd-C, EtOAc; (f) DIC, 2-chlorobenzyl isothiocyanate; (g) NH3, MeOH, 50 °C, overnight.

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C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
Table 3
Table 4
Aza-benzimidazole carboxamide analogs
In vitro data for compounds 65 to 79
R1
R7
R1
N
N
NH
NH
H2N
N
H2N
N
N
O
O
CI
CI
Compound
Structure of R1
ACCase, IC50
0.16
(lM)
a,b
Compound Structure of R1 Structure of R7
ACCase, IC50
M)
a,b
(
l
36
Cl
65
1.6
37
0.13
NO2
66
0.09
38
39
2.06
0.17
CH3
CH3
NO2
NH2
NO2
67
68
3.5
50
69
70
71
72
73
0.01
5
40
41
42
0.02
2.84
0.3
HO
NH2
Br
O
HO
HO
HO
HO
HO
0.13
0.17
0.26
O
O
SCH3
O
S
HO
O
43
44
45
3.01
2.75
40
O
S
O
O
74
75
76
77
0.06
0.1
O
F
F
F
O
S
O
HO
HO
HO
HO
O
O
O
S
NH2
0.7
46
47
0.35
0.27
O
O
S
NH
NH
0.38
OH
O
48
49
0.02
0.19
OH
OH
O
S
78
79
0.8
2.1
O
a
Values are means of duplicate experiments on two separate weightings.
[ATP] = 100 lM.
b
O
S
NH
HO
Cl
O
compounds supports inhibition of ACCase activity as the mecha-
nism-of-action as shown for 69 in Table 7. The optimized com-
pounds were also shown to have broad spectrum enzyme
inhibition activity and are selective for bacterial AccC. No activity
was found against eukaryotic carboxylases and human kinases
(data not shown).
a
Values are means of duplicate experiments on two separate weightings.
[ATP] = 100 lM.
b
Combining X-ray crystallography with computer-aided modeling
The AccC ATP-competitive inhibitors we report herein represent
a class with a novel mode of action and broad spectrum enzyme
inhibition activity. We utilized ALIS and mixture-based optimiza-
tion during the early hit optimization stage to provide informative
affinity SAR which translated well into the biochemical assay.
achieved a ꢀ360-fold improvement in enzyme inhibition and
>100-fold improvement in cell-based activity (0.2 lg/mL MIC;
HS294 E. coli strain). Additionally, the aza-benzimidazole carbox-
amide scaffold was discovered, providing a new chemotype for fur-
ther discovery efforts.

C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
NO2
6513
NO2
NO2
NO2
NR1H
NH2
Cl
Cl
NR1H
b
c
a
d
O
O
HO
O
NO2
NO2
NO2
O
e
O
O
O
51
53
NH2
50
NO2
52
R1
N
R1
NH2
R1
N
N
NH
NH
g, h
O
O
H2N
NH
N
N
O
N
O
Cl
Cl
O
55
56
54
Cl
i
g, h
f
R1
N
SR'
R7
R1
N
R1
N
NO2
NH
NH
O
NH
O
N
N
H2N
N
O
O
Cl
O
60
58
Cl
Cl
57
g, h
R7
j
R1
N
R'
O S O
g, h
R1
N
NH
H2N
N
NH
O
N
O
59
Cl
g, h
O
Cl
NR3R4
61
NH2
NH2
O S O
R1
R1
O S O
O S O
Cl
k
a-c
N
N
NH
NH
MeO
MeO
HO
N
N
NO2
O
O
O
Cl
Cl
64
63
62
Scheme 4. Synthesis of compound 65 to 79. Reagents and conditions: (a) TMS–CHN2, MeOH; (b) R1NH2, i-Pr2NEt, DMF, 90 °C, overnight; (c) H2, Pd/C; (d) DIC, 2-chlorobenzyl
isothiocyanate; (e) Zn powder, NH4Cl; (f) CuBr or CuCl, t-BuONO, CH3CN; (g) HCl, H2O–dioxane, 90 °C overnight; (h) (NH4)2CO3, i-Pr2NEt, EDC, HOBt, DMF; (i) NaNO2, HCl,
NaSR0; (j) oxone; (k) R3R4N, DEAD, PPh3, MeOH.
Figure 5. Depiction of the X-ray crystal structure of 70 bound to AccC. Water serves as an H-bond bridge between the inhibitor and two amino acid residues (Glu276,
Glu288). PDB code 3JZI.

