New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis

Article abstract of DOI:10.1016/j.bmcl.2011.06.082

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagel

Full text of DOI:10.1016/j.bmcl.2011.06.082

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5583–5588
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis
Xin Hao a, Tam Nguyen a, Daniel B. Kearns b, Carolynn C. Arpin a, Ray Fall a, Tarek Sammakia a,
⇑
a Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA
b Department of Biology, Indiana University, Bloomington, IN 47405, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and
here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate
pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacte-
rials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures,
p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming,
was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and
only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-
spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify
more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was
found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoiso-
Received 16 April 2011
Revised 15 June 2011
Accepted 17 June 2011
Available online 25 June 2011
Keywords:
Bacillus subtilis
Bacillus anthracis
Colony spreading
p-Nitrophenylglycerol (NPG)
p-Methylsulfonylphenylglycerol (p-SPG)
mers showed that the 1R-anti-isomer was the most active with an average IC50 of 16
For B. anthracis, the colony-spreading IC50 values for 1R-anti-SPG and 1R-anti-NPG are 12
g mLÀ1) and >150
M, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony
lM (3–5
l
g mLÀ1).
M (2–
l
4
l
l
spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures.
Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead
to an understanding of the mechanism(s) of colony spreading as well as design and development of more
potent inhibitors for the control of B. anthracis surface cultures.
Ó 2011 Elsevier Ltd. All rights reserved.
Bacteria use a variety of motility mechanisms to colonize sur-
faces. These include swimming and swarming, both dependent
on flagella for motility, and twitching, gliding and sliding, more di-
verse flagella-independent mechanisms of motility (reviewed by
Harshey1). We have been interested in sliding motility in aflagel-
late Bacillus subtilis strains2 as this type of multicellular movement
on laboratory media can exhibit an unusual tendril-like growth
mode that precedes extensive web-like colony formation. We have
suggested that the formation of tendrils could be a growth strategy
for nutrient-deprived cells to colonize natural surfaces such as
plant roots that contain large populations of Bacillus species.3
When sufficient nutrients are available, the tendrils then spread
and form an entire colony or biofilm.
Notably, Henrichsen defined sliding motility as flagella-
independent surface translocation driven by expansive forces in
the growing colony combined with some type of cell-surface
mechanism to lower the friction between the cells and substrate.4
Henrichsen demonstrated this type of motility with Bacillus
anthracis (10 strains were examined), as well as with many other
Gram-negative and Gram-positive bacteria. In the case of B. sub-
tilis wild type strains, biosurfactants such as lipopeptides termed
surfactins,5 are secreted from the cells. We have shown that dis-
ruption of the srfAA gene of B. subtilis prevents surfactin produc-
tion and that the mutant does not produce tendrils or extensive
films;6 these growth phenotypes are rescued by addition of surf-
actin. These results confirm the essential role of extracellular sur-
factants in sliding motility in this species, as proposed by
Henrichsen.
Interestingly, B. anthracis not only lacks flagellar motility,4 but
there apparently have been no reports of biosurfactant properties
of its cells, capsule surfaces or extracellular products. This raises
the question of how this bacillus species lowers the surface tension
of the substrate to allow sliding motility. In addition, Henrichsen4
observed that sliding motility in B. anthracis and other bacteria oc-
curs as a monolayer of cells. This is clearly not the case with the
sliding motility we have studied in B. subtilis as the tendrils and
surface colonies are multicellular.2 A similar observation has been
discussed by Kaito and Sekimizu7 in their studies of flagella-
independent colony spreading in Staphylococcus aureus strains. Re-
cently, Stewart et al.8 discovered a type of sliding motility in the
Gram-negative species, Legionella pneumophila, and demonstrated
that this motility is dependent on type II protein secretion. This
suggests that a secreted L. pneumophila protein may lower surface
tension to allow colony expansion. At present, the exact mecha-
nism of sliding motility in Bacillus species is uncertain.
⇑
Corresponding author.
