Mechanistic insight into inhibition of two-component system signaling

Article abstract of DOI:10.1039/c2md20308a

Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses. Targeting the highly conserved kinase domain in these systems represents a promising strategy for the design of a broad-spectrum antibiotic; however, development of such compounds has been marred by an incomplete understanding of the conserved binding features within the active site that could be exploited in molecule design. Consequently, a large percentage of the available TCS inhibitors demonstrate poor target specificity and act via multiple mechanisms, with aggregation of the kinase being the most notable. In order to elucidate the mode of action of some of these compounds, molecular modeling was employed to dock a suite of molecules into the ATP-binding domain of several histidine kinases. This effort revealed a key structural feature of the domain that is likely interacting with several known inhibitors and is also highly conserved. Furthermore, generation of several simplified scaffolds derived from a reported inhibitor and characterization of these compounds using activity assays, protein aggregation studies and saturation transfer differential (STD) NMR suggests that targeting of this protein feature may provide a basis for the design of ATP-competitive compounds. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2md20308a

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CONCISE ARTICLE
Mechanistic insight into inhibition of two-component
system signaling†
Cite this: DOI: 10.1039/c2md20308a
a
a
a
ab
*
Samson Francis, Kaelyn E. Wilke, Douglas E. Brown and Erin E. Carlson
Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple
environmental stimuli to adaptive responses. Targeting the highly conserved kinase domain in these
systems represents a promising strategy for the design of a broad-spectrum antibiotic; however,
development of such compounds has been marred by an incomplete understanding of the conserved
binding features within the active site that could be exploited in molecule design. Consequently, a large
percentage of the available TCS inhibitors demonstrate poor target specificity and act via multiple
mechanisms, with aggregation of the kinase being the most notable. In order to elucidate the mode of
action of some of these compounds, molecular modeling was employed to dock a suite of molecules
into the ATP-binding domain of several histidine kinases. This effort revealed a key structural feature of
the domain that is likely interacting with several known inhibitors and is also highly conserved.
Received 1st July 2012
Furthermore, generation of several simplified scaffolds derived from
a reported inhibitor and
Accepted 9th November 2012
characterization of these compounds using activity assays, protein aggregation studies and saturation
transfer differential (STD) NMR suggests that targeting of this protein feature may provide a basis for
the design of ATP-competitive compounds.
DOI: 10.1039/c2md20308a
www.rsc.org/medchemcomm
structural features that govern functionality in this family of
signaling proteins. As a result, several sites have been identi-
Introduction
Two-component systems (TCSs) are signal transduction path-
ways that are ubiquitous in bacterial systems.1,2 Consisting of a
histidine kinase (HK) and a response regulator (RR), these
systems regulate diverse processes ranging from chemotaxis3 to
virulence.4 Extracellular events activate HKs, inducing auto-
phosphorylation of a conserved histidine (His) residue (Fig. 1).
The phosphate group is subsequently transferred to an aspar-
tate (Asp) on a cognate RR, eliciting an adaptive response such
as quorum sensing,5 antibiotic resistance,6 or the production of
virulence factors in pathogenic bacteria.7 Furthermore, the
essentiality of some of these systems in the promotion of viru-
lence traits8,9 and activation of resistance mechanisms10 makes
them attractive drug targets.
ed for the action of chemical compounds to disrupt the
intracellular signaling activities of these systems. Intervention
by such a compound could occur at key junctions in the
cascade (Fig. 1) including receipt of the activation signal,
binding of ATP, autophosphorylation of the HK (HKꢀP),
dephosphorylation of HKꢀP, phosphotransfer from HKꢀP to
the RR, and binding of phosphorylated RR (RRꢀP) to the gene
promoter.
The search for inhibitors capable of interrupting TCS signal
transduction has yielded several classes of synthetic
compounds through
a combination of high-throughput
screening,18 rational design and structure-based design.19
To date, although ꢀ50 000 TCS proteins have been identi-
ed from genomic sequences, most have not been character-
ized.11 However, with the availability of X-ray crystal structures
for a variety of HKs12–14 and RRs,15,16 and recently an HK–RR
complex,17 much knowledge has been gained about the
aDepartment of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington,
Indiana, USA. E-mail: carlsone@indiana.edu; Tel: +1 812-855-3665
bDepartment of Molecular and Cellular Biochemistry, Indiana University, 212 S.