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C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6507–6514
Table 5
Supplementary data
Cell-based activity of compounds optimized for enzyme inhibition potency11
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.057.
Compound
ACCase IC50
(
l
M)
E. coli HS294, MIC (lg/mL)
69
40
48
74
71
73
0.01
0.02
0.05
0.06
0.13
0.26
0.2
0.8
1.6
1.6
0.4
References and notes
12.5
1. (a) Annis, D. A.; Athanasopoulos, J.; Curran, P. J.; Felsch, J. S.; Kalghatgi, K.; Lee,
W. H.; Nash, H. M.; Orminati, J.-P.; Rosner, K. E.; Shipps, G. W., Jr. Int. J. Mass
Spectrom. 2004, 238, 77; (b) Annis, D. A.; Nickbarg, E. B.; Yang, X.; Ziebell, M. R.;
Whitehurst, C. E. Curr. Opin. Chem. Biol. 2007, 11, 518.
2. (a) Gould, I. M. Int. J. Antimicrob. Agents 2008, 32S, S2; (b) Chastre, J. Clin.
Microbiol. Infect. 2008, 14, 3.
Table 6
Mechanism-of-action analysis using E. coli HS294 sensitized for AccC inhibitors11
3. (a) Miller, J. R.; Dunham, S.; Mochalkin, I.; Banotai, C.; Bowman, M.; Buist, S.;
Dunkle, B.; Hanna, D.; Harwood, J.; Huband, M. D.; Karnovsky, A.; Kuhn, M.;
Limberakis, C.; Liu, J. Y.; Mehrens, S.; Mueller, W. T.; Narasimhan, L.; Ogden, A.;
Ohren, J.; Vara Prasad, J. V. N.; Shelly, J. A.; Skerlos, L.; Sulavik, M.; Thomas, V.
H.; VanderRoest, S.; Wang, L. A.; Wang, Z.; Whitton, A.; Zhu, T.; Stover, C. K.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1737; (b) Mochalkin, I.; Miller, J. R.;
Narasimhan, L.; Thanabal, V.; Erdman, P.; Cox, P. B.; Prasad, J. V. N. V.; Lightle,
S.; Huband, M. D. Chem. Biol. 2009, 4, 473.
Compound
ACCase
E. coli HS294,
E. coli HS294/accB (AccC
sensitized strain), MIC (lg/mL)
IC50
(lM) MIC (
l
g/mL)
69
40
73
0.01
0.02
0.26
0.8
0.8
12.5
0.1
<0.05
<0.05
3
Trimethoprim Not
active
0.1
4. Harder, M. E.; Beacham, I. R.; Cronan, J. E., Jr.; Beacham, K.; Honegger, J. L.;
Silbert, D. F. Proc. Natl. Acad. Sci. 1972, 69, 3105.
5. (a) Chapman-Smith, A.; Morris, T. W.; Wallace, J. C.; Cronan, J. E., Jr. J. Biol. Chem.
1999, 274, 1449; (b) Cronan, J. E., Jr. J. Biol. Chem. 2001, 276, 37355.
6. Cronan, J. E.; Waldorf, G. L. Prog. Lipid Res. 2002, 41, 407.
7. Freiberg, C.; Brunner, N. A.; Schiffer, G.; Lampe, T.; Pohlmann, J.; Brands, M.;
Raabe, M.; Habich, D.; Ziegelbauer, K. J. Biol. Chem. 2004, 279, 26066.
8. Annis, D. A.; Shipps, G. W., Jr.; Deng, Y.; Popovici-Mueller, J.; Siddiqui, M. A.;
Curran, P. J.; Gowen, M.; Windsor, W. T. Anal. Chem. 2007, 79, 4538.
9. (a) Huang, K.-T.; Sun, C.-M. Bioorg. Med. Chem. Lett. 2002, 12, 1001; (b) Lahue, B.
R.; Ma, Y.; Shipps, G. W., Jr.; Seghezzi, W.; Herbst, R. Bioorg. Med. Chem. Lett.
2009, 19, 3405.
Table 7
Pathway specificity of compound 69
Target pathway11
E. coli HS294, IC50 (lg/mL)
Fatty acid Biosynthesis
DNA Biosynthesis
RNA Biosynthesis
0.5
>20
>20
>20
Protein biosynthesis
10. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
Acknowledgments
11. (a) Soriano, A.; Radice, A. D.; Herbitter, A. H.; Langsdorf, E. F.; Stafford, J. M.;
Chan, S.; Wang, S.; Liu, Y.-H.; Black, T. A. Anal. Biochem. 2006, 349, 268;
(b) Hecht, D. W. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically, Approved Standard-Seventh Edition; Clinical
and Laboratory Standard Institute, 2006.; (c) Enzyme and cell-based assay
procedures are described in the Supplementary data.
We thank Dr. Xianshu Yang for performing the initial ALIS
screening and Mr. Michael Starks for analytical support. We also
thank Dr. Paul McNicholas and Nathaniel Brown for supporting
some of the pathway assays.
12. James, E. S.; Cronan, J. E., Jr. J. Biol. Chem. 2004, 279, 2520.