The benzimidazole based drugs show good activity against T. gondii but poor activity against its proposed enoyl reductase enzyme target

Article abstract of DOI:10.1016/j.bmcl.2013.12.066

The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD⁺ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD⁺ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC₅₀ for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.12.066

Bioorganic & Medicinal Chemistry Letters 24 (2014) 911–916
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The benzimidazole based drugs show good activity against T. gondii
but poor activity against its proposed enoyl reductase enzyme
target q
Craig Wilkinson a, Martin J. McPhillie b, Ying Zhou c, Stuart Woods d, Gustavo A. Afanador e,
Shaun Rawson a, Farzana Khaliq d, Sean T. Prigge e, Craig W. Roberts d, David W. Rice b, Rima McLeod c,
Colin W. Fishwick f, Stephen P. Muench a,
⇑
a School of Biomedical Sciences, University of Leeds, Leeds, UK
b Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
c Department of Ophthalmology and Visual Sciences, Pediatrics (Infectious Diseases), Committees on Genetics, Immunology, and Molecular Medicine, Institute of Genomics and
Systems Biology, and The College, The University of Chicago, Chicago, IL 60637, United States
d Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
e Johns Hopkins School of Public Health, Rm. E5132, 615 N. Wolfe St., Baltimore, MD 21205, United States
f School of Chemistry, University of Leeds, Leeds, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been
intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors target-
ing the NAD+ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD+
and its availability in the natural environment makes the NADH complex form of ENR an attractive target.
Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella
ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhib-
itory activity at 1 lM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the
ENR enzyme and as such we believe that there may be significant off target effects for this family of
inhibitors.
Received 8 November 2013
Revised 16 December 2013
Accepted 17 December 2013
Available online 22 December 2013
Keywords:
Enoyl reductase
Triclosan
Toxoplasma
Benzimidazole
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Fatty acid biosynthesis (FAS) is essential for cellular mainte-
nance and survival making it an attractive target for drug design.
Importantly, there are distinct differences between the eukaryotic
and prokaryotic FAS pathways.1 The Eukaryotic system, termed
FAS I, uses one large polypeptide complex, but Prokaryotes use a
series of discrete monofunctional enzymes termed the FAS II path-
way to achieve the same goal.2 Enoyl acyl-carrier protein reductase
(ENR), which carries out the final stage of FAS II synthesis, has been
the main focus of drug development programs in this area. This has
resulted in a range of potent ENR inhibitors being developed such
as isoniazid, the diazaborine family and the common antibacterial
triclosan.3–6 The potency and easy synthesis of triclosan has re-
sulted in it being found in a range of common household items
such as toothpastes, mouthwashes and chopping boards. Common
to all of these potent ENR inhibitors is that they bind to the NAD+
complexed form of ENR, despite the NADH co-factor having a high-
er affinity than NAD+ for ENR and likely being equivalently preva-
lent in nature.7 Moreover, the NADH/NAD+ ratio has been shown to
have a significant effect on the potency of isoniazid, an ENR inhib-
itor in Mycobacterium tuberculosis.8 There are very few examples of
inhibitors which have been designed against the NADH form of
ENR. However, a benzimidazole family of inhibitors designed as
such has modest activity against Francisella tularensis (F. tularensis)
displaying an IC50 value of 300 nM.9,10 The co-crystal structure of a
benzimidazole/NADH/ENR complex shows the inhibitor makes no
p-stacking interactions with the bound cofactor, a feature common
to triclosan and its derivatives, but instead makes a hydrogen bond
to the conserved catalytic Tyr and NADH cofactor.10 The potency of
the benzimidazole family has been shown for F. tularensis, Esche-
richia coli, Bacillus anthracis, Yersinia pestis, Staphylococcus aureus
and MRSA, but to date no antiparasitic testing has been carried
out.9
Despite its eukaryotic nature, Toxoplasma gondii (T. gondii) uses
a typical FAS II pathway making it susceptible to inhibitors of
ENR.11 This pathway is located in the apicoplast organelle and
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
⇑
Corresponding author. Tel.: +44 (0)113 3434279.
