A bactericidal guanidinomethyl biaryl that alters the dynamics of bacterial FtsZ polymerization

Article

pubs.acs.org/jmc

A Bactericidal Guanidinomethyl Biaryl That Alters the Dynamics of

Bacterial FtsZ Polymerization

Malvika Kaul,^†Ajit K. Parhi,^‡Yongzheng Zhang,^‡Edmond J. LaVoie,^§Steve Tuske,^∥Eddy Arnold,^∥

John E. Kerrigan,^⊥and Daniel S. Pilch*^,†

^†Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,

Piscataway, New Jersey 08854-5635, United States

^‡TAXIS Pharmaceuticals, Inc., North Brunswick, New Jersey 08902, United States

^§Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854-8020,

United States

^∥Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University,

Piscataway, New Jersey 08854-5627, United States

^⊥Cancer Informatics Core, Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, United States

ABSTRACT: The prevalence of multidrug resistance among

clinically signiﬁcant bacterial pathogens underscores a critical need

for the development of new classes of antibiotics with novel

mechanisms of action. Here we describe the synthesis and evaluation

of a guanidinomethyl biaryl compound {1-((4′-(tert-butyl)-[1,1′-

biphenyl]-3-yl)methyl)guanidine} that targets the bacterial cell

division protein FtsZ. In vitro studies with various bacterial FtsZ

proteins reveal that the compound alters the dynamics of FtsZ self-

polymerization via a stimulatory mechanism, while minimally

impacting the polymerization of tubulin, the closest mammalian

homologue of FtsZ. The FtsZ binding site of the compound is

identiﬁed through a combination of computational and mutational

approaches. The compound exhibits a broad spectrum of bactericidal

activity, including activity against the multidrug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and

vancomycin-resistant Enterococcus (VRE), while also exhibiting a minimal potential to induce resistance. Taken together, our

results highlight the compound as a promising new FtsZ-targeting bactericidal agent.

viability, (ii) highly conserved among signiﬁcant bacterial

pathogens, and (iii) absent in humans. Bi and Lutkenhaus

identiﬁed FtsZ as the ﬁrst bacterial cell division protein, while

showing that it adopts a ring-like structure (termed the Z-ring)

at midcell.³²FtsZ forms the Z-ring via a process of GTP-

dependent self-polymerization.³³⁻³⁶The Z-ring plays a key role

in constriction of the cell membrane and serves as a scaﬀold for

recruitment of other components of the cell division machinery

(the divisome).^33,34,36Thus, FtsZ resides at the top of the

hierarchy of divisome assembly.

As part of a program to develop new chemical classes of

FtsZ-targeting antibacterial agents, we have synthesized libraries

of a broad array of heterocyclic molecules, including benzo[c]-

phenanthridines and dibenzo[a,g]quinoliziniums.^37,38In the

current study, we describe the chemical synthesis and

characterization of a guanidinomethyl biaryl compound that

acts as a stimulator of FtsZ self-polymerization and exhibits

potent bactericidal activity. The interactions of the compound,

INTRODUCTION

■

Multidrug resistance in bacterial pathogens has emerged as a

major global public health problem.¹⁻⁸Examples of multidrug-

resistant (MDR) strains of Gram-positive and Gram-negative

bacteria include methicillin-resistant Staphylococcus aureus

(MRSA), vancomycin-resistant Enterococcus (VRE), and

extended spectrum β-lactamase (ESBL)-producing Escherichia

coli and Klebsiella pneumoniae. The growing threat from MDR

bacterial pathogens highlights a critical need to expand our

currently available arsenal of antibiotics. Despite this critical

need, many of the new antibiotics that have reached the clinic

in the past 50 years have been next-generation derivatives of

existing drugs and thus suﬀer from the same propensity as their

predecessors to become targets of resistance pathways.⁹

Consequently, there is an urgent need for the development

of new classes of antibiotics that exhibit novel mechanisms of

action.

The bacterial cell division protein FtsZ has been recently

highlighted as an important new antibacterial target yet to be

clinically exploited.⁹⁻³¹FtsZ is particularly appealing as an

antibacterial target due to it being (i) essential for bacterial

Received: September 4, 2012

Published: October 10, 2012

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a

Scheme 1. Methods Used in the Preparation of the Guanidinomethyl Biaryl Analogues 13−15

a

Reagents and conditions: (a) 4-tert-Butylphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, dioxane:H₂O (3:1), 82−84%, (b) NaBH₄, EtOH, 70−75%, (c)

MsCl, Et₃N, DCM, 90% (for 7) and 70% (for 8), (d) NBS, CCl₄, light, 52%, (e) Diboc guanidine, DMF, K₂CO₃, quantitative, and (f) TFA:DCM

(1:1), quantitative.

and two of its aza-substituted analogues, with FtsZ proteins

from S. aureus, E. coli, and Enterococcus faecalis are investigated,

and the target site on the protein is identiﬁed using a

combination of computational, spectroscopic, mutational, and

biochemical analyses. The antibacterial activities of the

compounds are assessed against a broad array of both Gram-

positive and Gram-negative pathogens, including MDR strains

of S. aureus, E. faecalis, E. coli, and K. pneumoniae.

CHEMISTRY

■

The commercially available halogenated aryl aldehydes 1, 2,

and aryl bromide 3 were treated with 4-(tert-butyl)-

phenylboronic acid using standard Suzuki conditions to provide

the biaryl compounds 4, 5, and 6 in over 80% yield. Reduction

Figure 1. Chemical structures and calculated lipophilicities (ClogP) of

13−15. The indicated ClogP values were calculated using the weighted

of 4 and 5 using NaBH₄followed by reaction with excess MsCl

provided the corresponding chlorides 7 and 8 in very good

yield. Bromination of 6 in the presence of light with N-

bromosuccinimide in carbon tetrachloride furnished the 2-

(bromomethyl)aryl intermediate in moderate yield. These

chloro and bromomethyl intermediates were then treated

with 1,3-bis(tert-butoxycarbonyl)guanidine to give 10, 11, and

12. Removal of the bis-N-Boc protecting groups with

triﬂuoroacetic acid provided the desired guanidinomethyl

analogues 13−15 in quantitative yield.

method (VG = KLOP = PHYS = 1) in the Marvin 5.7 Software Suite

(ChemAxon, Ltd.), with Cl⁻and Na⁺/K⁺concentrations being set at

0.1 mol/dm³. The atomic numbering and ring lettering are indicated in

the structure of 13.

being shown in Figure 2. Note that addition of each of the three

FtsZ proteins increases the anisotropy of 13 in a concentration-

dependent manner, with the magnitude of the increase being

similar in the presence of SaFtsZ and EcFtsZ, while being

comparatively lower in the presence of EfFtsZ. This

observation is indicative of 13 binding to each host protein,

while also suggesting that the rotational diﬀusion of the

compound is greater when bound to EfFtsZ than to SaFtsZ or

EcFtsZ. The FtsZ-induced changes in 13 anisotropy (r) were

analyzed with the following 1:1 binding formalism to yield

compound−protein dissociation constants (K_d):

RESULTS AND DISCUSSION

■

Binding Aﬃnity of 13 for S. aureus, E. coli, and E.

faecalis FtsZ. We sought to determine whether the

synthesized compound 13 shown in Figure 1 interacts with

bacterial FtsZ proteins, and if so, to deﬁne the binding aﬃnity

of the compound for the host proteins. For these

determinations, we exploited the ﬂuorescence properties of

13, which exhibits an emission maximum at 318 nm when

excited with middle UV light at a wavelength of 245 nm.

Puriﬁed S. aureus, E. coli, and E. faecalis FtsZ (denoted SaFtsZ,

EcFtsZ, and EfFtsZ, respectively) were used as model bacterial

FtsZ proteins in these experiments. The ﬂuorescence

anisotropy of 13 was measured in the presence of increasing

FtsZ concentrations, with the resulting anisotropy proﬁles

r_∞− r

r = r₀+

⁰[([C]_tot+ [P]_tot+ K_d)

2

−

([C]_tot+ [P]_tot+ K_d)²− 4[C]_tot[P]_tot

]

(1)

In this relationship, r₀and r are the anisotropies of the

compound in the absence and presence of protein, respectively,

r_∞is the anisotropy of the compound in the presence of an

inﬁnite protein concentration, and [C]_totand [P]_totare the total

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13 on the polymerization dynamics of puriﬁed SaFtsZ, EcFtsZ,

and EfFtsZ was assessed. Figure 3 shows the time-dependent

A

₃₄₀proﬁles of SaFtsZ (panel A), EcFtsZ (panel B), and EfFtsZ

(panel C) in the presence of 13 at concentrations ranging from

0 to 40 μg/mL. Note that 13 increases both the kinetics and

extent of SaFtsZ, EcFtsZ, and EfFtsZ polymerization, with the

magnitude of these stimulatory eﬀects increasing with

increasing compound concentration. It has been previously

reported that the antistaphylococcal substituted benzamide

PC190723 exerts a similar stimulatory impact on SaFtsZ

polymerization.^15,24,25However, unlike 13, PC190723 does not

aﬀect the polymerization of EcFtsZ.²⁵

There is a marked increase in 13-induced stimulation of

EcFtsZ polymerization when the compound concentration is

increased from 30 to 40 μg/mL (Figure 3B), with the same

being true for the induced stimulation of EfFtsZ polymerization

when the concentration of 13 is increased above 20 μg/mL

(Figure 3C). This behavior is likely due to an increase in the

extent of FtsZ polymer bundling at the higher compound

concentrations. The presence of large bundles of FtsZ polymers

may also underlie the noisy nature of the A₃₄₀proﬁles of SaFtsZ

and EfFtsZ at the higher concentrations of 13 (Figure 3A,C).

Structure−Activity Relationship (SAR) of 13−15: Aza

Substitution of 13 Diminishes Stimulation of FtsZ

Polymerization. The impacts of 14 and 15 on SaFtsZ,

EcFtsZ, and EfFtsZ polymerization were also determined for

subsequent comparison with the corresponding behavior of 13.