Hawthorne Drive, Bloomington, Indiana, USA
† Electronic supplementary information (ESI) available: Supporting gures and
tables are provided depicting the library of known inhibitors, additional
modeling studies, kinase activity assays, protein aggregation studies, sequence
alignments and experimental methods. See DOI: 10.1039/c2md20308a
Fig. 1 The two-component system signaling (TCS) cascade. Upon detection of
an appropriate signal, autophosphorylation occurs at a conserved His residue of
the HK, followed by phosphoryl group transfer to an Asp residue of the RR. A
typical function for the RR is gene regulation.
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Fig. 2 Molecules reported to inhibit TCS activity.
While these initiatives have generated large libraries of
inhibitors, examples of which are shown in Fig. 2, the mech-
anisms of action of most of these agents remain poorly
understood. For example, RWJ-49815 (30; Fig. 3), a represen-
tative HK inhibitor, was initially reported to exert its inhibitory
activity by targeting the catalytic and ATP-binding domain (CA)
of several HKs;20 however, subsequent studies have shown that
this compound inhibits HK autokinase activity non-speci-
Fig. 3 Compounds bearing guanidine moieties. (A) Subset of compounds pre-
dicted to interact with an active site Asp using molecular modeling. (B) Simplified
variants of compounds 26, 27, 30 and 33. Further deconstruction of these
cally.21 In fact, 30, along with a large majority of compounds compounds resulted in the fragment-like compound 50.
reported to inhibit TCS activity, do so through aggregation of
the protein.22
Clearly, attaining specicity in the development of
compounds capable of disrupting TCS signaling remains a
Results and discussion
To examine the probable ways in which previously reported
challenging task. In recent years, there has been a heightened
interest in deactivating TCS transduction by targeting the CA
domain of the HK.22,23 Similar to other classes of kinases, the
catalytic core within HKs has been reported to exhibit a high
degree of homology11,24 in both Gram-positive and Gram-nega-
tive bacteria. Exploiting such a feature could yield an inhibitor
with the potential to handicap multiple TCSs in a single path-
ogen, perhaps providing an additional avenue towards
addressing antimicrobial drug resistance. Therefore, a better
understanding of the interactions that take place between the
CA domain and possible ligands is paramount. Herein, we
report a chemical scaffold with the potential to interact with a
conserved structural feature in the CA of HKs. This scaffold was
identied, optimized and validated through a combination of
molecular modeling, kinase inhibition and aggregation
experiments, and ligand-observed NMR studies.
inhibitors may be interacting with an HK, we sourced a diverse
set of compounds with reported activity against TCS signaling
from the literature. In all, forty-ve inhibitors with IC50 values in
the range of 0.4 mM to 1000 mM were identied and added to a
virtual library for screening (compounds 1–38, 41–47; Table
S1†).18,20,23,25–38 This library was appended with a subset of 1000
randomly selected drug-like compounds from the ZINC small-
molecule database to expand library diversity and enhance
screening for actives.39,40 In the selection of suitable receptors
from the RSCB Protein Data Bank (PDB),41 preference was given
to those that had been co-crystallized with a nucleotide and/or
ligand. These included the virulence sensor PhoQ from Salmo-
nella typhimurium (PDB: 3CGY)42 and Escherichia coli (PDB:
1ID0),14 the chemotaxis HK CheA (PDB: 1I58)43 and the kinase
HK853 (PDB: 2C2A),13 both from Thermotoga maritima.
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The combined 1045 compound set was docked into the
active sites of the selected receptors using the Surex-Dock
module44 of SYBYL.45 The fragments-constraints feature of
Surex was implemented and utilized a portion of the co-crys-
tallized ligand as a guide during docking (Fig. S1†). In order to
gauge docking accuracy, the ATP analogue AMP-PNP (5), was
also included in the compound database. This nucleotide
derivative ranked among the top 0.5% of all top-scoring
compounds for one of the receptors, PhoQ (PDB: 1ID0), with the
predicted binding pose of its adenine moiety closely matching
that observed crystallographically (Fig. S2†). This ‘positive
control’ helped to verify the docking and scoring parameters.