E-mail address: s.p.muench@leeds.ac.uk (S.P. Muench).
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.066

912
C. Wilkinson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 911–916
Table 1
not surprisingly apicomplexan parasites that also retain an apicop-
last such as Plasmodium (species) and Eimeria (species) possess this
pathway and are known to be susceptible to ENR targeted drugs.11–
Data collection and refinement statistics
TgENR apo
13
Although FAS II and ENR play an important role in liver-stage
Data collection statistics
Space group
Wavelength used (Å)
Resolution range (Å)
Unique reflections
Multiplicity
Completeness of data (%)
Mean I/r (I)
Multiplicity
Rmerge (%)
development of P. falciparum it is not essential for the survival of
blood stages where triclosan would therefore appear to have an
off-target effect.14,15
The development of a novel set of ENR/NADH inhibitors against
F. tularensis has provided a new potential therapeutic avenue for
the development of T. gondii inhibitors. Here we demonstrate the
benzimidazole family of compounds shows no inhibition of T. gon-
C121
0.97949
30.0–2.0 (2.03–2.0)
129,052 (6302)
3.6 (3.7)
99.7 (99.6)
6.5 (2.8)
3.6 (3.7)
0.13 (0.5)
dii ENR (TgENR) at 1
lM. However, the same inhibitors show
Refinement Statistics
Resolution limits (Å)
Rcryst (%)
Rfree (%)
Rmsd values
promising activity with a MIC50 value of between 1 and 10
lM
30.0–2.0
16.9
19.9
against two different strains of T. gondii parasites cultured
in vitro. The ability of these compounds to curtail T. gondii growth,
but not affect ENR activity, suggests that they have an off-target ef-
fect. Consistent with this idea, a structurally similar compound
Chlormidazole is active against fungi which have a type I and not
a type II fatty acid biosynthesis pathway and lack an ENR homo-
logue, indicating an alternative primary target in fungi, most likely
14 alpha methylase. Although T. gondii lacks this enzyme, data
mining of the PubChem compound library has shown a number
of similar scaffolds with different targets which may explain some
of the off-target affects evident against T. gondii.
Bond length (Å)
0.015
1.54
Bond angle (deg.)
Ramachandran plota
Most favoured (%)
Additionally allowed (%)
Generously allowed (%)
Disallowed (%)
Molecules in asymm. unit
Protein atoms
Co-factor atoms
97.9
2.1
0.0
0.0
6
13,302
264
415
Johnson et al. reported that the 3,4-dichloro substituted benz-
imidazole 1 was potent against F. tularensis ENR (FtENR) com-
plexed with NADH.9 Hit compound 1 and two derivatives 2 and 3
were synthesized from commercially available 5,6-dimethylbenz-
imidazole and substituted benzyl bromides using NaH and KI in
DMF, in moderate to good yields (Scheme 1). HPLC determined
purity to be >95% for each compound.
The fibroblast host cell toxicity assays, inhibition assays and
parasite replication assays were performed as previously de-
scribed.16–20
Water molecules
Mean B-values (Å2)
Protein Mainchain/sidechain
Co-factors
9/13
17
15
Water molecules
Data was collected at station I02 at the Diamond synchrotron to 2.0 Å. Values in
parentheses are for the highest resolution shell.
Ramachandran values were determined in PROCHECK.30
a
Cell toxicity assays were carried out in PC3-Luc cells. Confluent
been deposited within the protein data bank, accession number
4O1M.