Recall that 14 and 15 are aza-substituted analogues of 13, with

14 containing a single aza substitution at position 4 and 15

containing two aza substitutions at positions 2 and 4 (see

Figure 1). At equivalent concentrations of 40 μg/mL, the extent

to which the three compounds stimulate the polymerization of

all three host FtsZ proteins follows the hierarchy: 13 > 14 > 15

(see Figure 3D−F). Thus, the monoaza substitution in 14

adversely impacts the ability of the compound to stimulate FtsZ

polymerization relative to the unsubstituted compound 13, with

the corresponding adverse impact of the diaza substitution in

15 being even greater still. Note that each aza substitution

results in a decrease in compound hydrophobicity, as reﬂected

by the decreasing ClogP values of 13 (ClogP = 3.96), 14

(ClogP = 2.82), and 15 (ClogP = 2.50). Signiﬁcantly, this

ClogP hierarchy is identical to that deﬁned above for the extent

of FtsZ polymerization stimulation by the three compounds.

This correlation implies that the ability to stimulate FtsZ

polymerization is inﬂuenced by the hydrophobicity of the

compound, with increasing hydrophobicity resulting in

enhanced stimulation of FtsZ polymerization. We have

observed a similar pattern with other classes of heterocyclic

compounds.^37,38

Figure 2. Fluorescence anisotropies of 5 μM 13 as a function of

■

●

○

increasing concentrations of SaFtsZ ( ), EcFtsZ ( ), or EfFtsZ ( ).

The solid lines reﬂect the nonlinear least-squares ﬁts of the data with

eq 1. Experiments were conducted at 25 °C in solution containing 50

mM Tris·HCl (pH 7.4), 50 mM KCl, and 2 mM magnesium acetate.

concentrations of compound and protein, respectively. Note

that the 1:1 binding formalism yields excellent ﬁts (R²> 0.99)

of all three 13 anisotropy proﬁles (the solid lines in Figure 2).

The K_dvalues obtained from these ﬁts are listed in Table 1. The

Table 1. Aﬃnity of 13 for S. aureus, E. coli, and E. faecalis

a

FtsZ at 25 °C

FtsZ

K_d(μM)

S. aureus

E. coli

3.5 0.5

3.8 0.3

21.8 4.6

E. faecalis

a

Solution conditions were 50 mM Tris·HCl (pH 7.4), 50 mM KCl,

and 2 mM magnesium acetate.

interactions of 13 with SaFtsZ and EcFtsZ are associated with

similar K_dvalues of 3.5

0.5 μM and 3.8

0.3 μM,

respectively, the diﬀerence between the two values being within

the experimental uncertainty. The corresponding interaction of

EfFtsZ with 13 is associated with a K_dvalue of 21.8 4.6 μM,

indicating that the aﬃnity of 13 for the FtsZ protein from E.

faecalis is approximately 6-fold lower than the corresponding

aﬃnity of the compound for the FtsZ proteins from S. aureus

and E. coli. Taken together, these results indicate that 13 binds

to the three FtsZ proteins with a stoichiometry of one

compound molecule per protein and aﬃnities in the range of

3.5 0.5 to 21.8 4.6 μM. Although we also investigated the

binding of 14 and 15 to the three FtsZ proteins, the weak

nature of the binding interactions precluded the quantitative

assessment of K_dvalues for these compounds, due to large

experimental uncertainties.

13 Stimulates the Polymerization Dynamics of

SaFtsZ, EcFtsZ, and EfFtsZ in a Concentration-Depend-

ent Manner. We next sought to examine whether the binding

of 13 to FtsZ had an impact on the self-polymerization activity

of the protein. To assay this FtsZ function, we utilized a

microtiter plate-based light-scattering assay in which FtsZ

polymerization is detected in solution by a time-dependent

increase in light scattering, as reﬂected by a corresponding

increase in solution absorbance at 340 nm (A₃₄₀). Using this

light-scattering assay, the impact of increasing concentrations of

Also included in the polymerization proﬁles shown in Figure

3D−F are four non-FtsZ-targeting control drugs for each FtsZ

protein investigated. Clindamycin and oxacillin were used as

non-FtsZ-targeting controls in the SaFtsZ polymerization

assays, as these agents exhibit potent antistaphylococcal activity.

For similar reasons of antibacterial speciﬁcity, ampicillin and

vancomycin were used as the controls agents in the

polymerization assays of EcFtsZ and EfFtsZ, respectively. The

four control antibiotics target either the bacterial cell wall

(oxacillin, ampicillin, and vancomycin) or bacterial protein

synthesis (clindamycin).^39,40As expected, and in marked

contrast to 13, equivalent concentrations of the non-FtsZ-

targeting control drugs exert a negligible impact on FtsZ

polymerization.

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Figure 3. Concentration dependence and structure−activity relationship of the impact of the biaryl compounds on the polymerization of SaFtsZ (A

and D), EcFtsZ (B and E), and EfFtsZ (C and F), as determined by monitoring time-dependent changes in absorbance at 340 nm (A₃₄₀) at 25 °C.

(A−C) The time-dependent A₃₄₀polymerization proﬁles of each target FtsZ in the presence of 13 at the indicated concentrations. (D−F) Time-

dependent A₃₄₀polymerization proﬁles of each target FtsZ in the presence of vehicle (DMSO) only or 40 μg/mL of 13, 14, 15, or the indicated

comparator drugs. Experimental conditions for all the FtsZ polymerization studies were 10 μM protein, 50 mM Tris·HCl (pH 7.4), 50 mM KCl, 2

mM magnesium acetate, 1 mM CaCl₂(for SaFtsZ) or 10 mM CaCl₂(for EcFtsZ and EfFtsZ), and 1 mM GTP.

13 Promotes the Assembly and Bundling of FtsZ

Polymers As Revealed by Electron Microscopy. We

visualized the FtsZ polymers induced by 20 μg/mL 13 using

transmission electron microscopy (EM). Negatively stained

electron micrographs of 13-induced polymers of SaFtsZ and

EcFtsZ are shown in Figure 4. Inspection of these images

reveals that 13 induces long ﬁlamentous polymers in both

SaFtsZ and EcFtsZ. However, the widths of the ﬁlaments

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uniform width of approximately 4 to 6 nm (with the arrow in

Figure 4B highlighting a representative 5-nm ﬁlament) and

likely reﬂect single EcFtsZ polymers that are not bundled

together. Note that, under the experimental conditions

employed, neither SaFtsZ nor EcFtsZ polymers were

observable in the absence of added compound.

Viewed as a whole, the EM data suggest that, at 20 μg/mL,

13 promotes both the assembly and bundling of SaFtsZ

polymers, while promoting only the assembly, and not the

bundling, of EcFtsZ polymers. This diﬀerence in the impact of

13 on SaFtsZ versus EcFtsZ polymerization and bundling may

account for the diﬀerence in the magnitudes of the light-

scattering (A₃₄₀) signals of the two proteins induced by the

compound (compare Figure 3A,B). At each equivalent

concentration of 13, the extent of light scattering is greater

for SaFtsZ than for EcFtsZ, which may reﬂect the presence of

thick ﬁlaments of bundled polymers in the SaFtsZ solution that

are absent from or reduced in the EcFtsZ solution. Thick

ﬁlaments of bundled FtsZ polymers would scatter light more

eﬀectively than thin ﬁlaments of single FtsZ polymers.

The Interaction of GTP with SaFtsZ, EcFtsZ, and

EfFtsZ Is Minimally Aﬀected by the Presence of 13. The

studies described above indicate that 13 binds to bacterial FtsZ

and stimulates its self-polymerization in the presence of GTP.

These ﬁndings prompted further investigation into the nature

of the FtsZ binding site targeted by 13. Our initial

investigations focused on determining whether 13 targeted

the GTP binding pocket of FtsZ. Toward this end, we

developed a ﬂuorescence anisotropy competition assay to assess

the impact, if any, of 13 binding on the interaction of SaFtsZ,

EcFtsZ, and EfFtsZ with GTP. Note that in this assay, the

anisotropy of a ﬂuorescent nonhydrolyzable GTP analogue

(composed of GTPγS conjugated to the ﬂuorescent dye

BODIPY and, hereafter, denoted as BoGTPγS) was monitored

rather than the anisotropy of 13. Speciﬁcally, the impact of

increasing concentrations of 13 on the anisotropy of FtsZ-

bound BoGTPγS was recorded and compared with the

corresponding anisotropy of unbound (FtsZ-free) BoGTPγS.

Upon binding to each target FtsZ, the anisotropy of

BoGTPγS increases by approximately 5- to 6-fold (Figure 5).

If 13 targets the nucleotide binding pocket of FtsZ, then it

should compete with the binding of BoGTPγS, thereby

resulting in the release of the FtsZ-bound nucleotide analogue

and a concomitant decrease in BoGTPγS anisotropy. For

comparative purposes, unlabeled nonﬂuorescent GTP was

included as a positive control in these assays. Inspection of the

anisotropy proﬁles in Figure 5A−C reveals that unlabeled GTP

induces a marked, concentration-dependent decrease in the

anisotropy of FtsZ-bound BoGTPγS, consistent with the

expected GTP-induced release of FtsZ-bound BoGTPγS. By

contrast, 13 does not signiﬁcantly alter the anisotropy of

BoGTPγS bound to SaFtsZ, EcFtsZ, or EfFtsZ, even at a 40-

fold excess of 13 (40 μM) relative to BoGTPγS (1 μM). These

observations suggest that 13 binding to SaFtsZ, EcFtsZ, and

EfFtsZ does not induce the release of bound GTP and are

therefore inconsistent with the nucleotide binding pocket of

FtsZ serving as the target site for the compound.

Figure 4. Transmission electron micrographs of 13-induced ﬁlaments

of (A) SaFtsZ and (B) EcFtsZ. Polymerization of the proteins was

induced by addition of 20 μg/mL 13 under the experimental

conditions described in the legend to Figure 3. The ﬁlaments were

negatively stained with a solution of 1% phosphotungstic acid (PTA)

and visualized at 80 kV on a Philips CM12 transmission microscope

interfaced with an AMT XR111 camera. The red, white, and yellow

arrows in panel A highlight ﬁlaments 5, 11, and 25 nm wide,

respectively. The red arrow in panel B highlights a 5 nm wide ﬁlament.

observed for SaFtsZ vary widely from approximately 4 to 25

nm, while the widths of the EcFtsZ ﬁlaments have a narrow

range of approximately 4 to 6 nm. The arrows in Figure 4A

highlight SaFtsZ ﬁlaments approximately 5 nm (red arrow), 11

nm (white arrow), and 25 nm (yellow arrow) in width.