E-mail address: Sammakia@colorado.edu (T. Sammakia).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.082

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X. Hao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5583–5588
In the course of studying the tendril to surface colony transfor-
collapse method where 5 lL water drops are placed near the edges
mation of B. subtilis, we searched for various inhibitors of this type
of colony expansion. We reasoned that such inhibitors might be
colony-spreading or biofilm inhibitors. There are relatively few
known small molecule biofilm inhibitors in Gram-positive bacte-
ria,9 and apparently few reports of colony-spreading inhibitors.
An exception is described by Ogasa et al.,10 where the widely used
tetrazolium dye, 2,3,5-triphenyltetrazolium chloride (TTC), can
prevent colony overgrowth in enumeration of food and clinical
microbial species.
In this article, we describe experiments that take advantage of
flagella-less mutants of B. subtilis with a hag gene disruption in
an undomesticated parent as described previously.2,11 Lacking fla-
gella, these hag mutants can, apparently, only colonize surfaces by
the sliding mechanism described above. As described herein, we
have found a commercially available small molecule inhibitor of
colony spreading in hag mutants, and have used it as the lead mol-
ecule to synthesize a family of related compounds, and identified a
more potent inhibitor of Bacillus surface colony spreading. This
family of compounds also inhibits the colony spreading of B.
anthracis Sterne, an avirulent strain derived from a pathogenic an-
thrax-causing parent.12
of growing colonies or tendrils.6 Surfactin, a potent lipopeptide
surfactant, is needed for sliding motility in B. subtilis as mentioned
above.
The stereochemistry of the commercial sample of NPG used in
the experiment shown in Figure 1 was not indicated by the sup-
plier, but upon analysis by NMR and chiral HPLC (ChiralcellÒ OD
column), it was found to be a racemic mixture of the anti diastereo-
mer (Fig. 2, compounds 2 and 3). We wished to determine if the
activity of NPG is stereospecific, and therefore synthesized the four
possible isomers by the routes shown in Schemes 1 and 2. The syn-
thesis of the two syn enantiomers began with aldehyde 4, which
was subjected to the List-modification of the Knovenagel conden-
sation15 to provide
sponding allylic alcohol (6) was accomplished with DIBAL-H, and
dihydroxylation with either AD-mix- or AD-mix-b provided the
a,b-unsaturated ester 5. Reduction to the corre-
a
individual syn enantiomers, 1S,2S-NPG (1S-syn-, 7) and 1R,2R-
NPG (1R-syn-, 8), respectively, in high enantiomeric excess.16
The 1R,2S (1R-anti-) and 1S,2R (1S-anti-) NPG isomers 2 and 3
were also prepared starting with aldehyde 4 which was treated
with vinylmagnesium bromide to provide the corresponding allylic
alcohol and then subjected to Sharpless kinetic resolution17 using
Few colony-spreading inhibitors have been described in the lit-
erature, so we tested dozens of compounds for inhibition of surface
colony spreading by a B. subtilis hag gene mutant (DS64). Examples
of its colony spreading phenotypes are shown in Figure 1. The MK
agarose plates used represent a semi-solid medium with complete,
defined nutrients,2 and when inoculated with the hag mutant in
the center and incubated overnight (37 °C), a robust surface colony
forms (Fig. 1, left). However, when the plates contained relatively
either D- or L-diisopropyl tartrate to provide the R- and S-allylic
alcohols, 9 and 10, respectively, in high enantiomeric excess. The
R-allylic alcohol (9) was then subjected to Sharpless asymmetric
epoxidation using
in high enantiomeric excess. Similarly, subjection of S-allylic alco-
hol 10 to Sharpless asymmetric epoxidation using -diisopropyl
L-diisopropyl tartrate to provided epoxide 11
D
tartrate provided epoxide 12 in high enantiomeric excess. Epoxide
opening of 11 and 12 with water under acidic conditions occurred
at the terminal carbon to provide 1R-anti-NPG (2) and 1S-anti-NPG
(3). Related epoxide openings have been studied by Jäger et al.18
and found to also occur at the terminal position. These general
schemes were also used to synthesize analogs of NPG starting with
different aldehydes as described in the Supplementary data.