cells were incubated with compounds 1–3 at 10 nM, 100 nM, 1
and 10 M concentration in phenol red free DMEM (supplemented
with 10% FCS, 1% -glutamine and 1% penicillin streptomycin). At
48 and 96 h the cells were supplemented with 150 g/ml -lucif-
lM
l
Iterative cycles of model building and refinement were carried
out to 2.0 Å resolution in COOT and REFMAC5 with PDB_REDO
optimizing the refinement procedure, with resulting Rfact and Rfree
values of 0.17 and 0.20, respectively.21–23 The resulting, refined
map showed clear and continuous electron density for the bound
NADH cofactor. However, there was only a small region of strong
positive Fobs–Fcalc density within the proposed binding site
(Fig. 1A). Moreover, the position of this density is not consistent
with the proposed binding site for the benzimide inhibitor
(Fig. 1B). The inhibitor could not be refined either in a similar posi-
tion to that seen in FtENR or in an alternative conformation. The
features seen within the Fobs–Fcalc map within the ENR binding site
are consistent with the density associated with water and bound
crystallization additives (Fig. 1C). Despite data for several crystalli-
zation conditions being collected and processed no evidence for
benzimidazole binding could be seen, with all structures showing
features consistent with those seen in Figure 1.
L
l
D
erin potassium salt and imaged for 1 min in an IVIS Spectrum
(Perkinelmer, USA).
To investigate if the benzimidazole compounds had a similar
binding mode in TgENR as described for FtENR, co-crystallisation
experiments were conducted.10 In the first instance crystals were
grown in the presence of 1.6 mM inhibitor, which was more than
sufficient for the FtENR/NADH/Benzimidazole crystal structure.
However, this was insufficient to produce a TgENR/NADH/Benz-
imidazole crystal complex. Instead, 3.2 mM of inhibitor was used
and crystals were grown in the Morpheus crystal screen from
Molecular Dimensions. Several different crystallisation conditions
yielded diffracting crystals from which data was collected to iden-
tify any bound inhibitor. The highest diffracting crystals grew in
condition G6 (0.1 M Na-Formate, NH4-Acetate, Na3-Citrate, NaK-
Tartrate, Na-Oxamate, 1 M Sodium HEPES, MOPS pH7.5, 30% v/v
P500MME_P20K). The crystals were flash frozen and data were col-
lected on beamline I02 at the Diamond SRS. Full data collection and
processing statistics are found in Table 1. The coordinates have
Since the inability to co-crystalise TgENR with the benzimid-
azole inhibitors is not in of itself evidence that the family are poor
inhibitors, enzyme and cellular assays were used to investigate
their potency. The enzyme assays showed no inhibitory activity
Scheme 1. Reagents and conditions: (a) NaH, KI, DMF, 0 °C warming to rt, 16 h, 40–60%.

C. Wilkinson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 911–916
913
Figure 1. (A) Refined TgENR/NADH crystal structure with the 2Fobs–Fcalc density map (blue) and 1Fobs–1Fcalc density map (green) contoured at 1.6
r
and 4.0
r
, respectively. (B)
The same view as in (A) but with the FtENR/NADH/benzimide complex superimposed in red to show the predicted position of the benzimide inhibitor. (C) Representative
density of the refined TgENR/NADH structure with waters and glycerol modeled into the inhibitor binding site with the map contoured at 1.5 . For A–C, nitrogen, oxygen,
r
chlorine, phosphorous and carbon are colored blue, red, green, orange and yellow (TgENR)/red (FtENR), respectively. (D) Overlay of FtENR (blue sidechains) and TgENR (green
sidechains) inhibitor binding sites with bound inhibitor (yellow), NADH cofactor (green) shown in stick format. The position of the critical Tyr residue which forms a
hydrogen bond to the bound inhibitor is shown along with those residues which are significant different between species.
even at 1
l
M concentration for compounds 1–3 which showed that
strain (type 2) T. gondii stably transfected with the yellow fluores-
cence gene (RH-YFP). This allows for parasite viability to be di-
rectly recorded from the relative fluorescence intensity. The
fluorescence intensities were measured after 72 h using a Synergy
H4 Hybrid Multi-Mode Microplate Reader with excitation/emis-
sion wavelength at 510/540 nm. Compounds 1, 2 and 3 were effec-
the benzimidazole compounds are very poor inhibitors of TgENR.
This discrepancy between binding of benzimidazole to TgENR
and FtENR was investigated by comparing the two binding sites.