Previously reported crystal structures of both Gram-positive

and Gram-negative bacterial FtsZ proteins have revealed

molecular widths ranging from approximately 45 to 60 Å in a

perpendicular direction to the protoﬁlament axis.^15,41−44It is,

therefore, likely that the 5-nm ﬁlament corresponds to a single

SaFtsZ polymer, the 11-nm ﬁlament corresponds to two SaFtsZ

polymers bundled together, and the 25-nm ﬁlament corre-

sponds to a bundle of four to ﬁve SaFtsZ polymers. In contrast

to the SaFtsZ ﬁlaments, the EcFtsZ ﬁlaments have a relatively

Computational Studies Suggest That 13 Binds SaFtsZ

at a Site Distal from the GTP Binding Pocket. As our

ﬂuorescence anisotropy competition assays ruled against the

GTP binding pocket as the target site for 13, we probed for

other potential FtsZ binding sites for the compound using a

computational approach. In this computational approach

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found that the compound preferentially targeted a pocket in

SaFtsZ located near the C-terminal domain of the protein, at a

site distinct from the GTP binding pocket (see Figure 6).

Signiﬁcantly, this pocket is in the same vicinity as the purported

binding site of the FtsZ polymerization stimulator

PC190723.^15,24,25Figure 6B,C presents expanded views of 13

bound to SaFtsZ. Note that residues E185 and I228 form key

interactions with the compound, the ﬁrst being an electrostatic

contact between the acidic side chain of E185 and the basic

guanidinomethyl functionality of the compound and the second

being a van der Waals contact between the branched alkyl side

chain of I228 and phenyl ring A of the compound.

Site-Directed Mutagenesis of the E185 and I228

Residues of SaFtsZ Reduce Aﬃnity for 13, as well as

the Extent to Which the Compound Stimulates SaFtsZ

Polymerization. If 13 does indeed target the SaFtsZ site

indicated by our computational studies, then mutations

intended to disrupt the contacts between 13 and the E185

and I228 residues should diminish the aﬃnity of the compound

for the protein. To this end, we made two diﬀerent mutant

forms of SaFtsZ, one with the E185 residue mutated to alanine

(E185A) and the other with the I228 residue mutated to

arginine (I228R). We then used the 13 ﬂuorescence anisotropy

assay described above to determine the binding aﬃnity of the

compound for each of the mutant proteins. As observed for the

wild-type protein, addition of either of the mutant proteins to

13 increased the anisotropy of the compound in a

concentration-dependent manner (Figure 7). However, the

magnitudes of the increases at equivalent protein concen-

trations were signiﬁcantly lower for the mutants relative to the

wild-type protein. The FtsZ-induced changes in 13 anisotropy

were analyzed with eq 1 to yield the K_dvalues listed in Table 2.

Signiﬁcantly, 13 binds to the E185A and I228R mutant proteins

(K_d= 38.3

5.4 and 32.9

6.2 μM, respectively) with an

approximately 10-fold lower aﬃnity than the wild-type protein

(K_d= 3.5 0.5 μM). These results validate the 13 target site

on SaFtsZ identiﬁed by the computational studies. The reduced

aﬃnity of 13 for the E185A mutant relative to the wild-type

protein correlates with the intended disruption of favorable

electrostatic contacts between the basic guanidino functionality

on the compound and the carboxylate group on E185.

Similarly, the reduced aﬃnity of 13 for the I228R mutant

relative to the wild-type protein likely includes contributions

from the intended abrogation of favorable hydrophobic

contacts between ring A of the compound and I228. However,

it is also possible that the reduced aﬃnity for the I228R mutant

may include contributions from unfavorable electrostatic

repulsive forces between the basic guanidino functionality on

the introduced arginine and that on the compound.

Previous studies have shown that a G196A mutation in

SaFtsZ confers resistance to the FtsZ polymerization stimulator

PC190723.^15,24,25Our computational studies indicate that the

G196 residue of SaFtsZ does not form any contacts with bound

13. Thus, a G196A mutation should not impact the aﬃnity of

13 for the protein. To test this hypothesis, we made a third

mutant SaFtsZ protein containing the G196A mutation and

assessed its aﬃnity for 13. This assessment yielded a K_dvalue

(5.0 0.7 μM) similar in magnitude to that associated with the

interaction of 13 with the wild-type protein, lending further

support to the computationally deﬁned 13 binding site on

SaFtsZ.

Figure 5. Fluorescence anisotropies of SaFtsZ-bound (A), EcFtsZ-

bound (B), and EfFtsZ-bound (C) BoGTPγS (1 μM BoGTPγS, 5 μM

FtsZ) as a function of increasing concentrations of nonﬂuorescent

unlabeled GTP ( ) or 13 ( ). The dashed line represents the

anisotropy value of 1 μM BoGTPγS in the absence of FtsZ (FtsZ-free

BoGTPγS). Experimental conditions were as described in the legend

to Figure 2.

●

○

(detailed in Experimental Section), we ﬁrst developed a

homology model for SaFtsZ using a reported crystal structure

of Bacillus subtilis FtsZ,¹⁵which has an 81% sequence identity

with SaFtsZ. We then used the Autodock Vina docking

algorithm to dock 13 to the structural model of SaFtsZ and

We next sought to determine whether the E185A and I228R

mutations impact the polymerization activity of SaFtsZ using

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Figure 6. continued

molecular docking program. The E185 and I228 residues of the

protein are indicated in magenta and yellow, respectively. 13 and the

bound nucleotide are depicted as a stick models and color coded by

atom (carbon in green, nitrogen in blue, oxygen in red, phosphorus in

orange, and hydrogen in white). (B, C) Expanded views of 13 bound

to SaFtsZ. In panel B, the dashed lines reﬂect hydrogen bonds

between the basic guanidinomethyl functionality of the compound and

the acidic side chain of E185. In panel C, the protein is depicted in its

solvent-accessible surface and color coded according to its electrostatic

potential (blue for positive, red for negative, and white for neutral).

Figure 7. Fluorescence anisotropies of 5 μM 13 as a function of

●

○

increasing concentrations of wild-type ( ), I228R mutant ( ), or

■

E185A mutant ( ) SaFtsZ. The solid lines reﬂect the nonlinear least-

squares ﬁts of the data with eq 1. Experimental conditions were as

described in the legend to Figure 2.

Table 2. Impact of the E185A and I228R Mutations on the

a

Aﬃnity of 13 for SaFtsZ at 25 °C

SaFtsZ

K_d(μM)

wild-type

E185A

I228R

3.5 0.5

38.3 5.4

32.9 6.2

a

Solution conditions were as described in the footnote to Table 1.

the light-scattering assay described above. Figure 8 shows the

time-dependent A₃₄₀proﬁles of wild-type (panel A), E185A

(panel B), and I228R (panel C) SaFtsZ in the absence of

compound (the black proﬁles in each panel). The extent of

polymerization exhibited by the E185A mutant protein is

reduced in comparison to the wild-type protein, while the

extent of polymerization exhibited by the I228R mutant protein

is slightly higher than that exhibited by the wild-type protein. In

addition, the shape of the time-dependent A₃₄₀proﬁle of the

I228R mutant protein diﬀers from the corresponding shape of

wild-type proﬁle in that it rises to a maximal value at

approximately 15 min and then decreases, a behavior that

likely reﬂects a time-dependent depolymerization from larger to

smaller polymeric structures on the part of the I228R mutant

protein. These collective results indicate that both the E185R

and I228R mutations do impact the intrinsic polymerization

activity of SaFtsZ to some degree, with the nature of this

impact depending on the mutation.

Figure 6. Structural homology model of SaFtsZ in complex with 13

derived as detailed in Experimental Section. (A) SaFtsZ is schemati-

cally depicted according to its secondary structural elements (α-helices

in red, β-strands in cyan, and loops in gray), with the GTP binding

pocket and T7 loop (which is involved in nucleotide hydrolysis when

the proteins are polymerized) indicated. The 13−FtsZ complex shown

reﬂects the highest scoring complex obtained using the Autodock Vina

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Figure 8. Concentration dependence of the impact of 13 on the polymerization of wild-type (WT) (A), E185A mutant (B), and I228R mutant (C)

SaFtsZ, as determined by monitoring time-dependent changes in A₃₄₀. The time-dependent A₃₄₀proﬁles of 10 μM protein are shown in the absence

(black) and presence of 13 at concentrations of 10 (red) or 20 (green) μg/mL. Experimental conditions were as described in the legend to Figure 3.

Table 3. Antibacterial Activities of 13−15 against S. aureus and E. faecalis

MIC (μg/mL)

a

compound or

control agent

S. aureus 8325-4 S. aureus ATCC 49951 S. aureus ATCC 33591

S. aureus Mu3

E. faecalis ATCC

19433 (VSE)

E. faecalis ATCC

51575 (VRE)

(MSSA)

(MRSA)

13

1.0

8.0

1.0

8.0

1.0

16.0

1.0

8.0

4.0

32.0

>64.0

8.0

4.0

64.0

14

15

64.0

0.063

0.031

0.125

0.5

32.0

0.25

0.063

0.25

0.5

64.0

32.0

>64.0

64.0

oxacillin

clindamycin

erythromycin

vancomycin

>64.0

2.0

>64.0

2.0

>64.0

1.0

a

Mu3 is a clinical MRSA isolate also identiﬁed as being a hetero-glycopeptide-intermediate S. aureus (hetero-GISA) strain.

We also explored how the E185A and I228R mutations aﬀect

(MIC >64.0 μg/mL) but are also cross-resistant to other

clinical antibiotic classes, including the lincosamides (e.g.,

clindamycin) and the macrolides (e.g., erythromycin). Sig-

niﬁcantly, 13 exhibits potent activity against all four S. aureus

strains, with an MIC value of 1 μg/mL versus each of the

strains. This MIC value associated with 13 is similar to the

corresponding MIC values associated with the clinical

glycopeptide antibiotic vancomycin (MIC values ranging from

0.5 to 2.0 μg/mL). Although 13 is not as potent as oxacillin,

clindamycin, and erythromycin against the two MSSA strains

tested, it is worthy to note that it exhibits potent antibacterial

activity against both the MRSA strains for which all three

comparator antibiotics are ineﬀective. This observation under-

scores the potential value of 13 as a promising lead compound

with antimicrobial activity against the MDR pathogen MRSA.

A comparison of the MIC values of 13−15 for both MSSA

and MRSA reveals that the antistaphylococcal potencies of the

compounds follow the hierarchy: 13 > 14 > 15 (Table 3). Note

the correlation between this hierarchy and that deﬁned above

with regard to stimulation of SaFtsZ polymerization (Figure

3D). This gratifying concordance is consistent with FtsZ

serving as the antibacterial target of these compounds.