All 4 NPG isomers were tested on MK agarose plates like those
high levels of commercial nitrophenylglycerol (NPG; 75 l
g mLÀ1),
only surface tendrils form (Fig. 1, right). NPG has long been used
to inhibit the flagella-dependent swarming of Proteus strains (re-
viewed by Williams13), and it is thought to act by disruption of a
signal cascade in these Gram-negative bacteria, also including Ser-
ratia marcescens.14 It was a surprise that a flagellar swarming
inhibitor for Gram-negative bacteria would inhibit flagella-
independent colony-spreading in a Gram-positive bacterium.
This effect of NPG on Bacillus growth on the MK agarose med-
ium was not due to growth inhibition, as measured in MK broth,
or reduction of surfactin release, as measured by a water drop
shown in Figure 1. Each isomer was tested at 75 l
g mLÀ1 as shown
in Figure 3. In this experiment we used a marker-less hag mutant
(DS1677), constructed to eliminate possible polar effects of the
hag strain with an MLS antibiotic marker inserted in strain DS64.
As seen in Figure 3, left, the DS1677 strain produced web-like col-
onies indistinguishable from those produced by DS64 (Fig. 1). Un-
der these conditions, only the 1R-anti-NPG (2) showed substantial
inhibition of surface colony formation (Fig. 3) and as in the case of
commercial NPG, surface tendrils (but not colony formation) are
observed. No surface colony spreading inhibition was evident with
the 1S-anti- or 1S-syn-NPG isomers (3 or 7), and these plates
showed the same behavior as the control with web-like surface
colony formation being observed. Weak activity was visible with
1R-syn-NPG (8), where a much thinner, but entire surface colony
was observed with no distinct tendrils.
Having the four NPG stereoisomers also allowed us to assess the
stereospecificity of NPG inhibition of swarming in Proteus. Using
the plate swarm assay described in the Supplementary data, only
the 1R-anti- (2) and 1R-syn-NPG (8) isomers had activity; at the
levels tested (75
of the 1S-anti- (3) or 1S-syn-NPG (7) isomers (data not shown).
OH
l
g mLÀ1 in the plate) there was no visible effect
OH
OH
O2N
OH OH
OH OH
p-nitrophenylglycerol (NPG, 1)
2
2
3
3
1
1
Figure 1. The film spreading of B. subtilis hag mutant (DS64; Table S1 in the
Supplementary data), but not tendril formation, is inhibited by NPG (1). MK agarose
OH
OH
O2N
O2N
plates with (right) and without (left) 75 l
g mLÀ1 NPG (1) were inoculated in the
2
, 1R,2S- (1R-anti)
3, 1S,2R- (1S-anti)
center with the hag strain and grown for 24 h at 37 °C. These results are typical of
triplicate determinations. Note that tendril growth precedes film formation with
and without NPG.
Figure 2. Stereochemistry of commercial NPG.

X. Hao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5583–5588
5585
O
O
a
b
H
OMe
O2N
O2N
c
4
5
OH OH
2
3
1
OH
OH
O2N
7, 1S,2S- (1S-syn)
OH OH
O2N
2
3
6
1
d
OH
O2N
8, 1R,2R- (1R-syn)
Figure 3. 1R-anti-NPG, but not the 1S-syn or 1S-anti stereoisomers, inhibits surface
colony formation of a B. subtilis hag mutant (DS1677; Table S1 in the Supplemen-
tary data). The experiment was performed in triplicate as described in Fig. 1 using
Scheme 1. Synthesis of 1S,2S- (1S-syn-) and 1R,2R- (1R-syn-) NPG. Reagents and
conditions: (a) 2-(methoxycarbonyl)acetic acid, DMAP, piperidine, DMF; (b) DIBAL-
MK agarose plates with 75 l
g mLÀ1 of each compound in DMSO, including
H, Et2O; (c) AD-mix-
a
, 1:1 t-BuOH/water; (d) AD-mix-b, 1:1 t-BuOH/water.
commercial NPG (Acros, Inc.). Control plates contained 75
l
g mLÀ1 of DMSO, the
solvent used for each of the NPGs.