A notable aspect of many of the compounds in the inhibitor
subset, particularly those in the top 10% (as surveyed across all
utilized receptors), was the manner in which they interacted
with a deeply buried Asp residue in the receptor active sites
(Fig. S3†). Hydrogen bonding interactions have been observed
between a phenolic hydrogen in radicicol and the analogous
Asp in the HK PhoQ from Salmonella typhimurium in a co-crys-
tallized structure.42 We were particularly drawn to a series of
high-scoring (0.1–7.2% across all receptors) guanidine-bearing
compounds (26, 27, 30, 33; Fig. 3A), that when docked, were
predicted to interact with this active site Asp through a salt-
bridge (Fig. S4†). For instance, a representative docked pose of
the diphenyl compound 27 reveals that a strong interaction is
established between this molecule's guanidine-like head group
and Asp411 within the active site of HK853 (Fig. 4). Intriguingly,
compound 28 (Fig. 3A), a close derivative of 30 that lacks the
guanidine moiety and instead displays an amine, did not score
as well and ranked in the range of 11.5–35% across all four
receptors. Other noteworthy interactions include a p–p stack-
ing between the aromatic ring of 27 with Tyr384, as well as other
complementary hydrophobic interactions that occur on the
outskirts of the binding site.
Fig. 5 Evolutionary profile of active site residues in HKs. Derived from the
sequence alignment of 150 HK homologs and rendered onto HK853 from
Thermotoga maritima. Red colored residues/areas indicate high degrees of
evolutionary conservation while blue regions indicate variations in conservation.
Inset: Co-crystallized structure of ADP in the CA domain of HK853 (ref. 13) illus-
trating residues that are involved in binding of the nucleotide. The measured
˚
distance between the exocylic N of ADP and D411 is 2.83 A.
alignment (MSA) of 150 HK homologs (Fig. S5†)48 rendered onto
the crystal structure of HK853 from Thermotoga maritima
(Fig. 5), we generated a map of the highly conserved residues in
the ATP-binding domain. This deeply situated Asp residue,
along with a pair of distal active-site residues, Asn380 and
Leu446, play key roles in the binding of the nucleotide to the CA
domain (Asn in N Box, Leu in G2 box; Fig. 5, inset) and may
provide viable contacts for appropriately designed ATP-
competitive inhibitors.
Salt-bridge mediated interactions between guanidine-like
motifs and Asp residues situated in an active site have been
observed before in serine proteases, thrombin,24 and even
utilized successfully as starting points for inhibitor design,49
prompting us to further investigate this scaffold. However, the
compound that was reported to be the most potent of this
collection, 30, has also been shown to elicit protein aggregation
as part of its mechanism of action.21,22 Shoichet and co-workers
reported the triphenyl-bearing compound, clotrimazole, as also
Previous active site mapping efforts indicated that this Asp
residue, which is present in the G1 box, is conserved across HKs
and is involved in ATP binding.46,47 Using the multiple sequence
Fig. 6 Gel-based aggregation studies using native-PAGE. (A) NH125 (3) induces
aggregation of the protein starting at concentrations as low as 50 mM as evi-
denced by the disappearance of protein bands and (B) compound 50 does not
aggregate HK853 even at concentrations as high as 1 mM.
Fig. 4 Predicted binding pose of compound 27 in the active site of HK853. The
guanidine moiety of 27 forms a salt-bridge with D411. The ligand is also projected
to participate in p–p stacking interactions with Y384.
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being a strong protein aggregator, suggesting that large hydro- detergent Triton X-100. Examination of NH125 (3) in these
phobic moieties are likely involved in this undesirable assays also revealed that protein aggregates appear at concen-
outcome.50 Therefore, we postulated that more simplied vari- trations where it inhibits autophosphorylation (Fig. 6A and S8†).
ants of compounds 26, 27, 30 and 33 devoid of the rigid, We postulated that the aggregation potential of these
hydrophobic rings would be better suited for study, with the compounds, all three of which contain a long alkyl tail, could be
goal of identifying a scaffold that does not cause protein attributed to this moiety. Therefore, to further the study, we
aggregation (48, 49; Fig. 3B).
pursued the variant, 50 (Fig. 3B), that lacked the alkyl chain.