We also investigated the activities of 13−15 against a second

Gram-positive bacterial species, E. faecalis, using both

vancomycin-sensitive and vancomycin-resistant strains (VSE

and VRE, respectively) as models (Table 3). The VSE strain

(ATCC 19433) is sensitive to all four comparator antibiotics

examined (vancomycin, oxacillin, clindamycin, and erythromy-

cin), with MIC values ranging from 1 to 8 μg/mL. By contrast,

the VRE strain (ATCC 51575) is resistant to all four

comparator agents (MIC ≥64 μg/mL). Signiﬁcantly, 13 is

equally active against VSE and VRE (MIC = 4 μg/mL). The

SAR for 13−15 against E. faecalis follows the same hierarchy

the stimulation of SaFtsZ polymerization by 13, with the results

of these assessments being shown in Figure 8. At a

concentration of 10 μg/mL, 13 exerts a stimulatory impact

on wild-type SaFtsZ polymerization but has essentially no eﬀect

on the polymerization of either mutant protein. Only when 13

is present at a concentration of 20 μg/mL does it stimulate the

polymerization of the mutant proteins, but, even then, the

magnitude of the stimulation is markedly lower than that

observed for wild-type SaFtsZ . As noted above with regard to

impact on 13 binding aﬃnity, the G196A mutation had a

negligible eﬀect on the stimulation of SaFtsZ polymerization by

13 (not shown). Taken together, these results indicate that the

interactions of 13 with the E185 and I228 residues of SaFtsZ

play an important role not only in facilitating the binding

interaction but also in compound-induced stimulation of

polymerization. Recall that reduction of 13 hydrophobicity

via mono and diaza substitution of ring A (to yield 14 and 15,

respectively) resulted in reduced FtsZ binding as well as

reduced stimulation of FtsZ polymerization. On the basis of our

mutational studies, it is likely that this behavior stems, at least in

part, from the key hydrophobic interactions formed between

I228 and compound ring A.

SAR of 13−15 with Regard to Antibacterial Activity

against S. aureus, E. faecalis, and E. coli. The antibacterial

activities of 13−15 against S. aureus were evaluated using two

MSSA strains (8325-4 and ATCC 49951) and two MRSA

strains (ATCC 33591 and Mu3). The ATCC 49951 MSSA

strain is a heavily encapsulated (CP⁺⁺) strain denoted as a

mucoid strain.⁴⁵Mu3 is a MRSA clinical isolate also classiﬁed as

being a hetero-glycopeptide-intermediate S. aureus (hetero-

GISA).⁴⁶As shown in Table 3, the two MRSA strains examined

are not only resistant to the antistaphylococcal drug oxacillin

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noted above for S. aureus (13 > 14 > 15) and also correlates

well with the corresponding SAR for the stimulation of EfFtsZ

polymerization (Figure 3F).

expression of RND-type eﬄux pumps such as AcrAB by the

Gram-negative bacteria.⁴⁹

In addition to their SAR against the Gram-positive bacteria

discussed above, the corresponding SAR of 13−15 against the

Gram-negative bacteria E. coli was also assessed. Three diﬀerent

E. coli strains were used in these assessments (summarized in

Table 4), an AcrAB eﬄux pump mutant strain (N43) and its

Table 5. Activity of 13 against Other Gram-Positive and

Gram-Negative Bacteria

bacterial strain

ATCC no.

MIC (μg/mL)

S. pyogenes (Strep A)

S. agalactiae (Strep B)

B. subtilis

19615

12386

23857

19606

13883

700603

2.0

16.0

4.0

Table 4. Antibacterial Activities of 13−15 against E. coli

A. baumannii

16.0

32.0

16.0

MIC (μg/mL)

K. pneumoniae

a

b

a

compound or control

agent

ATCC BAA-201

(ESBL)

N43

K. pneumoniae (ESBL)

W4573

(acrA1)

a

ATCC 700603 is an ESBL-producing strain of K. pneumoniae that

13

16.0

64.0

16.0

64.0

>64.0

4.0

2.0

8.0

expresses the SHV-18 lactamase.

14

15

>64.0

32.0

2.0

13 is Bactericidal against S. aureus and E. coli. The

antibacterial activity of 13 highlighted by the results in Tables

3−5 prompted further investigation as to whether this activity is

bactericidal or bacteriostatic in nature. To this end, the MBC

values of 13 against S. aureus 8325-4 (MSSA) and E. coli

W4573 were determined and subsequently compared with

corresponding MIC values. The bactericidal antistaphylococcal

drug vancomycin and the bacteriostatic drug erythromycin were

used as comparator controls in the S. aureus determinations,

with the bactericidal aminoglycoside neomycin being used as a

comparator control in the E. coli assays.^39,50As per CLSI

standards, a MBC/MIC ratio of 1 to 2 is considered indicative

of bactericidal behavior.⁵¹By contrast, an MBC/MIC ratio ≥8

is viewed as being indicative of bacteriostatic behavior. As

expected, the control bactericidal agents vancomycin and

neomycin yielded MBC/MIC ratios of 2, with a corresponding

ratio of 256 being observed for the bacteriostatic agent

erythromycin (see Table 6). Signiﬁcantly, 13 exhibits identical

ampicillin

amikacin

2.0

1.0

a

ATCC BAA-201 is an extended spectrum β-lactamase (ESBL)-

b

producing strain that expresses the TEM-3 lactamase. N43 is an

AcrAB eﬄux pump mutant strain of W4573.

wild-type homologue (W4573),⁴⁷as well as an extended

spectrum β-lactamase (ESBL)-producing strain (ATCC BAA-

201) that expresses the TEM-3 lactamase. The β-lactam

antibiotic ampicillin and the aminoglycoside amikacin were

used as comparator control agents in these assays, as these

drugs have known activities versus E. coli. Note that the ESBL-

producing strain is resistant (MIC >64.0 μg/mL) not only to

the β-lactam drug ampicillin but also to amikacin. By contrast,

13 is active (MIC of 16.0 μg/mL) against the ESBL-producing

strain, while also exhibiting a similar activity against W4573. It

is also worthy of emphasis that the activity of 13 against N43 is

8-fold more potent (MIC = 2.0 μg/mL) than its activity against

W4573, an observation indicating that the compound is a

substrate for the AcrAB eﬄux transporter. This ﬁnding provides

insights into potential strategies to improve the potency of 13

versus AcrAB-expressing E. coli, including cotreatment of the

compound with AcrAB eﬄux pump inhibitors (EPIs), such as

phenylalanyl arginyl β-naphthylamide (PAβN).⁴⁸

Further inspection of the MIC data in Table 4 reveals that

the SAR of 13−15 for all three E. coli strains follows the same

hierarchy of activity observed for S. aureus and E. faecalis (13 >

14 > 15), as well as the corresponding SAR for the stimulation

of EcFtsZ polymerization (Figure 3E). As with 13, the activities

of both 14 and 15 are more potent against N43 than W4573,

indicating that these compounds are also substrates of the

AcrAB eﬄux pump.

Table 6. Comparison of MIC and MBC Values for 13 in S.

aureus and E. coli

MBC/

MIC

a

compound or control agent MIC (μg/mL) MBC (μg/mL)

S. aureus 8325-4

13

1.0

0.5

1.0

1

2

vancomycin

erythromycin

0.125

32.0

256

E. coli W4573

16.0

13

16.0

2.0

1

2

neomycin

1.0

a

Vancomycin and neomycin are included as control bactericidal

agents, while erythromycin is included as a control bacteriostatic agent.

Activity of 13 against Other Gram-Positive and Gram-

Negative Bacteria. The antibacterial activity of 13 was further

explored against other Gram-positive and Gram-negative

bacteria. The Gram-positive organisms examined were

Streptococcus pyogenes (Strep A), Streptococcus agalactiae

(Strep B), and B. subtilis, while the Gram-negative organisms

tested included Acinetobacter baumannii and two diﬀerent

strains of K. pneumoniae, one of which is an ESBL-producing

strain that expresses the SHV-18 β-lactamase. Although 13 is

active against all the bacterial strains examined (MIC = 2.0 to

32.0 μg/mL), its potency is, in general, greater against the

Gram-positive than the Gram-negative bacteria (see Table 5).

This diﬀerence in potency could be due, at least in part, to the

MBC and MIC values (i.e., an MBC/MIC ratio of 1) for both

S. aureus and E. coli, indicative of a bactericidal mode of action

against both bacterial species. Thus, 13 exhibits bactericidal

behavior against both Gram-positive and Gram-negative

bacteria.

Kinetics of the Bactericidal Activity of 13 against S.

aureus. With the bactericidal nature of 13 having been

established, the kinetics of killing was next investigated in S.

aureus 8325-4. Time-kill curves reveal that the rate at which 13

kills S. aureus is dependent on compound concentration, with

higher concentrations resulting in greater rates of kill (Figure

9A). At a concentration of 8 × MIC (8 μg/mL), 13 reduces the

bacterial number (as indicated by the number of colony

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incubation, aliquots from wells containing the highest

compound concentration and still showing turbidity (i.e.,

bacterial growth) are used to inoculate a new microtiter plate

containing serial 2-fold compound dilutions. This procedure is

then repeated for multiple cycles, and the compound MIC is

determined for each cycle. An increase in MIC with successive

cycles is indicative of induced resistance. The RNA synthesis

inhibitor rifampicin was used as a comparator control agent in

these studies (the results of which are shown in Figure 10),

Figure 10. Probing for the emergence of S. aureus 8325-4 (MSSA)

resistance to 13 and the comparator agent rifampicin by monitoring

MIC over repeated cycles of incubation in the presence of compound.

since this antibiotic is known to induce spontaneous mutants in

S. aureus at a rate of 10⁻⁶to 10⁻⁸.^52,53Note that the MIC of

rifampicin increased more than 500-fold within 8 incubation

cycles. In striking contrast, even after 12 incubation cycles, the

MIC of 13 did not change to any signiﬁcant degree, an

observation consistent with the compound having a minimal

potential to induce resistance in S. aureus.

Figure 9. Time−kill curves for S. aureus 8325-4 (MSSA). Each data

point reﬂects the average of two independent measurements, with the

error bars reﬂecting the standard deviation from the mean. (A)

Bacteria were treated with vehicle (DMSO) only or 13 at

concentrations ranging from 1 to 8 × MIC (1 to 8 μg/mL). (B)

Bacteria were treated with vehicle only, 13, vancomycin, or

erythromycin. When present, all agents were used at a concentration

corresponding to 8 × MIC.