OH
b
O2N
O
a
9
H
O2N
OH
c
4
O2N
10
OH
OH
OH OH
d
d
c
2
3
9
1
O
O
OH
O2N
O2N
11
3
, 1S,2R- (1S-anti)
Figure 4. An example of the colony-spreading assay using a B. subtilis hag deletion
strain (Table S1 in the Supplementary data). Three microliters of an overnight LB
culture of hag DS1677 was spotted on TSB agar that had been dried for 3–4 h. The
plates (prepared in duplicate) contained the indicated amounts of 1R-anti-SPG, and
were allowed to grow at 32 °C for 16 h. Measurement of colony diameters allowed
OH OH
2
3
b
1
10
OH
O2N
determination of the IC50—in this case 3–5 l .
g mLÀ1
O2N
2
, 1R,2S- (1R-anti)
12
Scheme 2. Synthesis of 1S,2R- (1S-anti-) and 1R,2S- (1R-anti-) NPG. Reagents and
conditions: (a) vinylmagnesium bromide, THF; (b) Ti(i-PrO)4, -diisopropyl tartrate,
-diisopropyl tartrate, t-BuOOH, CH2Cl2; (d) H2O,
NPG isomers with significant activity were 1R-anti- and 1R-syn-
(2 and 8), with colony spreading inhibition of 25% and 16%, respec-
D
t-BuOOH, CH2Cl2; (c) Ti(i-PrO)4,
L
tively, at 30
inhibition of colony-spreading was determined to be approxi-
mately 122–132 M (25–27
g mLÀ1 in different independent
l
g mLÀ1 in the plates. For 1R-anti-NPG (2), its IC50 for
acetic acid.
Notably, in the type of colony growth inhibition assays shown
in Figures 1 and 3, it is not feasible to calculate IC50 values. That
is, it is difficult to find the inhibitory transition point between ten-
dril and colony growth. To calculate IC50 values for the NPG iso-
mers (and other analogs), we employed a colony-spreading assay
(S1) based on that used by Kaito and Sekimizu7 in studies with
Staphylococcus aureus. In this assay, a bacterial inoculum of hag
1677 is placed at the center of a suitable soft agar medium, and
after a defined period of incubation the diameter of the colony is
measured. We found that a medium of Tryptic soy broth solidified
with agar (7 g LÀ1) gave the most reproducible results. Typical re-
sults are shown in Figure 4. Each colony’s average diameter was
measured and the results of duplicate determinations for the
NPG stereoisomers are summarized in Table 1. Again, the only
l
l
experiments, each performed in duplicate). The stereochemistry
of the glycerol side chain is obviously important in the interaction
of the NPG stereoisomers with the cellular target, with the 1R,2R/S
stereochemistry presumably providing the best binding to this un-
known target.
With 1R-anti-NPG established as a lead compound, we wished
to determine its pharmacophore and develop more potent inhibi-
tors. Specifically, we studied the role of the nitro group and deter-
mined whether other polar groups on the aromatic ring could be
used in its place. In addition, we probed the effect of locating the
polar group at the meta-, or ortho-positions, and determined
whether the hydroxyl groups are required for activity. Our results
are shown in Table 1.