A number of sequence changes are observed including significant
changes in the loop region Ser242–Asp249 (TgENR numbering). A
further change which may affect binding is the substitution of
Met/Ala at the base of the binding pocket (Fig. 1D). The Methionine
residue in FtENR makes packing interactions with the bound inhib-
itor and appears to be important for stabilising the inhibitor
(Fig. 1D). The replacement with Ala in TgENR reduces the ability
of this residue to co-ordinate benzimidazole binding. This is an
important aspect of binding, as unlike with other ENR inhibitors
the benzimidazole compound is not coordinated by forming stack-
ing interactions with the bound co-factor.10 A further change is the
presence of Phe242 (TgENR numbering) which creates steric hin-
drance within the binding site, however we have previously shown
that this residue is capable of flipping out of the binding site, to re-
move the steric hindrance.24 These subtle changes will likely fur-
ther reduce the potency of the benzimidazole family of inhibitors
which have been shown to have only modest activity >100,000–
300 nm against FtENR.10 In the absence of a crystal structure we
conducted docking simulations using AutoDock 4.2 of compound
1 to see if it could bind in a mode similar to that observed in
FtENR.25 The predicted binding mode of 1 within TgENR was sim-
ilar to that of FtENR with no significant steric clashes when Phe242
is flipped out of the active site. However, it is clear from the en-
tive against type 1 parasites with MIC50 values of 2.5
>10 M, respectively (Fig. 2A). Type 2 T. gondii were also inhibited
with compounds 1 and 3 at 10 M ((p < 0.005) (Fig. 2B)). The dif-
lM, 4 lM and
l
l
ference in activity of compound 2 maybe due to subtle differences
in the two types of strains used. This activity is far in excess of
what was expected based on the effects of the same compounds
on TgENR enzyme, suggesting that they have an additional off-tar-
get effect against T. gondii.
The promising parasite replication assay data shows that these
compounds may be a promising lead for novel T. gondii inhibitors.
To investigate their potential further, compounds 1, 2 and 3 were
tested for cellular toxicity on both fibroblast and prostate cancer
cells (Fig. 2C and D). Importantly, none of the compounds showed
significant toxicity, in either of the two different assays even at a
concentration of 10 lM against mammalian cells (Fig. 2C and D).
The low toxicity paired with the good MIC50 values make this fam-
ily of inhibitors a promising avenue for inhibitor design, however
the poor IC50 values makes TgENR an unlikely target. To further de-
velop the benzamidazole family we looked for possible targets
other than ENR in the PubChem database.
The PubChem search showed that similar small compound scaf-
folds have been developed which target Sterol 14 alpha-demethyl-
ase, Galanin receptor 3, Pteridine Reductase 1, transient receptor
potential cation channel C4 (TRPC4) and aldosterone synthase (Ta-
ble 2). The crystal structure for the Trypanosoma brucei Pteridine
Reductase (3GN2) shows that the additional amine group of the
zyme assay data that this is not a potent interaction with 1 lM
and 3.2 mM being insufficient for inhibition and co-crystallisation,
respectively.
