NB2001, a novel antibacterial agent with broad-spectrum activity and enhanced potency against β-lactamase-producing strains

Article abstract of DOI:10.1128/AAC.46.5.1262-1268.2002

Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance resulting from enzyme overexpression. β-Lactamase overexpression is a common mechanism of bacterial resistance to β-lactam antibiotics. We present here the results for one of the β-lactamase ECTA compounds, NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold. Unlike conventional β-lactam antibiotics, where hydrolysis of the β-lactam ring inactivates the antibiotic, hydrolysis of NB2001 by β-lactamase releases triclosan. Evidence supporting the proposed mechanism is as follows. (i) NB2001 is a substrate for TEM-1 β-lactamase, forming triclosan with a second-order rate constant (k_cat/K_m) of greater than 77,000 M^-1 s^-1. (ii) Triclosan is detected in NB2001-treated, β-lactamase-producing Escherichia coli but not in E. coli that does not express β-lactamase. (iii) NB2001 activity against β-lactamase-producing E. coli is decreased in the presence of the β-lactamase inhibitor clavulanic acid. NB2001 was similar to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum antibacterial agent and demonstrate the potential of ECTA in overcoming β-lactamase-mediated resistance.

Full text of DOI:10.1128/AAC.46.5.1262-1268.2002

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 1262–1268
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.5.1262–1268.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 5
NB2001, a Novel Antibacterial Agent with Broad-Spectrum Activity
and Enhanced Potency against ␤-Lactamase-Producing Strains
Qing Li,1* Jean Y. Lee,1 Rosario Castillo,1 Mark S. Hixon,1 Catherine Pujol,1
Venkata Ramana Doppalapudi,1 H. Michael Shepard,1 Geoffrey M. Wahl,2
Thomas J. Lobl,1 and Ming Fai Chan1†
NewBiotics, Inc., San Diego, California 92121,1 and Salk Institute, La Jolla, California 92037-10992
Received 16 April 2001/Returned for modification 26 July 2001/Accepted 18 January 2002
Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance
resulting from enzyme overexpression. ␤-Lactamase overexpression is a common mechanism of bacterial
resistance to ␤-lactam antibiotics. We present here the results for one of the ␤-lactamase ECTA compounds,
NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold.
Unlike conventional ␤-lactam antibiotics, where hydrolysis of the ␤-lactam ring inactivates the antibiotic,
hydrolysis of NB2001 by ␤-lactamase releases triclosan. Evidence supporting the proposed mechanism is as
follows. (i) NB2001 is a substrate for TEM-1 ␤-lactamase, forming triclosan with a second-order rate constant
(kcat/Km) of greater than 77,000 M؊1 s؊1. (ii) Triclosan is detected in NB2001-treated, ␤-lactamase-producing
Escherichia coli but not in E. coli that does not express ␤-lactamase. (iii) NB2001 activity against ␤-lactamase-
producing E. coli is decreased in the presence of the ␤-lactamase inhibitor clavulanic acid. NB2001 was similar
to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including
MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis,
Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enter-
obacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum
antibacterial agent and demonstrate the potential of ECTA in overcoming ␤-lactamase-mediated resistance.
Increased resistance of bacterial infections to antibiotic
treatment has been extensively documented (30) and has now
become a generally recognized medical problem, particularly
with nosocomial infections (9, 21, 23, 29).
␤-Lactam antibiotics are among the most widely used anti-
microbial agents. Mechanistically, they act by covalently bind-
ing to penicillin-binding proteins in the cytoplasmic membrane
of bacteria, thereby inhibiting peptidoglycan transpeptidase
and eventually leading to cell death (32).
cessors, and many are not orally active. A third strategy is to
develop ␤-lactamase inhibitors that have been successful pri-
marily with class A enzymes. However, resistance to ␤-lacta-
mase inhibitors via point mutations in the ␤-lactamase gene
has been reported (25). Further, some ␤-lactamase inhibitors
(e.g., clavulanic acid and sulbactam) induce ␤-lactamase ex-
pression, which diminishes their effectiveness (10, 15). Thus, a
new approach is needed to intercept the established cycle of
drug resistance.