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X. Hao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5583–5588
We found that substitution of the para-nitro group of com-
pound 2 for a para-methyl sulfonyl group (SPG, compound 13) pro-
NPG (Table 1, entries 9 and 10; these substrates were prepared
as mixtures of all four stereoisomers). Other polar groups at the
para position, such as carbomethoxy (19), and thiomethyl (20),
provided compounds that were not active (Table 1, entries 11
and 12; these substrates were also prepared as mixtures of all four
stereoisomers). Surprisingly, the para-sulfonamide substituted
compound (21) was essentially inactive, while the parent com-
pound bearing a proton in place of the nitro group was of compa-
rable activity to NPG (Table 1, entries 13 and 14; these substrates
were prepared as mixtures of isomers).
vided a significantly more active compound (95% inhibition with
15 l lM) vs 25% inhibition with 30 l lM),
g mLÀ1 (60 g mLÀ1 (140
compare Table 1, entries 1 and 5). We next studied the effect of
varying the stereochemistry of the hydroxyl groups in the SPG ser-
ies. Consistent with our findings in the NPG series, the 1R-anti sub-
strate (13) was the most active, while the 1R-syn substrate (14)
was second most active (Table 1, entries 5 and 6). The 1S-anti-
(15) and 1S-syn- (16) compounds were essentially inactive (Table
1, entries 7 and 8).
Substitution of the para-nitro group with other polar substitu-
ents including hydroxy (17), and mesyloxy (18), provided com-
pounds of comparable or slightly reduced activity as compared to
Variation of the position of the sulfone was then studied and the
corresponding meta- and ortho- methylsulfones were prepared as
mixtures of all four stereoisomers. The meta-substituted com-
pound (23) showed significantly lower activity (10.5% inhibition),
Table 1
Screening of NPG and SPG analogs for inhibition of colony spreading of a B. subtilis hag mutant (DS1677a)
b,c
b,c
Entry
Compound
% Inhibition of colony spreading
Entry
Compound
% Inhibition of colony spreading
OH
OH
OH
OH
OH
OH
OH
1
24.8
12
0
OH
OH
MeS
O2N
20d
2
OH
OH
OH
2
3
4
5
6
7
8
16.1
1.5
0
13
14
15
16
17
18
19
4.5
12.9
10.5
0
OH
OH
O2N
H2NO2S
21d
8
OH
OH
OH
OH
OH
O2N
22
3
OH
OH
MeO2S
OH
OH
OH
O2N
23
7
OH
OH
OH
OH
95.3
69.1
1.6
0
OH
OH
MeO2S
MeO2S
MeO2S
MeO2S
SO2Me
13
14
15
16
24
OH
OH
OH
OH
OH
21.3
10.1
0
OH
MeO2S
MeO2S
MeO2S
25
OH
OH
OMe
OMe
OH
OH
26
OH
OMe
OH
OH
27

X. Hao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5583–5588
5587
Table 1 (continued)
b,c
b,c
Entry
Compound
% Inhibition of colony spreading
Entry
20
Compound
% Inhibition of colony spreading
OH
OMe
OH
OMe
OH
9
12.9
18.5
OH
OMe
HO
MeO2S
17d
28
29
OH
OMe
OH
10
11
11.5
21
22
0
0
OH
OH
MsO
MeO2S
18d
OH
OH
0
DMSO control
OH
MeO2C
19d
a
Results from colony expansion experiments like those in Figure 3, where each compound was tested in duplicate or triplicate (S1). Where replicates varied by more than
10% in average colony diameter, the assay for that compound was repeated.
b
NPG isomers were added at a concentration of 30
All other compounds were added at concentration of 15
Mixture of 4 stereoisomers.
l
g mLÀ1
.
c
l
g mLÀ1
.
d
while the ortho-substituted compound (24) was inactive (Table 1,
entries 15 and 16).
We then studied the effect of variation of the hydroxyl groups in
the 1R-anti-SPG series, and found that the presence of the hydroxyl
groups provided the most active compounds. Thus, the 1R-vinyl
alcohol substrate (25) that lacks hydroxyl groups at the 2- and 3-
positions while active, provided significantly reduced inhibition
(Table 1, entry 17, 21% inhibition), while methylation of any or
all of the hydroxyl groups provided compounds of low or no activ-
ity (Table 1, entries 18–21). These results indicate that the optimal
compounds in this series bear a para-methylsulfone substituent on
the aromatic ring, and hydroxyl groups on the aliphatic chain. The
stereochemistry of the hydroxyl groups is important for activity,
especially at the 1-position where the R-configuration is required
while at the 2-position the S-configuration is favored.