We used our previously disclosed assay, which utilizes the Given the small size of this compound, we were unsurprised
uorescent nucleotide BODIPY-FL-ATP-gS,51 to evaluate the that even at concentrations nearing ꢀ10 mM, inhibition of
activity of HK853 from Thermotoga maritima upon subjection to autophosphorylation by compound 50 was not detected in our
the designed molecules. Inhibition of HK autophosphorylation activity assay (Fig. S10†). Gel-based aggregation studies,
was observed with both 48 and 49 and the commercially avail- however, using 50 conrmed that this compound did not
able HK inhibitor, NH-125 (3, Fig. 2 and S9†). The activity of induce aggregation of HK853 (Fig. 6B) suggesting that if this
compounds 48 and 49 was modest, with inhibition seen starting component does bind to the protein, it could provide a viable
from 600 mM to 1 mM (Fig. S6†). Competition was observed starting point for the design of an ATP-competitive inhibitor
between BODIPY-FL-ATP-gS and the test compounds (Fig. S7†), (Fig. S11 and S12†). The fragment-like nature of compound 50
suggesting that either the lead molecules also bind in the active presented signicant challenges in assessing its potential
site or that they aggregate the protein reversibly. When sub- interactions with a protein receptor using standard biological
jected to in-gel aggregation studies (Fig. S8†), we conrmed that assays. Therefore, we utilized a ligand-observed NMR spec-
even the simplied scaffolds were still operating in a non- troscopy technique, saturation transfer differential (STD),52 to
specic fashion by inducing aggregation of the protein, a characterize the interactions between 50 and the receptor
phenomenon that was easily reversed by addition of the HK853 (Fig. 7). STD-NMR has become an invaluable tool in
Fig. 7 Assessment of binding using saturation transfer differential (STD) experiments (A) 1H spectrum of 50 and HK853, (B) 1H STD-NMR spectrum of 50 and HK853,
(C) 1H NMR spectrum of 50, ADP and HK853, (D) 1H STD-NMR spectrum of 50, ADP and HK853, (E) 1H STD-NMR spectrum of 50 and mutated (D411A) HK853, (F) 1
spectrum of tyramine and HK853 and (G) 1H STD-NMR spectrum of tyramine and HK853.
H
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compound 50 was determined (Fig. 8) to be 2.41 (ꢁ0.25) mM,
affording it a ligand efficiency (LE) of 0.27. If the guanidine-
bearing group of 50 binds specically in the ATP-binding site,
adenosine diphosphate (ADP), the kinase reaction product,
would disrupt this interaction. Thus, STD-NMR was repeated
with protein and an equimolar mixture of ADP and 50. This
yielded resonances corresponding only to those of the nucleo-
tide indicating a competitive relationship between ligand 50
and ADP (compare Fig. 7C and D). Additionally, mutation of the
active-site Asp residue of the protein to Ala resulted in signi-
cant attenuation of ligand 50's affinity for HK853 as judged by
the diminishment of its resonances in the STD-NMR spectrum,
which demonstrated the importance of this residue in ligand
binding (compare Fig. 7B and E). We also examined the ability
of tyramine, an analogous structure that contains an amine
instead of a guanidine, to interact with the histidine kinase.
Extremely weak ligand signals were observed, indicating that
little to no binding was taking place (compare Fig. 7F and G).
These data are consistent with the previous nding that the
guanidine-functionalized compound 30 is 10-fold more potent
than the amine-containing analogue (28; Table S1†). Collec-
tively, our results are strongly suggestive that the simplied
fragment 50 is binding in the nucleotide-binding domain of
HK853 and is likely interacting with the protein via a salt-bridge
with Asp411 in the active site.
Fig. 8 Determination of the Kd values of compounds 50 and 51 using STD-NMR.
Using the initial growth rates approach53 and mathematical fitting to a Langmuir
isotherm, the Kd values of 50 and 51 were found to be 2.41 (ꢁ0.25) mM and 0.58
(ꢁ0.06) mM, respectively.
characterizing ligand–receptor complexes and has seen great
utility in fragment-based drug discovery. Based on the nuclear-
Overhauser effect, STD-NMR relies upon the selective saturation
of the receptor in order to elicit resonances from any interacting
ligands, distinguishing between binders and non-binders. In
the presence of protein (HK853), resonances corresponding to
compound 50 were observed in the STD-NMR spectrum, con-
rming a ligand–protein association (compare Fig. 7A and B).
By utilizing the STD initial growth rates approach,53 the Kd for
With fragment 50 as a starting point, we sought to determine
if a molecule with increased affinity could be designed without
generation of a compound that causes protein aggregation. Our
modeling studies suggest that 50 is mimicking the adenosine
portion of ATP. We anticipated that appending fragment 50
with a phosphate mimic, such as a sulfonyl group, could
increase compound potency. Structural data indicates that the
phosphate of ADP interacts with a highly conserved residue,
N380 (Fig. 5), which could also be expected to bond to a sulfonyl
moiety. Accordingly, we synthesized the modied fragment 51
(Fig. 9A) and proceeded to assess both its inhibitory and
binding potentials using our activity-based assay and STD-
NMR, respectively. Indeed, the evolved fragment 51 was found
to not only inhibit autophosphorylation of HK853 (Fig. S13†)
without inducing aggregation of the protein (Fig. S14†), but to
also register a binding event to the receptor that is competitive
with ADP (compare Fig. 9B and C). NMR Ranking experiments
based on the STD factor values (fSTD)
54 of ADP, tyramine, 50 and
51 (Table 1) reected the increased affinity of 51 for HK853
[Kd ¼ 0.58 (ꢁ0.06) mM; Fig. 8]. These data also indicate, as
previously discerned, that binding of the guanidine-containing
Table 1 STD factor values of compounds binding to HK853
a
Entry
Compound
fSTD
1
2
3
4
Tyramine
ADP
50
16.6
73.3
28.4
32.8
Fig.