Consistent with the stepwise broth microdilution approach,

the large inoculum approach also indicated that 13 is associated

with a minimal potential for inducing resistance in S. aureus. In

this latter approach, a large inoculum of ∼3 × 10⁹CFU/mL of

S. aureus was prepared and plated onto selective tryptic soy agar

(TSA) plates containing either 13 or rifampicin at concen-

trations 4- to 32-fold higher than their MIC values. The plates

were incubated at 37 °C overnight and examined after 24 h.

Colonies were observed in 100% of the control rifampicin-

containing plates, and the ratio of the number of colonies

observed to total number of CFUs plated yielded a mutational

frequency of (2.5 0.3) × 10⁻⁸, a value similar to that reported

previously for rifampicin.⁵⁴In contrast to rifampicin, none of

the 13-containing plates developed colonies, yielding a

forming units, CFUs) by three logs within 2 h and by four logs

within 6 h. Note that this rate of kill is approximately 100-times

greater than that associated with an equivalent concentration (8

× MIC) of vancomycin and more than 1000-times greater than

the corresponding kill rate of erythromycin (Figure 9B).

13 Exhibits a Minimal Potential for Inducing

Resistance in S. aureus. In addition to the bactericidal

kinetics of 13, the potential for the compound to induce the

emergence of resistance in S. aureus was also assessed using two

techniques: (1) a stepwise broth microdilution approach and

(2) a large inoculum approach. In the stepwise broth

microdilution approach, microtiter wells containing 2-fold

increasing concentrations of compound are inoculated with S.

aureus 8325-4 bacteria (at 10⁵CFU/mL). After 24 h of

mutational frequency of (<3.1

0.8) × 10⁻⁹. Collectively,

both the stepwise broth microdilution and large inoculum

experiments demonstrate a minimal potential on the part of 13

for inducing resistance in S. aureus, a desirable property for an

antistaphylococcal agent.

13 Has a Negligible Impact on the Polymerization of

Mammalian Tubulin. FtsZ and eukaryotic tubulin share only

a weak sequence identity, yet their three-dimensional structures

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Figure 11. (A) Comparison of the crystal structure of the β-tubulin−taxol complex (PDB 1JFF)⁵⁹with the homology model of SaFtsZ in complex

with 13. The proteins, nucleotides, and compounds are depicted as described in Figure 6A. The orientation of protoﬁlament growth is indicated. (B)

Comparison of the impact of 13 and the antineoplastic drugs paclitaxel (taxol) and nocodazole on the polymerization of microtubule-associated

protein (MAP)-rich porcine β-tubulin (70% β-tubulin, 30% MAPs) at 37 °C. The time-dependent A₃₄₀proﬁles of 2 mg/mL porcine β-tubulin are

presented in the presence of vehicle (DMSO) only (black), 40 μg/mL 13 (red), 25 μg/mL taxol (blue), or 10 μg/mL nocodazole (green).

Experimental conditions for the tubulin polymerization studies were 80 mM PIPES·NaOH (pH 7.0), 2 mM MgCl₂, 1 mM EGTA, and 1 mM GTP.

are similar (see Figure 11A).^{41−44,55,56}Due to this structural

similarity, tubulin is considered the closest eukaryotic

homologue of FtsZ. The antineoplastic drug paclitaxel (taxol)

is known to exert its antitubulin activity by stimulating tubulin

self-polymerization,^57,58another example of a compound

having a detrimental impact on a protein by interfering with

concentration. Thus, the profound stimulatory impact of 13 on

bacterial FtsZ polymerization (Figure 3) is not observed with

mammalian tubulin, implying that 13 is not likely to exhibit

antitubulin-mediated toxicity in mammalian cells.

CONCLUDING REMARKS

■

the dynamics of protein self-polymerization. Lowe and co-

̈

Three guanidinomethyl biaryl analogues have been synthesized

and pharmacologically evaluated as FtsZ-targeting antibacterial

agents. Regarded as a whole, the studies described herein

highlight 13 as a lead compound with potent bactericidal

activity against known MDR bacterial pathogens of acute

clinical importance and reinforce the importance of FtsZ as a

new antibacterial target yet to be exploited in the clinic. An

important next step in the development of 13 and related

biaryls into clinically useful agents will be an assessment of

eﬃcacy in appropriate in vivo models of infection.

workers have reported the structure of β-tubulin bound to

taxol, thereby revealing the drug binding site in the protein.⁵⁹

Interestingly, taxol binds tubulin at a site located in an

equivalent domain to that identiﬁed by our computational

studies as the binding site of 13 in SaFtsZ (Figure 11A). The

similarity between the target binding sites of taxol and 13,

coupled with the similar stimulatory impact the two

compounds have on their target proteins, suggests that the

two compounds may disrupt the functions of their protein

targets via a similar molecular mechanism.

Considering the structural similarity between tubulin and

FtsZ, we sought to determine whether 13 exhibits any cross-

reactivity with tubulin. If cross-reactive, 13 would likely be

associated with toxicity in tubulin-expressing mammalian cells,

which, in turn, could potentially limit the clinical utility of the

compound. To explore the impact, if any, of 13 on tubulin self-

polymerization, we used a light-scattering assay similar to that

described above for FtsZ polymerization, but using porcine β-

tubulin containing 30% microtubule-associated proteins

(MAPS) instead of FtsZ. Taxol and a second antineoplastic

drug nocodazole were used as positive controls in these assays,

the former drug being a known stimulator of tubulin

polymerization^57,58and the latter drug being a known inhibitor

of tubulin polymerization.⁶⁰Figure 11B shows the time-

dependent A₃₄₀proﬁles of porcine β-tubulin in the absence and

presence of 13 (at 40 μg/mL), taxol (at 25 μg/mL), or

nocodazole (at 10 μg/mL). Taxol exhibits its expected

stimulatory impact on tubulin polymerization dynamics, with

this stimulation being suﬃcient in magnitude to render the

nucleation phase (i.e., the ∼4-min lag period observed in the

presence of vehicle only) unobservable within the time frame of

the experiment. In addition, nocodazole also exhibits its

expected inhibitory impact on tubulin polymerization. In

striking contrast to the control agents, 13 exerts a negligible

impact, despite it being present at a comparatively higher

EXPERIMENTAL SECTION

■

General Chemistry Methods. Column chromatography refers to

ﬂash chromatography conducted on disposable normal phase

Teledyne ISCO columns with a CombiFlash Rf Teledyne ISCO

using the solvent systems indicated. Proton and carbon nuclear

magnetic resonance (¹H and ¹³C NMR, respectively) were recorded

using either a Bruker 400 MHz or a Varian 300 MHz Unity Inova

spectrometer in the deuterated solvent indicated, with chemical shifts

reported in δ units downﬁeld from tetramethylsilane (TMS). Coupling

constants are reported in hertz (Hz). 4-Chloro-6-methylpyrimidine

and 4-chloropicolinaldehyde were obtained from Combi-Blocks, LLC.

All other starting materials and reagents were obtained from Aldrich.

Solvents were purchased from Fisher Scientiﬁc and were A.C.S. or

HPLC grade. Methylene chloride was freshly distilled from calcium

hydride. All other solvents were used as provided without further

puriﬁcation. All stock solutions of ﬁnal compounds were prepared in

DMSO and stored at −20 °C prior to their use in any experiment.

General Method A: Suzuki Coupling. A 100-mL round-bottom

ﬂask equipped with a magnetic stirrer, a condenser, and a nitrogen in/

outlet adapter was charged with halogenated compounds (8.10 mmol),

4-tert-butylphenylboronic acid (1.93 g, 9.70 mmol), water/dioxane (10

mL/30 mL), and K₂CO₃(2.23 g, 16.2 mmol). The resulting solution

was degassed for 5 min, whereupon Pd(PPh₃)₄(180 mg, 0.162 mmol)

was added. The reaction mixture was warmed to 100 °C and stirred for

3 h. After cooling to room temperature, the reaction mixture was

diluted with EtOAc (100 mL), washed with saturated NaHCO₃(30

mL) and brine (30 mL), and dried over Na₂SO₄. The organic layer

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was concentrated in a rotovapor and puriﬁed on silica gel. Elution with

EtOAc/hexanes solvent system aﬀorded the title compounds.

δ: 7.69−7.35 (m, 8H), 4.81 (m, 2H), 1.40(s, 9H). ¹³C NMR (100

MHz, CDCl₃) δ: 150.4, 141.4, 138.0, 128.9, 126.8, 126.3, 125.7, 65.5,

34.5, 31.4.

General Method B: Aldehyde Reduction. A 50-mL round-

bottom ﬂask equipped with a magnetic stirrer was charged with biaryl-

carbaldehyde (2.94 mmol) and ethanol (95%, 10 mL), and NaBH₄

(112 mg, 2.94 mmol) was added in several portions. The reaction

mixture was stirred at room temperature for 1 h. Acetone (1 mL) was

added to the reaction mixture. After 20 min, the reaction mixture was

concentrated, and the residue was partitioned between EtOAc (50

mL) and 1 N HCl (15 mL). The organic layer was washed with

saturated NaHCO₃(15 mL) and brine (15 mL), dried over Na₂SO₄,

concentrated in a rotovapor, and puriﬁed on silica gel. Elution with

10% EtOAc/hexanes aﬀorded the reduced compounds in good yield.

General Method C: Preparation of Chlorides. A 25-mL round-

bottom ﬂask equipped with a magnetic stirrer under nitrogen was

charged with alcohol (2.50 mmol), CH₂Cl₂(10 mL), and triethyl-

amine (0.70 mL, 5.00 mmol). Methanesulfonyl chloride (0.39 mL,

5.00 mmol) was added via a syringe over 5 min. The resulting reaction

mixture was stirred at room temperature overnight. The reaction

mixture was then diluted with CH₂Cl₂(30 mL), washed with saturated

NaHCO₃(15 mL) and brine (15 mL), dried over Na₂SO₄,

concentrated in a rotovapor, and puriﬁed on silica gel. Elution with

hexanes aﬀorded the products as white solids.

General Method D: Reaction with Diboc-Guanidine. A 25-mL

round-bottom ﬂask equipped with a magnetic stirrer, a condenser, and

a nitrogen in/outlet adapter was charged with chloro or bromomethyl

intermediates (130 mg, 0.50 mmol), DMF (2 mL), K₂CO₃(103 mg,

0.75 mmol), and 1,3-bis(tert-butoxycarbonyl)guanidine (143 mg, 0.55

mmol). The reaction mixture was stirred at 50 °C for 2 h. The reaction

mixture was then diluted with EtOAc (40 mL), washed with water (10

mL), 10% LiCl (10 mL) and brine (10 mL), dried over Na₂SO₄,

concentrated, and puriﬁed on silica gel. Elution with 5% EtOAc/

hexanes aﬀorded the title compounds as white solids.