A common mechanism for bacterial resistance to ␤-lactam
antibiotics is via production of ␤-lactamases, which catalyze the
hydrolysis of the amide bond of the ␤-lactam ring, resulting in
ring opening and antibiotic inactivation. Numerous ␤-lactama-
ses exist, encoded either by chromosomal genes or by trans-
ferable genes located on plasmids or transposons (3, 17). Four
molecularly distinct ␤-lactamase classes (A, B, C, and D) have
been defined based on amino acid and nucleotide sequence
analysis (18). Of these, class A, to which the plasmid-mediated
TEM-1 enzyme belongs, is the most prevalent in clinical iso-
lates (26).
The classical approach to circumvent resistance has been to
develop new ␤-lactam antibiotics resistant to ␤-lactamase hy-
drolysis. However, new ␤-lactamases have risen to meet the
challenge of new synthetic ␤-lactam derivatives (11, 14, 18, 25).
A second approach has been to develop novel antibiotics.
However, these agents are often more toxic than their prede-
Enzyme-catalyzed therapeutic activation (ECTA) is a novel
prodrug strategy to overcome drug resistance resulting from
enzyme overexpression. In this approach, specific enzyme sub-
strates are designed for conversion by the overexpressed en-
zyme into cytotoxic agent(s). Based on the ECTA concept,
␤-lactamase can be used as an engine to generate antibacterial
agents in drug-resistant microorganisms. We describe here the
design, synthesis, and in vitro evaluation of the ␤-lactamase
ECTA compound NB2001. The formation of triclosan from
NB2001 via ␤-lactamase hydrolysis is demonstrated. The de-
pendence of antibacterial activity on ␤-lactamase is demon-
strated in an Escherichia coli model system, where NB2001
was more active against a TEM-1 ␤-lactamase-overproduc-
ing strain than a nonproducing strain; its antibacterial activity
against the former was antagonized by ␤-lactamase inhibitors.
Further in vitro studies showed NB2001 to be active against
major gram-positive and gram-negative pathogens.
MATERIALS AND METHODS
* Corresponding author. Mailing address: NewBiotics, Inc., 11760-E
Sorrento Valley Rd., San Diego, CA 92121. Phone: (858) 259-8600,
ext. 227. Fax: (858) 259-7634. E-mail: qli@newbiotics.com.
† Present address: Palomar Research, Inc., Encinitas, CA 92093-
1989.
Chemicals. Chemical reagents and anhydrous solvents were purchased from
Aldrich Chemicals (Milwaukee, Wis.). 7-Aminocephalosporanic acid was pur-
chased from Sigma (St. Louis, Mo.) or Antibiotico (Milan, Italy), and cephalo-
thin, oxacillin sodium salt, gentamicin sulfate, ampicillin sodium salt, and van-
comycin hydrochloride were from Sigma. Nitrocefin was from Calbiochem (San
1262

VOL. 46, 2002
ENZYME-CATALYZED ANTIMICROBIAL ACTIVATION
1263
FIG. 1. Outline of synthesis and chemical structure of NB2001 and its precursors. For details of this synthesis, see Materials and Methods.
Diego, Calif.). Sulbactam sodium was from Morepen Laboratories, Ltd. (New
Delhi, India). Lithium clavulanate was a generous gift from Smithkline Beecham
(West Sussex, United Kingdom), and triclosan was kindly provided by KIC
Chemicals (Armonk, N.Y.).
was 0.37 g (64%). 1H NMR (dimethyl sulfoxide [DMSO]-d6, 500 MHz): ␦ 3.10
(1H, d, J ϭ 16.5 Hz), 3.75 (2H, qAB, J ϭ 15.17, 4.75 Hz), 4.80 (1H, d, J ϭ 12.0
Hz), 4.97 (1H, d, J ϭ 12.0 Hz), 5.03 (1H, d, J ϭ 4.0 Hz), 6.80 (1H, d, J ϭ 8.7 Hz),
6.92 to 6.95 (2H, m), 7.09 to 7.07 (1H, m), 7.16 (1H, d, J ϭ 8.7 Hz), 7.29 to 7.31
(1H, m), 7.31 to 7.37 (1H, m), 7.72 (1H, d, J ϭ 2.8 Hz), and 9.11(1H, d, J ϭ 8.3
Hz). Infrared analysis (neat) 1,767, 1,715, 1,662, and 1,495 cmϪ1. The purity of
NB2001 was 98.2% as determined by high-pressure liquid chromatography
(HPLC).