9
Assessment of binding using saturation transfer differential (STD)
51
experiments (A) compound 51, (B) 1H spectrum of 51, ADP and HK853 and (C) 1
STD-NMR spectrum of 51, ADP and HK853.
H
a
Aromatic peak resonances.
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fragments is tighter than the related compound, tyramine, that deprotection step in a stirred solution of 1 mL dichloromethane
is instead functionalized with an amine, establishing the and 1 mL TFA. Evaporation of the solvents in vacuo followed by
importance of this group.
chromatography through a plug of neutral alumina (0.1 : 2
MeOH : CHCl3) afforded 48 (5.1 mg, 0.018 mmol, 91% yield). 1H
NMR (400 MHz, CD3OD) d 7.16 (d, J ¼ 8.3 Hz, 2H), 6.87 (d, J ¼
8.3 Hz, 2H), 3.94 (t, J ¼ 6.4 Hz, 2H), 3.41 (t, J ¼ 7.0 Hz, 2H), 2.81
Conclusions
The catalytic and ATP-binding domains of bacterial HKs (t, J ¼ 6.9 Hz, 2H), 1.82–1.71 (m, 2H), 1.51–1.43 (m, 2H), 1.41–
represent an ideal target for the action of new antibiotics. 1.32 (m, 6H), 0.91 (t, J ¼ 6.1 Hz, 3H). 13C NMR d, 15.0, 24.3, 27.8,
Although challenging, the design of ATP-competitive molecules 30.8, 31.1, 33.6, 35.8, 44.5, 69.7, 116.4, 131.4, 160.2 MS (m/z): [M
specic for this site can likely be achieved through a better + H]+ calcd for C16H27N3O 278.2232; found 278.2220.
understanding of its structural features. Using a combination of
1-(4-(DECYLOXY)PHENETHYL)GUANIDINE (49). Preparation of
molecular docking and sequence alignments, we have identi- compound 49 used a method similar to that of 48 (5.1 mg, 0.016
ed that the conserved Asp residue in the active site of HKs may mmol, 85% yield). 1H NMR (400 MHz, CD3OD) d 7.16 (d, J ¼ 7.9
interact with a portion of the compounds reported to have TCS Hz, 2H), 6.88 (d, J ¼ 8.5 Hz, 2H), 3.95 (t, J ¼ 6.5 Hz, 2H), 3.42 (t,
inhibitory activities. Also, by deconstructing a series of known J ¼ 7.0 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz, 2H), 1.79–1.73 (m, 2H), 1.51–
aggregators, we have identied a fragment (50) that likely binds 1.45 (m, 2H), 1.32 (s, 12H), 0.92 (t, J ¼ 6.4 Hz, 3H). 13C NMR d
with this residue, providing a basis for the design of selective 14.4, 23.7, 27.2, 30.43, 30.44, 30.5, 30.68, 30.73, 33.1, 35.1, 43.9,
HK inhibitors. Elaboration of this fragment into the sulfonyl 51, 69.0, 115.8, 130.8, 159.55 MS (m/z): [M + H]+ calcd for C19H34N3O
which may interact similarly to AMP, resulted in the identi- 320.2702; found 320.2715.
cation of a compound with increased affinity. Our studies
1-(4-HYDROXYPHENETHYL)GUANIDINE (50). To a 25 mL round-
suggest that rational design is a viable avenue for the generation bottomed ask charged with a magnetic stir bar and under a
of potent inhibitors that specically preclude autophosphory- nitrogen atmosphere was added 4-(2-aminoethyl)phenol (0.10 g,
lation in this family of proteins.