General Method E: Diboc Deprotection. A 10-mL vial was

charged with di-tert-butyl guanidine compounds (0.05 mmol), CH₂Cl₂

(1 mL), and TFA (1 mL). The sealed vial was stirred at room

temperature overnight. The solvent was then removed, and the residue

was puriﬁed on silica gel. Elution with MeOH:CHCl₃:ammonium

hydroxide (10:89:1) aﬀorded the title compounds 13−15 as white

solids. Compounds 13−15 were analyzed for purity by HPLC using a

Shimadzu LC-20AT Prominence chromatograph equipped with a

SPD-20A UV−vis detector monitoring absorbances at both 254 and

280 nm. Each compound was analyzed using a PrincetonSPHER-100

5-μm C18 reverse-phase column (150 mm × 4.6 mm), using gradients

of 0.1% TFA in water with increasing percentages of either 0.1% TFA

in acetonitrile or 0.1% TFA in methanol. Under both of these solvent

conditions, each compound was eluted over 12 min, using a ﬂow rate

of 1.0 mL/min and a gradient ranging from 10% to 90%. The purity of

each compound as determined by HPLC was found to be >95%.

Analytical Data. 4′-(tert-Butyl)-[1,1′-biphenyl]-3-carbaldehyde

(4). Prepared by general method A: white solid, mp = 48−50 °C;

¹H NMR (300 MHz, CDCl₃) δ: 10.12 (s, 1H), 8.13 (s, 1H), 7.88 (m,

(4-(4-t-Butyl)phenyl)pyridin-2-yl)methanol. Prepared by general

method B: white solid, mp = 103−105 °C; H NMR (300 MHz,

1

CDCl₃) δ: 8.57 (d, J = 3.0 Hz, 1H), 7.60 (d, J = 6.0 Hz, 2H), 7.58−

7.41 (m, 4H), 4.83 (s, 2H), 1.37 (s, 9H). ¹³C NMR (75 MHz, CDCl₃)

δ: 149.1, 135.3, 126.9, 126.3, 120.6, 118.4, 64.6, 34.9, 31.5. HRMS

calculated for C₁₆H₂₀NO (M + H)⁺, 242.1545; found, 242.1530.

4′-(tert-Butyl)-3-(chloromethyl)-1,1′-biphenyl (7). Prepared by

general method C: white solid, mp = 62−64 °C; ¹H NMR (400

MHz, CDCl₃) δ: 7.53−7.20 (m, 8H), 4.56 (s, 2H), 1.28 (s, 9H). ¹³C

NMR (100 MHz), CDCl₃) δ: 150.6, 141.7, 137.8, 129.1, 127.2, 126.8,

125.8, 46.3, 34.6, 31.8.

4-(4-(tert-Butyl)phenyl)2-(chloromethyl)pyridine (8). Prepared by

general method C: ¹H NMR (300 MHz, CDCl₃) δ: 8.60 (J = 6.0 Hz,

1H), 7.58−7.25 (m, 6H), 4.73 (s, 2H), 1.37 (s, 9H). ¹³C NMR (75

MHz, CDCl₃) δ: 157.3, 152.9, 150.1, 149.7, 135.1, 127.0, 126.4, 121.1,

120.8, 47.1, 35.0, 31.5.

4-(Bromomethyl)-6-(4-(tert-butyl)phenyl)pyrimidine (9). A mix-

ture of 4-(4-(tert-butyl)phenyl)-6-methylpyrimidine (1 mmol) and

NBS (1.3 mmol) in carbon tetrachloride (3.0 mL) was heated under

light for 30 min. The solids were ﬁltered, and the solvent was removed

to give the crude product. Puriﬁcation using 10% ethyl acetate in

hexane aﬀorded the product in 52% yield along with some

dibrominated product. ¹H NMR (300 MHz, CDCl₃) δ: 9.19 (s,

1H), 8.04 (d, J = 6.0 Hz, 2H), 7.84 (s, 1H), 7.54 (d, J = 6.0 Hz, 2H),

4.5 (s, 2H), 1.37 (s, 9H).

1-((4′-(tert-Butyl)-[1,1′-biphenyl]-3-yl)methyl)-[1,3-bis(tert-

Butoxycarbonyl)]guanidine (10). Prepared by general method D:

white solid, mp = 174−176 °C; ¹H NMR (400 MHz, CDCl₃) δ: 7.54

(m, 3H), 7.48 (m, 3H), 7.37 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz,

1H), 5.26 (s, 2H), 1.52 (s, 9H), 1.39 (s, 9H), 1.37 (s, 9H). ¹³C NMR

(100 MHz, CDCl₃) δ: 150. 3, 140.9, 139.3, 138.2, 128.5, 126.7, 125.8,

125.7, 125.6, 125.3, 84.0, 47.8, 34.5, 31.3, 28.3, 27.8.

1-((4-(4-(tert-Butyl)phenyl)pyridin-2-yl)methyl)-[1,3-bis(tert-

butoxycarbonyl)]guanidine (11). Prepared by general method D:

white solid, mp = 65−69 °C; ¹H NMR (300 MHz, CDCl₃) δ: 9.5 (bs,

2H), 8.47 (d, J = 6.0 Hz, 1H), 7.50−7.42 (m, 4H), 7.3 (m, 2H), 5.4 (s,

2H), 1.47 (s, 9H), 1.36 (s, 9H), 1.25 (s, 9H). ¹³C NMR (75 MHz,

CDCl₃) δ: 163.8, 161.2, 159.3, 155.2, 152.6, 149.6, 148.8, 135.6, 126.9,

126.3, 119.9, 118.0, 84.2, 79.2, 49.7, 34.9, 31.5, 28.5, 28.3, 27.9.

1-((6-(4-(tert-Butyl)phenyl)pyrimidin-4-yl)methyl)-[1,3-bis(tert-

butoxycarbonyl)]guanidine (12). Prepared by general method D:

white solid, mp = 152−154 °C; ¹H NMR (300 MHz, CDCl₃) δ: 9.54

(bs, 1H), 9.41 (bs, 1H), 9.16 (s, 1H), 8.01 (d, J = 5.4 Hz, 2H), 7.56−

7.53 (m, 3H), 5.38 (s, 2H), 1.48 (s, 9H), 1.37 (s, 1H), 1.31 (s, 1H).

¹³C NMR (75 MHz, CDCl₃) δ: 167.7, 164.1, 163.7, 160.8, 158.8,

154.9, 154.8, 134.0, 113.1, 84.7, 79.3, 49.0, 31.4, 28.5, 28.3, 27.8.

HRMS calculated for C₂₆H₃₈N₅O₄(M + H)⁺, 484.2924; found,

484.2908.

1-((4′-(tert-Butyl)-[1,1′-biphenyl]-3-yl)methyl)guanidine (13).

Preparation by general method E: white solid; mp = 134−136 °C;

¹H NMR (300 MHz, CDCl₃) δ: 7.38−7.35 (m, 6H), 7.15 (t, 1H), 7.06

(d, 1H), 4.1 (s, 2H), 1.28 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ:

158.4, 150.7, 141.6, 138.6, 138.0, 129.4, 127.0, 126.3, 125.9, 45.9, 34.7,

31.6. HRMS calculated for C₁₈H₂₄N₃(M + H)⁺, 282.1970; found,

282.1957.

2H), 7.63−7.52 (m, 5H), 1.40 (s, 9H). ¹³C NMR (75 MHz, CDCl₃)

δ: 151.4, 142.2, 137.2, 137.0, 133.2, 129.7, 128.6, 128.3, 127.0, 126.2,

34.9, 31.6.

4-(4-(tert-Butyl)phenyl)picolinaldehyde (5). Prepared by general

method A: oil; ¹H NMR (300 MHz, CDCl₃) δ: 10.15 (s, 1H), 8.81 (d,

J = 6.0 Hz, 1H), 8.21 (d, J = 3.0 Hz, 1H), 7.74 (dd, J = 6.0, 3.0 Hz,

1H), 7.65 (m, 2H), 7.54 (m, 2H), 1.37 (s, 9H). ¹³C NMR (75 MHz,

CDCl₃) δ: 193.8, 153.8, 153.4, 150.8, 149.7, 134.2, 126.9, 126.5, 125.5,

119.5, 35.0, 31.4. HRMS calculated for C₁₆H₁₈NO (M + H)⁺,

240.1388; found, 240.1381.

1-((4-(4-(tert-Butyl)phenyl)pyridin-2-yl)methyl)guanidine (14).

1

Prepared by general method E: white solid, mp = 88−91 °C; H

NMR (300 MHz, (CDCl₃) δ: 8.32 (s, 1H), 7.45−7.20 (m, 7H), 6.43

(bs, 2H), 4.32 (s, 2H), 1.27 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ:

159.1, 157.2, 152.8, 149.7, 149.3, 134.5, 126.9, 126.3, 120.6, 120.4,

47.5, 34.9, 31.4. HRMS calculated for C₁₇H₂₃N₄(M + H)⁺, 283.1923;

found, 283.1917.

4-(4-(tert-Butyl)phenyl)-6-methylpyrimidine (6). Prepared by

1

general method A: H NMR (300 MHz, CDCl₃) δ: 9.12 (s, 1H),

8.03−7.99 (m, 2H), 7.56−7.50 (m, 3H), 2.58 (s, 3H), 1.36 (s, 9H).

¹³C NMR (75 MHz, CDCl₃) δ: 167.5, 163.9, 158.9, 154.6, 134.1,

127.1, 126.2, 116.4, 35.1, 31.4, 24.6. HRMS calculated for C₁₅H₁₉N₂

(M + H)⁺, 227.1548; found, 227.1537.

1-((6-(4-(tert-Butyl)phenyl)pyrimidin-4-yl)methyl)guanidine (15).