Chemical analyses. Flash chromatography was performed on Merck grade 60
silica gel (230 to 400 mesh). Infrared spectra were measured neat on a Nicolet
Avatar 320 spectrophotometer and are reported as per-centimeter values. Proton
nuclear magnetic resonance spectra were recorded in a Varian Associates Gem-
ini (Bruker Instruments) spectrometer operating at 500 MHz, and chemical
shifts were reported relative to an internal tetramethylsilane reference at ␦ ϭ 0.0
ppm. Melting points are uncorrected.
Susceptibility testing. MICs of antimicrobial compounds were determined by
the broth microdilution method (range, 0.016 to 128 ␮g/ml) in 96-well microtiter
plates, according to National Committee for Clinical Laboratory Standards
guidelines (5). Stock solutions of test compounds were prepared in water or
DMSO, depending on their solubility. In the latter case, DMSO concentration in
the incubation mixture did not exceed 0.5%. The bacterial inoculum was 5 ϫ 105
CFU/ml, and growth was monitored by measuring the increase in the optical
density at 600 nm (OD600) with a microplate reader (Tecan SpectraFluor Plus).
MIC was defined as the lowest antibiotic concentration at which bacteria growth
(OD600 Ͼ 0.05, i.e., a value equal to visible growth) was inhibited after 18 to 24 h
of incubation at 35°C.
Bacterial strains. E. coli BL21(DE3) and E. coli N (Novablue) were obtained
from Novagen (Madison, Wis.). The E. coli/TEM-1 clone was generated by
transforming plasmid pcDNA3.1(Ϫ) (Invitrogen, San Diego, Calif.), which con-
stitutively expresses TEM-1 ␤-lactamase, in E. coli N. The bacterial strains used
for determining antimicrobial activity included Staphylococcus aureus strains
700698, 700699, 43300, 700787, 700788, 700789, 33591, 33592, 33593, 33594,
700260, 13301, 11632, and 14154 (American Type Culture Collection [ATCC],
Manassas, Va.). A total of 300 recent clinical isolates tested in the Microbiology
Reference Lab (MRL) in Herndon, Va., were procured from the MRL Bacterial
Culture Collection and included S. aureus oxacillin-susceptible (n ϭ 20), S.
aureus oxacillin-resistant (n ϭ 20), Staphylococcus epidermidis oxacillin-suscep-
tible (n ϭ 15), S. epidermidis oxacillin-resistant (n ϭ 15), Streptococcus pneu-
moniae (n ϭ 10), Enterococcus faecalis (n ϭ 15), Enterobacter cloacae (n ϭ 30),
Enterobacter aerogenes (n ϭ 30), E. coli (n ϭ 30), Klebsiella pneumoniae (n ϭ 30),
Pseudomonas aeruginosa (n ϭ 10), Haemophilus influenzae (n ϭ 30), and Morax-
ella catarrhalis (n ϭ 30) strains.
All bacteria were grown in cation-adjusted Mueller-Hinton (MH) broth, ex-
cept for H. influenzae, which was grown in Haemophilus Test Medium broth, and
Streptococcus pneumoniae, which was grown in cation-adjusted MH broth sup-
plemented with 2 to 5% lysed horse blood.
Bacterial cultures were tested for ␤-lactamase production by use of nitrocefin
according to the manufacturer’s instructions.