0.73 mmol) and triethylamine (0.10 mL, 0.73 mmol) to 10 mL of
dichloromethane. Aer stirring for 10 min, 1,3-di-boc-2-(tri-
uoromethylsulfonyl)guanidine (0.31 g, 0.80 mmol) was added
and allowed to stir for an additional 4 h. TLC conrmed the
formation of product and the solvents were removed in vacuo to
Experimental section
Synthetic methods
GENERAL INFORMATION. All materials and chemicals were yield a light brown crude oil, which was puried via column
purchased from EMD Chemicals, Sigma, Aldrich, J.T. Baker chromatography (2 : 1 hexanes : EtOAc) to afford 52, the pro-
(unless noted otherwise) and were used without further puri- tected variant of 50 (0.22 g, 0.58 mmol, 79% yield). 1H NMR (400
cation. Solvents were purchased as anhydrous and not further MHz, CD2Cl2) d 7.00 (d, J ¼ 8.4 Hz, 2H), 6.75 (d, J ¼ 8.5 Hz, 2H),
puried. 1H nuclear magnetic resonance (NMR) spectra were 3.53 (t, J ¼ 7.1 Hz, 2H), 2.75 (t, J ¼ 7.3 Hz, 2H), 1.48 (s, 9H), 1.43
recorded on a Varian I500 or a Varian VXR-400 instrument. (s, 9H). Compound 52 (0.10 g, 0.26 mmol) was taken up in 1 mL
Chemical shis are reported relative to residual solvent peaks in dichloromethane and 1 mL triuoroacetic acid and the reaction
parts per million. Apparent rst-order multiplicities are indi- mixture was stirred for 2 h at r.t. aer which the solvents were
cated by s, singlet; d, doublet; dd, doublet of doublets; t, triplet; removed in vacuo. Purication of the crude material was
dt, doublet of triplets; q, quartet; m, multiplet. Analytical TLC accomplished by passage through a plug of neutral alumina
was performed on Silica Gel 60 F254 plates, E. Merck by detec- (0.1 : 2 MeOH : CHCl3) to afford compound 50 (0.046 g, 0.256
tion with 254 nm UV light and then spray–heat development mmol, 97% yield). 1H NMR (400 MHz, CD3OD) d 7.04 (d, J ¼ 8.1
using a p-anisaldehyde–sulfuric acid reagent. Column chro- Hz, 2H), 6.71 (d, J ¼ 8.1 Hz, 2H), 3.36 (t, J ¼ 7.0 Hz, 2H), 2.75 (t,
matography was run by using silica gel (63–200 mesh).
J ¼ 7.0 Hz, 2H). 13C NMR d 33.7, 42.5, 115.0, 129.4, 155.9 MS (m/
1-(4-HEPTYLOXY)PHENETHYL)GUANIDINE (48). Compound 52 z): [M + H]+ calcd for C9H14N3O 180.1137; found 180.1133.
(0.011 g, 0.029 mmol), synthesized as described for compound
1-(4-METHYLSULFONYL)PHENETHYL)GUANIDINE (51). Prepara-
1
50,was added to a round-bottom ask equipped with a Teon tion of compound 51 used a method similar to that of 50. H
coated magnetic stir bar containing 15 mL of acetone. Potas- NMR (400 MHz, CD3OD) d 7.93 (d, J ¼ 8.1 Hz, 2H), 7.56 (d, J ¼
sium carbonate (0.040 g, 0.29 mmol) was added to the ask and 8.0 Hz, 2H), 3.53 (t, J ¼ 6.9 Hz, 2H), 3.12 (s, 3H), 3.02 (t, J ¼ 7.0
the reaction mixture stirred at reux for 1 h. 1-Bromoheptane Hz, 2H). 13C NMR d 34.3, 41.5, 43.7, 127.1, 129.9, 139.1, 144.7,
(6.2 mg, 0.035 mmol) was added, followed by NaI (2.2 mg, 0.014 156.9 MS (m/z): [M + H]+ calcd for C10H15N3O2S 242.0963 found
mmol) and the mixture stirred overnight at reux. The mixture 242.0965.
was cooled to room temperature and the solvent removed
in vacuo. The crude material was re-dissolved in 30 mL
dichloromethane (DCM) and an equal part water and extracted
Biochemical methods
3 times with DCM. The organic layers were pooled, dried over
GENERAL METHODS AND INFORMATION. Reagents were
MgSO4 and the solvent evaporated in vacuo. The crude product obtained from J.T. Baker, Mallinkrodt, Sigma, IBI, VWR, EMD
was further puried via column chromatography (2 : 0.6 Biosciences, Bio-Rad and Fisher. BODIPY-FL-ATPgS was
hexanes : EtOAc) to afford the Boc-protected derivative of 48 purchased from Invitrogen, NH125 from Tocris Bioscience, and
(9.7 mg, 0.020 mmol, 70% yield) which then underwent a 2 h BS3-d0 from Thermo Scientic.