In conjunction with TgENR enzyme assays, parasite replication
assays were also conducted using the RH strain (type 1) and Pru

914
C. Wilkinson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 911–916
Figure 2. Efficacy and toxicity of compound 1–3 against T. gondii. (A) Growth of RH-YFP in human Foreskin fibroblasts (HFF) measured as fluorescence intensity. HFF were
infected with differing numbers of RH-YFP tachyzoites as indicated, with uninfected control HFF also shown. HFF infected with 3200 RH-YFP tachyzoites treated with
pyrimethamine/sulfadiazine (p/s) or 0.1% DMSO are additional positive and negative controls, respectively (left panel). The effect of compound 1–3 against Toxoplasma
tachyzoites was determined by measurement of RH-YFP fluorescence as shown in the right hand panel. HFF were infected with 3200 RH-YFP and treated with compounds 1–
3 at the concentration indicated. Toxicity was measured using WST stain. (B) The effect of compounds 1–3 on Toxoplasma growth was determined by infecting confluent
fibroblasts with YFP expressing Pru strain T. gondii tachyzoites and measuring YFP florescence at 72 h. Compounds 1 and 3 effectively reduced parasite growth however at
10
was included as a positive control for cytotoxicity. After 96 h luciferin (150
the cells. Neither compounds 1–3, nor the vehicle control resulted in a reduction of bioluminescent activity from the PC3-Luc cells. (D) The viability of host HFF cells was
lM (p < 0.005). (C) Compounds 1–3 were incubated with confluent luciferase expressing PC3-Luc cells to determine their toxicity against prostate cancer cells. 1% Triton X
l
g/ml) was added and cytotoxicity was determined by a reduction in bioluminescent activity from
assessed by WST-1 staining after 72 h of incubation of indicated compounds at 10
medium served as a standard toxicity curve.
lM concentration. Effect of various concentrations of DMSO present in the HFF culture

C. Wilkinson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 911–916
915
Table 2
Activity data for the benzimidazole based scaffold inhibitors
Notebook
ID
Structure
IC50
Target
Organism
T. gondii
MIC50
300 nM
>1000 nM
Francisella tularensis
T. gondii
1
2
3
4
Enoyl-ACP Reductase
3
4
l
M
M
>1000 nM Enoyl-ACP Reductase
>1000 nM Enoyl-ACP Reductase
T. gondii
l
T. gondii
>10
ND
lM
400a nM
Pteridine Reductase 1
Trypanosoma brucei
140 nM
Galanin Receptor 3
Transient receptor potential cation channel C4
Homo Sapiens
Homo Sapiens
ND
ND
668b nM
5
6
7
(TRPC4)
Fungi, Mycobacteria, Leishmania &
Trypanosoma
Homo Sapiens
ND
Sterol 14
a
-demethylase
ND
ND
200 nM
Galanin Receptor 3
635 nM
107 nM
Steroid 11-beta-hydroxylase
Aldosterone synthase
Homo Sapiens
Homo Sapiens
ND
ND
188 nM
632 nM
Steroid 11-beta-hydroxylase
Aldosterone synthase
Homo Sapiens
Homo Sapiens
ND
ND
8
a
Value for Kiapp not IC50
.
b
Value for EC50 not IC50
.
bound inhibitor (4) is essential in the formation of two hydrogen
bonds with Asp161 which is absent in the benzamidizole com-
pound.26 This would make it an unlikely target within Toxoplasma.
The Galanin receptor and TRPC4 have been shown to be targets of
similar compounds 5 and 6 (Table 1). However, these two proteins
are not present within Toxoplasma, making them unlikely targets. A
further target is sterol 14 alpha-demethylase, which is inhibited by
the antifungal drug Chlormidazole (6) (Table 2). Sterol 14 alpha-
demethylase is found in a range of fungi and bacteria such as Myco-
bacteria species as well as the Leishmania and Trypanosoma para-
sites. However, it is absent from apicomplexan parasites with
BLAST searches revealing only poor matches to putative cyto-
chrome P450 enzymes in T. gondii. Moreover, T. gondii, as well as
other apicomplexan parasites lack a cholesterol biosynthesis path-
way, instead relying on scavenging mechanisms.27 Further studies
have found that different sterol 14 alpha-demthylase targeting
drugs were more effective against a different cytochrome P450
CYP121 in Mycobacteria.28 An additional sterol 14 alpha-demthy-
lase inhibitor itraconazole is effective against T. gondii, however,
an additional off target has not been found and it has been hypoth-
esized that there may be several general non-specific targets in T.
gondii which account for the cellular activity.29 A similar scaffold
has been shown to display activity against steroid 11-beta-hydrox-
ylase and aldosterone synthase (7, 8). Taken together this would
suggest that despite the benzimidazole inhibitor family offering
potential as a novel inhibitor against F. tularensis ENR it has a likely
off target effect which accounts for the good MIC50 value estab-
lished against the parasite in vitro but poor IC50 value observed
against Toxoplasma ENR.
potential for novel T. gondii inhibitors given their good anti par-
asitic activity and low toxicity against both prostate and HFF
cells.