Synthesis of NB2001. (i) Diphenylmethyl 7-(2-thienylacetylamino)-3-{[5-
chloro-2-(2,4-dichlorophenoxy)phenoxymethyl}-3-cephem-4-carboxylate (com-
pound II [Fig. 1]). A solution of diphenylmethyl 7-(2-thienylacetylamino)-3-
hydroxymethyl-3-cephem-4-carboxylate (compound I in Fig. 1) (28) (0.52 g, 1.0
mmol) and triclosan (0.36 g, 1.25 mmol) in anhydrous THF (10 ml) was cooled
to Ϫ20°C in a dry-ice bath under an argon atmosphere. A solution of triphenyl-
phosphine (0.32 g, 1.25 mmol) in anhydrous THF (5.0 ml) was then added by
using a syringe. After 10 min, a solution of diisopropyl diazodicarboxylate (0.25 g,
1.25 mmol) in THF (5.0 ml) was added slowly by using a syringe pump over
40 min. The reaction mixture was then poured into water and extracted twice
with ethyl acetate (25 ml each time). The combined ethyl acetate extracts were
washed sequentially with water and brine and dried over Na2SO4. Removal of
volatiles, followed by purification on a silica gel column with 5% ethyl acetate in
dichloromethane, provided compound II as light yellow solid (yield ϭ 0.32 g,
32%).
(ii) 7-(2-Thienylacetylamino)-3-[5-chloro-2-(2,4-dichlorophenoxy)phenoxym-
ethyl]-3-cephem-4-carboxylic acid (compound III in Fig. 1). Compound II (0.75
g, 0.94 mmol) was dissolved in anhydrous anisole (4.0 ml) and cooled in an ice
bath under an argon atmosphere. Trifluoroacetic acid (TFA; 9.0 ml) was then
introduced slowly over 10 min. After 45 min, the volatiles were removed under
reduced pressure with continuous stirring to give a light yellow residue. Ether (10
ml) was added, and the mixture was stirred at 0°C for 1 h. The resultant light
yellow precipitate was filtered, washed with cold ether (10 ml), and dried under
vacuum to obtain NB2001 as a light yellow solid (melting point, 105°C). The yield
Time-kill kinetics. Overnight liquid cultures of E. coli/ TEM-1 were diluted
100 times into MH broth and allowed to grow to exponential phase (OD600
ϭ
0.6). Bacteria were next diluted into fresh medium to give a working concentra-
tion of 106 CFU/ml. NB2001 or triclosan was added to a final concentration
corresponding to 2ϫ, 4ϫ, and 8ϫ MIC, and the suspension was incubated at
37°C. Aliquots (0.1 ml) were removed at 1-h intervals for up to 6 h of incubation.
Serial 10-fold dilutions down to 10Ϫ6 were prepared in saline and plated onto
agar plates. The plates were incubated for 24 h at 37°C to obtain CFU counts.
TABLE 1. Comparison of the ␤-lactamase TEM-1 kinetic
constants for NB2001 and nitrocefina
a
a
Substrate
kcat (sϪ1
)
Km (␮M)
kcat/Km (MϪ1 sϪ1
)
NB2001
Nitrocefin
0.50
3.3
6.5
15
7.7 ϫ 104
22 ϫ 104
a The assay conditions for NB2001 (fixed-time assay) and nitrocefin (contin-
uous assay) hydrolyses are described in Materials and Methods. The kinetic
constants kcat and Km for NB2001 were determined from a progress curve
analysis fit to the integrated Michaelis-Menten equation at 16 ␮M NB2001 and
12.5 nM TEM-1 ␤-lactamase.

1264
LI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
␤-Lactamase assays. Initial assessment of partially purified ␤-lactamase
TEM-1 was by use of the chromogenic substrate nitrocefin on a Tecan Spec-
trafluor Plus with a 495 cutoff filter on the excitation path. Nitrocefin in concen-
trations ranging from 2 to 100 ␮M, diluted from a 10 mM stock in DMSO, was
added to 100 mM potassium phosphate (pH 7.2) and 1 mM EDTA and incu-
bated at 37°C for 15 min. After thermal equilibrium, 0.25 ␮g of TEM-1 ␤-lac-
tamase/ml was added and the hydrolysis product, nitrophenol, was monitored at
486 nm. The extinction coefficient, ε, of nitrophenol (15,900 MϪ1 cmϪ1) was
determined from a standard curve of the total hydrolysis of nitrocefin and found
to be consistent with published values (1).