Prepared by general method E: white solid, mp = 78−81 °C; ¹H NMR

(300 MHz, CDCl₃) δ: 9.03 (s, 1H), 7.95 (d, J = 6.0 Hz, 2H), 7.69 (s,

1H), 7.43 (d, J = 6.0 Hz, 2H), 5.33 (bs, 2H), 4.36 (s, 2H), 1.29 (s,

(4′-(tert-Butyl)-[1,1′-biphenyl]-3-yl)methanol. Prepared by general

method B: white solid, mp = 52−54 °C; ¹H NMR (400 MHz, CDCl₃)

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9H). HRMS calculated for C₁₆H₂₂N₅(M + H)⁺, 284.1875; found,

284.1868.

plasmids were veriﬁed, they were each transformed into E. coli BL21

(DE3) bacteria, and the mutant proteins were puriﬁed using the

protocol described above for puriﬁcation of wild-type SaFtsZ.

Fluorescence Anisotropy Assays. All steady-state ﬂuorescence

anisotropy experiments were conducted at 25 °C on an AVIV

Model ATF105 spectroﬂuorometer (Aviv Biomedical, Lakewood, NJ)

equipped with a thermoelectrically controlled cell holder and

computer-controlled Glan-Thompson polarizers in both the excitation

and emission directions. A quartz ultramicro cell (Hellma, Inc.) with a

2 × 5 mm aperture and a 15 mm center height was used for all

measurements. The pathlengths in the excitation and emissions

directions were 1 and 0.2 cm, respectively. All anisotropy experiments

were conducted in triplicate, with the reported anisotropies reﬂecting

the average values.

Determination of Compound-FtsZ Binding Aﬃnities. Test

compound (5 μM) was combined with SaFtsZ, EcFtsZ, or EfFtsZ

(at concentrations ranging from 0 to 25 μM) in 150 μL of solution

containing 50 mM Tris·HCl (pH 7.4), 50 mM KCl, and 2 mM

magnesium acetate. After incubation for 5 min at 25 °C, the

ﬂuorescence emission intensities (I) of the compound were measured

with the excitation polarizer oriented vertically and the emission

polarizer oriented vertically (I_VV) or horizontally (I_VH). Compound

anisotropy (r) values were then determined using the following

relationship:

Comparator Antibiotics and FtsZ Proteins from E. coli and E.

faecalis. Vancomycin·HCl, ampicillin (sodium salt), neomy-

cin·3H₂SO₄·3H₂0, erythromycin, clindamycin·HCl, oxacillin (sodium

salt), and amikacin·2H₂SO₄were obtained from Sigma-Aldrich Co. E.

coli and E. faecalis FtsZ proteins were obtained from Cytoskeleton, Inc.

Expression and Puriﬁcation of Wild-Type and Mutant S. aureus

FtsZ. The ftsZ gene from S. aureus was ampliﬁed by polymerase chain

reaction (PCR) from the S. aureus genome obtained from ATCC

(ATCC 33591-D). The ﬁrst primer used for PCR was designed to

introduce a unique NdeI site at the 5′-end of the ftsZ gene, while the

second primer was designed to introduce a unique EcoRI site at the 3′-

end of the gene. The ampliﬁed gene product was digested with NdeI

and EcoRI and ligated into the pET-22b(+) cloning vector (Novagen-

EMD Chemicals, Inc.), which had been previously digested with the

same enzymes. The sequence of the ﬁnal recombinant plasmid

(pETSaFtsZ) was veriﬁed by sequence analysis and was used to

transform E. coli BL21 (DE3) cells.

A single colony of pETSAftsZ-transformed E. coli cells was used to

inoculate Luria−Bertani media containing 100 μg/mL of ampicillin

(LB-amp) and grown overnight at 37 °C. Twenty milliliters of the

overnight culture was diluted into 4 L of LB-amp and grown until an

optical density at 600 nm (OD₆₀₀) of 0.4, at which point FtsZ

production was induced by addition of isopropyl β-^D-1-thiogalactopyr-

anoside (IPTG) to a ﬁnal concentration of 1 mM. Following addition

of IPTG, the cultures were incubated for an additional 3 h at 37 °C.

Cells were then harvested by centrifugation at 4 °C and 4000g in a

swinging bucket Sorvall RC-3BP+ centrifuge with rotor H-6000 for 20

min. The bacterial cell pellet was washed with ice-cold 50 mM

Tris·HCl (pH 8.0) and 0.5 M NaCl and repelleted by centrifugation as

described above. The washed bacterial pellet was stored at −20 °C.

Approximately 15 g (wet weight) of cell pellet was resuspended in

40 mL of buﬀer containing 50 mM Tris·HCl (pH 8.0), 50 mM KCl, 1

mM EDTA, 1 mM EGTA, and 10% (v/v) glycerol (TKEGE buﬀer) to

which 0.5 mM PMSF and EDTA-free Complete protease cocktail

inhibitor was added, and the bacteria were lysed by sonication. The

crude lysate was clariﬁed into soluble and insoluble fractions by

centrifugation in a Sorvall RC 6+ centrifuge using a F21S-8 × 50y

rotor at 38 000g for 30 min at 4 °C. The soluble fraction was

ultracentrifuged at 76 500g in a Beckman L8−70 M Ultracentrifuge for

90 min at 4 °C. The supernatant was transferred to a beaker, and solid

ammonium sulfate was added to a ﬁnal concentration of 45% (w/v)

with stirring. The solution was centrifuged at 38 000g for 20 min at 4

°C and the 45% ammonium sulfate protein pellet collected. The pellet

was dissolved in 20 mL of TKEGE buﬀer and dialyzed overnight

against 2 L of TKEGE buﬀer.

I_VV− GI_VH

r =

I_VV+ 2GI_VH

(2)

G represents the instrument correction factor, and is given by the

ratio of the ﬂuorescence emission intensity acquired with the excitation

polarizer oriented horizontally and the emission polarizer oriented

vertically (I_HV) to that acquired with both the excitation and emission

polarizers oriented horizontally (I_HH). A G value was measured at the

start of each acquisition. The bandwidths were set at 5 nm in both the

excitation and emission directions, with the excitation and emission

wavelengths being set at 245 and 318 nm, respectively.

BoGTPγS Competition Assay. BoGTPγS (1 μM) (obtained from

Life Technologies Corp. as the sodium salt in a 5 mM stock solution)

was combined with 0 or 5 μM EcFtsZ or SaFtsZ and either 13 or GTP

(at concentrations ranging from 0 to 40 μM) in 150 μL of solution

containing 50 mM Tris·HCl (pH 7.4), 50 mM KCl, and 2 mM

magnesium acetate. After incubation for 5 min at 25 °C, ﬂuorescence

anisotropies for BoGTPγS were determined as described above for 13,

with the exception that the bandwidths were set at 4 nm in both the

excitation and emission directions and the excitation and emission

wavelengths were set at 488 and 510 nm, respectively.

FtsZ Polymerization Assay. Polymerization of SaFtsZ, EcFtsZ, and

EfFtsZ as well as all mutant SaFtsZ proteins was monitored using a

microtiter plate-based light-scattering assay in which changes in light

scattering are reﬂected by corresponding changes in absorbance at 340

nm (A₃₄₀). Test compound or comparator drug (at concentrations

ranging from 0 to 40 μg/mL) were combined with 1 mM GTP and 10

μM FtsZ in 100 μL of reaction solution. Reaction solutions contained

50 mM Tris·HCl (pH 7.4), 50 mM KCl, 2 mM magnesium acetate,

and either 1 mM CaCl₂(for the wild-type and mutant SaFtsZ

experiments) or 10 mM CaCl₂(for the EcFtsZ and EfFtsZ

experiments). Reactions were assembled in half-volume, ﬂat-bottom,

96-well microtiter plates, and polymerization was continuously

monitored at 25 °C by measuring A₃₄₀in a VersaMax plate reader

(Molecular Devices, Inc.) over a time period of 400 min.

Transmission Electron Microscopy (EM). At room temperature,

SaFtsZ and EcFtsZ polymerization reaction solutions were diluted

1:10 in polymerization buﬀer, and 25-μL drops of the resulting

dilutions were placed on glow-discharged, copper, 400 mesh,

Formvar/carbon-coated grids. Excess solution was wicked away with

ﬁlter paper. The grids were negatively stained with a solution of 1%

phosphotungstic acid for 1 min and blotted dry. The grids were then

digitally imaged at 80 kV on a Philips CM12 transmission microscope

interfaced with an AMT XR111 camera.

The dialyzed material was loaded onto a MonoQ 10/100 anion

exchange column at 1 mL/min and washed with 5 column volumes of

TKEGE buﬀer at 1 mL/min. The column was eluted at 4 °C with a 0−

60% (v/v) linear gradient (120 mL total volume) of the following two

buﬀers: (i) TKEGE (containing 50 mM KCl) and (ii) TKEGE

containing 1 M KCl instead of 50 mM KCl. The rate of elution was

maintained at 1 mL/min. Fractions were assayed for FtsZ by SDS-

PAGE analysis on a 10−15% Tris·HCl polyacrylamide gel. FtsZ elutes

at 250−350 mM KCl. The FtsZ-containing fractions were pooled and

dialyzed against 2 L of TKEGE buﬀer. The dialyzed fractions were

concentrated, if necessary, to 5 mL and loaded onto a Superdex-200

size exclusion column, with TKEGE buﬀer as the running buﬀer at

0.25 mL/min. SaFtsZ-containing fractions were detected by SDS-

PAGE as above. Peak fractions containing the pure SaFtsZ were

pooled and concentrated using Amicon Ultra centrifugal ﬁlters

(Millipore Corp.). Quantitation was performed spectrophotometrically

at 595 nm using a Bio-Rad colorimetric protein assay kit and bovine

serum albumin as the standard. Concentration of SaFtsZ was ∼8 mg/

mL and 90% pure by SDS-PAGE analysis.

The wild-type S. aureus FtsZ gene-expressing plasmid pETSaFtsZ

was used to generate the mutant proteins using the QuikChange Site-

Directed Mutagenesis Kit (Agilent Technologies) and appropriate

primers. Once the sequences of the mutant-protein expressing

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Computational Studies. The Modeler program (version 9.10) was

used to build the homology model of S. aureus FtsZ via the single

template approach.^61,62The model was built from the sequence of S.

aureus FtsZ [MRSA252] (protein accession no. YP_040573.1). A

BLAST search of nonredundant PDB sequences clustered at 95%

identity gave the crystal structure of B. subtilis FtsZ (PDB no. 2VXY)¹⁵

as the optimum template, with a sequence identity of 81% and a

resolution of 1.7 Å. The homology model with the lowest DOPE score

was selected, and the C-terminal residues from number 318 to the C-

terminus were removed. Analysis of the model using PROCHECK

revealed >95% of the residues to be in the most favored regions of the

Ramachandran plot.⁶³

the culture in duplicate on TSA plates. The initial culture was

aliquoted into tubes, each containing either a compound or

comparator drug at ﬁnal concentrations ranging from 0- to 16-times

MIC. An equivalent volume of DMSO was added to the vehicle

control tube. The cultures were then incubated at 37 °C with shaking.