Acknowledgments
The authors are grateful for the financial support provided by
NIAID U01 AI082180-01 and the Rooney-Alden, Taub, Engel, Pritz-
ker, Harris, Kapnick, Mussilami and Samuel families, the Mann-
Cornwell Family Foundation, The Wellcome Trust and the Rhona
Reid Charitable Trust. SPM is supported by an MRC Career Develop-
ment fellowship (G1000567). SR was supported by a studentship
from the Wellcome Trust (099752/Z/12/Z).
References and notes
1. Smith, S. FASEB J. 1994, 8, 1248.
2. Magnuson, K.; Jackowski, S.; Rock, C. O.; Cronan, J. E., Jr. Microbiol. Rev. 1993, 57,
522.
3. Baldock, C.; Rafferty, J. B.; Sedelnikova, S. E.; Baker, P. J.; Stuitje, A. R.; Slabas, A.
R.; Hawkes, T. R.; Rice, D. W. Science 1996, 274, 2107.
4. McMurry, L. M.; Oethinger, M.; Levy, S. B. Nature 1998, 394, 531.
5. Bhargava, N. H.; Leonard, P. A. Am. J. Infect. Control. 1996, 24, 209.
6. Rozwarski, D. A.; Grant, G. A.; Barton, D. H.; Jacobs, W. R., Jr.; Sacchettini, J. C.
Science 1998, 279, 98.
7. Lin, S. J.; Guarente, L. Curr. Opin. Cell. Biol. 2003, 15, 241.
8. Vilchèze, C.; Weisbrod, T. R.; Chen, B.; Kremer, L.; Hazbón, M. H.; Wang, F.;
Alland, D.; Sacchettini, J. C.; Jacobs, W. R. Antimicrob. Agents Chemother. 2005,
49, 708.
9. Hevener, K. E.; Mehboob, S.; Su, P. C.; Truong, K.; Bochi, T.; Deng, J.; Ghassemi,
M.; Cook, J. L.; Johnson, M. E. J. Med. Chem. 2012, 55, 268.
10. Mehboob, S.; Hevener, K. E.; Truong, K.; Bochi, T.; Santarsiero, B. D.; Johnson, M.
E. J. Med. Chem. 2012, 55, 5933.
Further studies will be required to determine the exact target
of the benzimidazole family of inhibitors and establish a clear
structure-activity relationship (SAR). However, they present the
11. McLeod, R.; Muench, S. P.; Rafferty, J. B.; Kyle, D. E.; Mui, E. J.; Kirisits, M. J.;
Mack, D. G.; Roberts, C. W.; Samuel, B. U.; Lyons, R. E.; Dorris, M.; Milhous, W.
K.; Rice, D. W. Int. J. Parasitol. 2001, 31, 109.

916
C. Wilkinson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 911–916
12. Lu, J. Z.; Muench, S. P.; Allary, M.; Campbell, S.; Roberts, C. W.; Mui, E.; McLeod,
R. L.; Rice, D. W.; Prigge, S. T. Parasitology 2007, 134, 1949.
13. Surolia, N.; Surolia, A. Nat. Med. 2001, 7, 167.
20. Cheng, G.; Muench, S. P.; Zhou, Y.; Afanador, G. A.; Mui, E. J.; Fomovska, A.; Lai,
B. S.; Prigge, S. T.; Woods, S.; Roberts, C. W.; Hickman, M. R.; Lee, P. J.; Leed, S.
E.; Auschwitz, J. M.; Rice, D. W.; McLeod, R. Bioorg. Med. Chem. Lett. 2013, 23,
2035.
21. Mushudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Cryst. D 1997, 53, 240.
22. Emsley, P.; Cowtan, K. Acta Cryst. D 2004, 60, 2126.
23. Joosten, R. P.; Joosten, K.; Cohen, S. X.; Vriend, G.; Perrakis, A. Bioinformatics
2011, 27, 3392.
24. Muench, S. P.; Stec, J.; Zhou, Y.; Afanador, J. A.; McPhillie, M. J.; Hickman, M. P.;
Lee, P. J.; Leed, S. E.; Auschwitz, J. M.; Prigge, S. T.; Rice, D. W.; McLeod, R.
Bioorg. Med. Chem. Lett. 2013, 23, 2035.
25. Goodsell, D. S.; Morris, G. M.; Olson, A. J. J. Mol. Recognit. 1996, 9, 1.
26. Mpamhanga, C. P.; Spinks, D.; Tulloch, L. B.; Shanks, E. J.; Robinson, D. A.; Collie,
I. T.; Fairlamb, A. H.; Wyatt, P. G.; Frearson, J. A.; Hunter, W. N. J. Med. Chem.
2009, 52, 4454.
14. Yu, M.; Kumar, T. R.; Nkrumah, L. J.; Coppi, A.; Retzlaff, S.; Li, C. D.; Kelly, B. J.;
Moura, P. A.; Lakshmanan, V.; Freundlich, J. S.; Valderramos, J. C.; Vilcheze, C.;
Siedner, M.; Tsai, J. H.; Falkard, B.; Sidhu, A. B.; Purcell, L. A.; Gratraud, P.;
Kremer, L.; Waters, A. P.; Schiehser, G.; Jacobus, D. P.; Janse, C. J.; Ager, A.;
Jacobs, W. R., Jr.; Sacchettini, J. C.; Heussler, V.; Sinnis, P.; Fidock, D. A.;
Viswanathan, L.; Freundlich, J. S. Cell Host Microbe. 2008, 4, 567.
15. Spalding, M. D.; Prigge, S. T. Cell Host Microbe. 2008, 4, 509.
16. Perozzo, R.; Kuo, M.; Sidhu, A. B.; Valiyaveettil, J. T.; Bittman, R.; Jacobs, W. R.,
Jr.; Fidock, D. A.; Sacchettini, J. C. J. Biol. Chem. 2002, 277, 13106.
17. Fomovska, A.; Huang, Q.; El Bissati, K.; Mui, E. J.; Witola, W. H.; Cheng, G.; Zhou,
Y.; Sommerville, C.; Roberts, C. W.; Bettis, S.; Prigge, S. T.; Afanador, G. A.;
Hickman, M. R.; Lee, P. J.; Leed, S. E.; Auschwitz, J. M.; Pieroni, M.; Stec, J.;
Muench, S. P.; Rice, D. W.; Kozikowski, A. P.; McLeod, R. Antimicrob. Agents
Chemother. 2012, 56, 2666.
27. Coppens, I.; Sinai, A. P.; Joiner, K. A. J. Cell. Biol. 2000, 149, 167.
28. McLean, K. J.; Marshall, K. R.; Richmond, A.; Hunter, I. S.; Fowler, K.; Kieser, T.;
Gurcha, S. S.; Besra, G. S.; Munro, A. W. Microbiology 2002, 148, 2937.
29. Martins-Duarte, E. S.; De Souza, W.; Vommaro, R. C. FEMS Microbiol. Lett. 2008,
282, 290.
18. Fomovska, A.; Wood, R. D.; Mui, E.; Dubey, J. P.; Ferreira, L. R.; Hickman, M. R.;
Lee, P. J.; Leed, S. E.; Auschwitz, J. M.; Welsh, W. J.; Sommerville, C.; Woods, S.;
Roberts, C.; McLeod, R. J. Med. Chem. 2012, 55, 8375.
19. Lai, B. S.; Witola, W. H.; El Bissati, K.; Zhou, Y.; Mui, E.; Fomovska, A.; McLeod,
R. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 14182.
30. Laskowski, R. A.; Macarthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl.
Crystallog. 1993, 26, 283.