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loading buffer was added prior to loading on the gels. Silver
staining was used for protein visualization.
GEL ELECTROPHORESIS. For sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE), tris–glycine gels
were used. The stacking gel was 4.5% acrylamide, and the
resolving gel was 10% acrylamide. For native-PAGE, tris–glycine
gels were prepared with 7.5% acrylamide. Electrophoresis
parameters were 180 V, 400 mA, and 60 W for 1 h.
IN-GEL FLUORESCENCE DETECTION. Aer SDS-PAGE, gels were
washed 3 times with water and scanned on a Typhoon Variable
Mode Imager 9210 (Amersham Biosciences) using 526 nm
AGGREGATION ANALYSIS OF NH125 (3), 48, 49, 50 AND 51 (SDS-
PAGE). Puried HK853 (0.44 mM) was prepared in 20 mM
HEPES, pH 7.5. Samples of 20 mL HK853 were mixed with 1 mL
2.18% Triton X-100 in HEPES (0.1% nal Triton X-100) or
HEPES alone. Various concentrations of analogues were added,
and reactions were incubated at RT for 4 h. The crosslinker
bis(sulfosuccinimidyl)suberate (BS3-d0) was suspended in
DMSO and was added at a 100-fold molar excess over the
protein. Total DMSO concentrations were no greater than 5% (v/
v). Crosslinking reactions were incubated another 2 h at RT and
quenched with a nal concentration of 20 mM ammonium
bicarbonate to bring individual samples to 25 mL. SDS-PAGE
sample loading buffer was added prior to loading on the gels.
Silver staining was used to visualize proteins.
(short-pass lter) detection for BODIPY (lex: 504 nm, lem
:
514 nm). Imaging of gels and/or integration of uorescence
bands were performed in ImageJ.
SILVER STAINING. All steps were carried out at RT with an
orbital shaker to ensure agitation, and solutions were discarded
aer each step. Aer SDS- or native-PAGE, gels were xed for 1 h
in 20% ethanol, 1% acetic acid. Gels were then washed in 20%
ethanol for 10 min. Aer pre-treating the gel for 1 min in 0.02%
sodium thiosulfate, gels were washed for 1 min in water. Gels
were incubated with 0.1% silver nitrate for 20 min and again
rinsed for 1 min in water. Developing solution (2% sodium
carbonate, 20 mL 37% formaldehyde) was incubated with gels
for approximately 10 min, or until protein bands were visible,
and development was halted with 5% acetic acid for 10 min.
Gels were then submerged in water.
BUFFERS. The reaction buffer used in activity assays with
BODIPY-FL-ATPgS was 50 mM Tris–HCl, 0.2 M KCl, 5 mM
MgCl2, nal pH 7.8. The 2ꢂ SDS-PAGE sample loading buffer
contained 125 mM Tris, pH 6.8, 20% glycerol (v/v), 4% SDS (w/
v), 5% BME (v/v), and 0.2% bromophenol blue (w/v). Native-
PAGE sample loading buffer contained 40 mM Tris, pH 7.5, 8%
glycerol (v/v), and 0.08% bromophenol blue (w/v). The electro-
phoresis running buffer for SDS-PAGE was diluted 10-fold from
Novex Tris-Glycine SDS Running buffer (10ꢂ; Invitrogen).
Native-PAGE electrophoresis buffer contained 83 mM Tris, pH
9.4, and 33 mM glycine.
ASSESSMENT OF AUTOPHOSPHORYLATION (COMPETITIVE ABPP).
All analogues were dissolved in DMSO, and the nal concen-
tration of DMSO in each sample was kept at or below 5% (v/v).
These concentrations of DMSO were found to have no effect
on the activity labeling. Puried HK853 (600 ng) in reaction
buffer was incubated with varied concentrations of analogues
for 30 min. Labeling was achieved by adding 1 mL BODIPY-FL-
ATPgS (B-ATPgS; 2 mM nal concentration) to each sample.
Individual mixtures were incubated at RT for 1 h in the dark
to prevent uorophore photobleaching. Aer incubation,
reactions were quenched with 2ꢂ SDS-PAGE loading buffer
before running on SDS-PAGE gels (samples were not heated).
Aer analyzing gels by uorescence, they were coomassie
stained.