The CFU/mL in each culture was determined over time by

withdrawing samples at 2, 4, and 6 h and plating appropriate serial

dilutions onto TSA plates. To avoid the possibility of carry-over eﬀects

in instances when no dilutions were made of the cultures, the samples

were centrifuged at 16 000g, the supernatant was removed, and the

bacterial pellet was resuspended in an equal volume of media before

plating. All TSA plates were incubated at 37 °C, and the CFU/mL at

each time point was determined by counting colonies after 24 h.

Assays for the Emergence of Resistance. Assays for the emergence

of resistance following prolonged compound exposure were conducted

using both stepwise broth microdilution and large inoculum

approaches. The stepwise broth microdilution approach assays for

an upward drift in MIC values with repeated incubation cycles, while

the large inoculum approach assays for the emergence of resistant

colonies on agar plates containing test compounds at concentrations

above the MIC value.

For the broth microdilution approach, microtiter plates containing

2-fold increasing concentrations of 13 or comparator drug (rifampicin)

were inoculated with S. aureus 8325-4 bacteria (at 10⁵CFU/mL in 0.1

mL total volume). After 24 h of incubation at 37 °C, 75 μL aliquots

from the wells containing the highest compound concentration and

still showing bacterial growth (i.e., the well containing a compound

concentration 2-fold lower than the MIC) were used to inoculate a

new microtiter plate containing serial 2-fold compound dilutions, and

the newly inoculated plates were incubated for 24 h at 37 °C. This

procedure was repeated for multiple cycles.

All molecular docking studies were performed using the Autodock

Vina (version 1.1.2) docking package.⁶⁴The ligand molecule was built

using the Sybyl-X version 1.3 (Tripos, Inc.) software package. Each

ligand model was reﬁned via conjugate gradient energy minimization

using the MMFF94 force ﬁeld.^65,66The reﬁned ligands and protein

receptor model were converted to pdbqt format using Autodock

Tools.⁶⁷Gasteiger−Huckel partial atomic charges were used for both

̈

the ligand and the protein receptor, although these partial atomic

charges are not needed by Vina, because it uses an internal

intermolecular energy scoring function based on Xscore.⁶⁸Vina uses

the Iterated Local Search global optimizer method for orientation and

conformer search of the ligands,⁶⁴with the receptor being kept rigid.

Initial docking studies used a grid box dimensioned to cover the entire

protein (56 Å × 54 Å × 58 Å) and a global search exhaustiveness of

15, with an energy range of 4 kcal/mol. Subsequent docking studies

used a grid box approximately centered in a pocket between residues

E305 and R29 and dimensioned to cover the entire pocket (32 Å × 20

Å × 22 Å). In these latter studies, an exhaustiveness of 10 was used for

the global search, with an energy range of 4 kcal/mol.

For the large inoculum approach, TSA plates containing either 13

or rifampicin at concentrations ranging from 4- to 32-times MIC were

prepared. A large inoculum of ∼3 × 10⁹CFU/mL S. aureus was spread

onto each plate. The colony count of the inoculum was veriﬁed by

plating serial dilutions of the culture onto nonselective TSA plates. All

plates were incubated at 37 °C overnight and examined after 24 h and

daily thereafter. Mutational frequency was calculated from the ratio of

the number of colonies observed on the selective plates to the total

number of plated bacteria.⁵⁴

Tubulin Polymerization Assay. Polymerization of MAP-rich

porcine β-tubulin containing 70% β-tubulin and 30% MAPs

(Cytoskeleton, Inc.) was monitored using a microtiter plate-based

light scattering assay similar to that described above for FtsZ

polymerization. Test compound or comparator drug was combined

with 1 mM GTP and 2 mg/mL porcine β-tubulin in 100 μL of

reaction solution containing 80 mM PIPES·NaOH (pH 7.0), 2 mM

MgCl₂, and 1 mM EGTA. Reactions were assembled in half-volume,

ﬂat-bottom, 96-well microtiter plates, and polymerization was

continuously monitored at 37 °C by measuring A₃₄₀in a VersaMax

plate reader over a time period of 60 min.

Minimum Inhibitory Concentration (MIC) Assays. MIC assays

were conducted in accordance with Clinical and Laboratory Standards

Institute (CLSI) guidelines for broth microdilution.⁵¹Brieﬂy, log-

phase bacteria were added to 96-well microtiter plates (at 10⁵CFU/

mL) containing 2-fold serial dilutions of compound or comparator

drug in cation-adjusted Mueller-Hinton (CAMH) broth at concen-

trations ranging from 64 to 0.016 μg/mL. The ﬁnal volume in each

well was 0.1 mL, and the microtiter plates were incubated aerobically

for 24 h at 37 °C. Bacterial growth was then monitored by measuring

OD₆₀₀using a VersaMax plate reader, with the MIC being deﬁned as

the lowest compound concentration at which growth was ≥90%

inhibited. The following bacterial strains were included in these assays:

S. aureus 8325-4 (MSSA from Dr. Glenn W. Kaatz, John D. Dingell VA

Medical Center, Detroit, MI), S. aureus ATCC 49951 (mucoid

MSSA), S. aureus ATCC 33591 (MRSA), S. aureus Mu3 (MRSA and

hetero-GISA clinical isolate from Dr. George M. Eliopoulos, Beth

Israel Deaconess Medical Center, Boston, MA), E. coli W4573 acrA⁺

and N43 acrA⁻(both from Dr. E. Lynn Zechiedrich, Baylor College of

Medicine, Houston, TX), E. coli ATCC BAA-201 (ESBL-producing

strain expressing the TEM-3 lactamase), S. pyogenes ATCC 19615, S.

agalactiae ATCC 12386, E. faecalis ATCC 19433 (VSE), E. faecalis

ATCC 51575 (VRE), B. subtilis ATCC 23857, A. baumannii ATCC

19606, K. pneumoniae ATCC 13883, and K. pneumoniae ATCC

700603 (ESBL-producing strain expressing the SHV-18 lactamase).

CAMH broth was supplemented with 2% NaCl in the MRSA

experiments, while being supplemented with 5% (v/v) deﬁbrinated

sheep’s blood (Becton Dickinson and Co.) in the S. pyogenes and S.

agalactiae experiments.

AUTHOR INFORMATION

■

Corresponding Author

*Tel: 732-235-3352; fax: 732-235-4073; e-mail: pilchds@

umdnj.edu.

Notes

The authors declare the following competing ﬁnancial

interest(s): Drs. Pilch and LaVoie are cofounders of TAXIS

Pharmaceuticals and therefore have a ﬁnancial interest in the

company.

Minimum Bactericidal Concentration (MBC) Assays. MBC assays

were conducted using the broth microdilution assay described in the

preceding section. After the 24 h incubation period, aliquots from the

microtiter wells were plated onto tryptic soy agar (TSA). The colonies

that grew after 24 h of incubation were counted using an Acolyte

colony counter (Synbiosis, Inc.), with MBC being deﬁned as the

lowest compound concentration resulting in a ≥3-log reduction in the

number of colony forming units (CFU).

ACKNOWLEDGMENTS

■

This study was supported by research agreements between

TAXIS Pharmaceuticals, Inc., and both the University of

Medicine and Dentistry of New Jersey (D.S.P.) and Rutgers,

The State University of New Jersey (E.J.L.). S. aureus 8325-4

Time−Kill Assays. Exponentially growing S. aureus 8325-4 bacteria

were diluted in CAMH broth to a ﬁnal count of 10⁵to 10⁶CFU/mL.

The colony count at time zero was veriﬁed by plating serial dilutions of

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(9) Lock, R. L.; Harry, E. J. Cell-Division Inhibitors: New Insights for

was a gift from Dr. Glenn W. Kaatz (John D. Dingell VA

Medical Center, Detroit, MI), S. aureus Mu3 was a gift from Dr.

George M. Eliopoulos (Beth Israel Deaconess Medical Center,

Boston, MA), and E. coli strains W4573 and N43 were gifts

from Dr. E. Lynn Zechiedrich (Baylor College of Medicine,

Houston, TX). We thank Raj Patel (UMDNJ-RWJMS, Core

Imaging Lab) for his assistance with the acquisition and analysis

of the EM data. The Bruker Avance III 400 MHz NMR

spectrometer used in this study was purchased with funds from

NCRR grant no. 1S10RR23698-1A1. Mass spectrometry was

provided by the Washington University Mass Spectrometry

Resource with support from the NIH National Center for

Research Resources grant no. P41RR0954.

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ABBREVIATIONS USED

■

MRSA, methicillin-resistant Staphylococcus aureus; MSSA,

methicillin-sensitive Staphylococcus aureus; GISA, glycopeptide

intermediate Staphylococcus aureus; VSE, vancomycin-sensitive

Enterococcus; VRE, vancomycin-resistant Enterococcus; S. aureus,

Staphylococcus aureus; E. faecalis, Enterococcus faecalis; E. coli,

Escherichia coli; B. subtilis, Bacillus subtilis; A. baumannii,

Acinetobacter baumannii; K. pneumoniae, Klebsiella pneumoniae;

S. pyogenes, Streptococcus pyogenes; S. agalactiae, Streptococcus

agalactiae; ESBL, extended spectrum β-lactamase; MDR,

multidrug-resistant; MIC, minimum inhibitory concentration;

MBC, minimum bactericidal concentration; SaFtsZ, Staph-

ylococcus aureus FtsZ; EcFtsZ, Escherichia coli FtsZ; EfFtsZ,

Enterococcus faecalis FtsZ; Strep A, Streptococcus A; Strep B,

Streptococcus B; BoGTPγS, boron-dipyrromethene-conjugated

GTPγS; A₃₄₀, absorbance at 340 nm; ClogP, calculated

log(partition coeﬃcient); ATCC, American Type Culture

Collection; CFU, colony forming units; TSA, tryptic soy

agar; CLSI, Clinical and Laboratory Standards Institute;

CAMH, cation-adjusted Mueller−Hinton; MAP, microtubule-

associated protein; EM, electron microscopy

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NOTE ADDED IN PROOF

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In the time since this work was conducted, crystal structures of

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A bactericidal guanidinomethyl biaryl that alters the dynamics of bacterial FtsZ polymerization

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