Figure 5. 1R-anti-SPG is an inhibitor of colony spreading in Bacillus anthracis Sterne.
Of the four SPG stereoisomers, each added at 75 g per disc, the 1R-anti-SPG isomer
is the most potent while the 1R-syn- isomer is still active, though less potent. The
l
As mentioned above, B. anthracis can colonize surfaces of labo-
ratory media by an unknown flagella-independent sliding mecha-
nism. With the results obtained with new colony-spreading
inhibitors of B. subtilis, it was logical to test them for colony-
spreading of B. anthracis; we used an avirulent B. anthracis strain12
for these experiments (Table S1 in the Supplementary data). As
with B. subtilis, we tried to use a colony-spreading assay based
on Tryptic soy broth and soft agar. When the agar level was re-
duced from 0.7% w/v, as used with B. subtilis, to 0.35% w/v and B.
anthracis Sterne cells were applied to relatively wet plates, an en-
tire giant colony formed overnight, analogous to that seen in S. aur-
eus and S. xylosus.7 As shown in Figure 5, when B. anthracis Sterne
colony growth was challenged with discs with each of the four SPG
stereoisomers, as with B. subtilis, the 1R-anti- and 1R-syn-SPG iso-
mers exhibited the greatest activity.
colony-spreading plates shown are Tryptic soy broth soft agar, described in the text,
and were inoculated in the center (white ‘X’) with 3 lL of a log phase B. anthracis
Sterne culture and grown overnight at 37 °C.
pathogenic infections of tissues. However, there is a dearth of liter-
ature on this point. Ironically, the lead inhibitor was discovered by
accident as a swarming inhibitor of certain Gram-negative bacte-
ria. Swarming is a multiflagellar motility phenotype, while the col-
ony spreading described here in B. subtilis hag strains and B.
anthracis is independent of the presence of flagella. When the cel-
lular target(s) of colony-spreading inhibitors are discovered it may
be possible to understand the relationship of swarming and colony
spreading inhibition by the NPG/SPG classes of compounds. In the
meantime, we have succeeded in synthesizing stereospecific ana-
logs of NPG, including one, para-1R-anti-SPG (13), that is a much
more effective inhibitor of multicellular colony expansion on
plates. It is interesting that 1R-anti-SPG inhibits colony spreading
on solidified media but is not bacteriostatic or bacteriocidal in li-
quid media. This is true for both B. subtilis and B. anthracis. This
strongly suggests that 1R-anti-SPG is acting to affect the expression
and/or function of some surface structure essential for colony
spreading. An important future goal is to discover the identity
and role of this surface structure.
For B. anthracis, the colony-spreading IC50 values for 1R-anti-
SPG and 1R-anti-NPG were found to be 12
>150 M, respectively. Thus, as with B. subtilis colony spreading,
lM (2–4 l
g mLÀ1) and
l
the 1R-anti-SPG is also much more effective than 1R-anti-NPG.
It is tempting to speculate that this work may have led to the
discovery of a new type of compound for the treatment of bacterial
infection: colony-spreading inhibitors. As summarized in the intro-
duction, the phenomenon of colony spreading on laboratory media
is widespread in bacteria, and conceivably could be related to

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X. Hao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5583–5588
3. Fall, R.; Kinsinger, R. F.; Wheeler, K. A. Syst. Appl. Microbiol. 2004, 27, 372.
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This work was supported by a seed money grant from the Grad-
uate School, University of Colorado, Boulder (to R.F. and T.S.) and in
part by the National Institutes of Health (GM-48498 to T.S.). Daniel
Kearns (Indiana University) was supported by NSF Grant MCB-
0721187. We thank Rebecca Kinsinger for help in the initial screen-
ing of colony-spreading inhibitors, and Colorado Serum, Denver,
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Supplementary data
Supplementary data (detailed bacterial colony spreading assays,
synthesis and characterization of all SPG and NPG isomers) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.06.082.
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