AGGREGATION ANALYSIS OF NH125 (3), 48, 49, 50 AND 51
(NATIVE-PAGE). Puried HK853 (0.44 mM) was prepared in 20
mM HEPES, pH 7.5. Samples of 20 mL HK853 were mixed with
1 mL 2.18% Triton X-100 in HEPES (0.1% nal Triton X-100) or
HEPES alone. Various concentrations of analogues were added,
and reactions were incubated at RT for 4 h. DMSO concentra-
tions were no greater than 5% (v/v). Native-PAGE sample
Computational methods
GENERAL INFORMATION. All molecular modeling operations
were performed using SYBYL X 2.0 on a quad-core Intel Core i3
workstation operating at 3.06 GHz equipped with 4 GB 1333
MHz DDR 3 RAM. Visualization of docked poses was accom-
plished using the latest available release of UCSF Chimera.
TARGET SELECTION. Four protein targets were retrieved from
the RSCB Protein Data Bank using the following accession
codes: 3CGY (virulence sensor PhoQ, Salmonella typhimurium)
and 1ID0 (virulence sensor, Escherichia coli), 1I58 (chemotaxis
HK CheA, Thermotoga maritima) and 2C2A (the kinase HK853,
Thermotoga maritima). The receptors were processed in order to
remove co-crystallized ligands and water molecules followed by
the addition of hydrogens using the Prepare Protein Structure
tool in SYBYL. Atom types were assigned using the AMBER
method and a staged-minimization was performed on the
hydrogens in the biopolymer using the AMBER7 FF99 force
eld.
COMPOUND LIBRARY. Compounds with reported activity
against TCS transduction were constructed using the SYBYL
sketcher, and their conformations were arrived at by optimiza-
tion via minimization with the Tripos force eld. Electrostatic
¨
charges were then assigned using the Gasteiger–Huckel
method. A larger set of 1000 drug-like compounds was obtained
from the ZINC small-molecule database and appended to the
database. The combined set was prepared for docking using the
“Sanitize” protocol in the Ligand Structure Preparation tool
found in SYBYL, which removed all counter ions.
DOCKING OF COMPOUNDS. The active sites of all receptors were
dened based on the coordinates of the co-crystallized ligand
and the Protomol Generation Mode in Surex using default
settings. A portion of the co-crystallized ligand was retained and
used as a guide for the placement of compounds into the active
site during docking.
SCORING. Upon completion of the docking screens, the
CScore module in SYBYL was used to rank the poses of docked
ligands. Although the primary criterion for inspecting high-
scoring members was the Surex assigned score, these highly
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ranked compounds were also visually inspected for favorable 20 J. F. Barrett, R. M. Goldschmidt, L. E. Lawrence, B. Foleno,
interactions with pertinent active-site residues. Compounds
that appeared to make interactions in the region leading into
the active site or had overly hydrophobic contacts with the
binding pocket were de-prioritized for further consideration.
R. Chen, J. P. Demers, S. Johnson, R. Kanojia,
J. Fernandez, J. Bernstein, L. Licata, A. Donetz, S. Huang,
D. J. Hlasta, M. J. Macielag, K. Ohemeng, R. Frechette,
M. B. Frosco, D. H. Klaubert, J. M. Whiteley, L. Wang and
J. A. Hoch, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 5317–5322.
21 E. C. Theodorou, M. C. Theodorou and D. A. Kyriakidis, Cell.
Signalling, 2011, 23, 1327–1337.
Acknowledgements
We thank D. Ma for assistance with the NMR spectrometers, C. 22 K. Stephenson, Y. Yamaguchi and J. A. Hoch, J. Biol. Chem.,
Dann and T. Stone for helpful discussions and F. Hardin, Jr for
2000, 275, 38900–38904.
generating HK853 protein. This work was supported by NIH 23 R. Gilmour, J. E. Foster, Q. Sheng, J. R. McClain, A. Riley,
R00GM82983, NIH DP2OD008592, a Pew Biomedical Scholar
Award (E.E.C.), and an Indiana University Quantitative and
Chemical Biology Training Fellowship (K.E.W.).
P.-M. Sun, W.-L. Ng, D. Yan, T. I. Nicas, K. Henry and
M. E. Winkler, J. Bacteriol., 2005, 187, 8196–8200.
ˇ
ˇ ´
ˇ ˇ
24 V. Skedelj, T. Tomasic, L. P. Masic and A. Zega, J. Med. Chem.,
2011, 54, 915–929.
25 J. E. Foster, Q. Sheng, J. R. McClain, M. Bures, T. I. Nicas,
K. Henry, M. E. Winkler and R. Gilmour, Microbiology,
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