Enzyme hydrolysis of NB2001 was determined by fixed-time assays of varied
concentrations of NB2001, followed by the addition of a 1% TFA quench. The
hydrolysis product, triclosan, was separated from NB2001 by use of an HP1100
series HPLC equipped with an Alltech Adsorbosphere HS(C18) 5-ml column (50
by 4.6 mm). The mobile phase was isocratic, containing 55% acetonitrile and
0.1% TFA, producing retention times of 20.4 and 24.1 min for triclosan and
NB2001, respectively. The flow rate was 1 ml minϪ1. Quantitation of triclosan
was based on the A260 integrated peak area compared to triclosan standards.
Detection of triclosan formed from NB2001 in E. coli/TEM-1. Overnight cul-
tures of both wild-type E. coli N and the ␤-lactamase-producing strain E. coli/
TEM-1 were diluted 100 times with fresh Luria broth (LB) medium, and cells
were grown at 37°C with shaking at 230 rpm for 4 h. Cells were then diluted with
fresh LB medium containing 100 ␮M NB2001 until the OD600 reached 0.2.
Culture aliquots were removed at different time intervals, and the cells were
pelleted by centrifugation at 10,000 ϫ g for 2 min at 4°C. The supernatant was
removed and combined with 500 ␮l of acetonitrile. The pelleted bacterial cells
were resuspended in 100 ␮l of phosphate-buffered saline (pH 8.0; Gibco-BRL)
and subjected to three cycles of freeze-thawing, followed by the addition of 500
␮l of acetonitrile. Both pellet and supernatant extracts were centrifuged at 10,000
ϫ g for 2 min at 4°C. Then, 550 ␮l of the resultant supernatant was transferred
into a new Eppendorf tube and vacuum dried. Dried pellets were resuspended in
50 ␮l of H2O, and 100 ␮l of acetonitrile was added. Finally, 75 ␮l of the resulting
solution was analyzed by HPLC as described above.
FIG. 2. Time course of TEM-1-catalyzed formation of triclosan
from NB2001. Assays were conducted at 37°C in 100 mM potassium
phosphate buffer (pH 7.2) with 1 mM EDTA and 0.5 ␮g of TEM-1
␤-lactamase/ml. A hydrolysis product (triclosan) was separated from
NB2001 by HPLC as described in Materials and Methods. In the
absence of TEM-1 ␤-lactamase, NB2001 was stable for at least 5 h at
37°C.
Preparation of TEM-1 ␤-lactamase. An N-terminal His-tagged TEM-1 con-
struct, TEM-1/pET28b(ϩ), was generated by subcloning TEM-1 into the NcoI
and HindIII sites of pET28b(ϩ) vector. TEM-1 was prepared by transforming
TEM-1/pET28b(ϩ) into E. coli BL21(DE3) (Novagen) strain. After induction
with IPTG (isopropyl-␤-D-thiogalactopyranoside), TEM-1 was purified by affinity
chromatography on a Ni2ϩ His-binding metal chelation resin (Novagen). The
Ni2ϩ His-binding metal chelation column was washed with 20 mM Tris (pH
7.9)–5 mM imidazole–0.5 M NaCl. TEM-1 was eluted with 20 mM Tris (pH 7.9),
100 mM imidazole, and 0.5 M NaCl at room temperature and dialyzed against
100 mM Tris (pH 8.0). Aliquots were stored at Ϫ80°C.
RESULTS
Design and synthesis of NB2001. The generic design of
␤-lactamase ECTA compounds includes a cephalosporin back-
bone with a prodrug form of an antibacterial agent incorpo-
rated at the 3Ј position of the cephem nucleus. Hydrolysis by
␤-lactamase triggers release of the antibacterial agent. One of
FIG. 3. Formation of triclosan from NB2001 in E. coli. E. coli N and ␤-lactamase-expressing E. coli/TEM-1 were treated with NB2001 under
the conditions described in Materials and Methods. Culture aliquots were removed at 0, 5, and 15 min. The amount of NB2001 and triclosan was
assessed by HPLC.

VOL. 46, 2002
TABLE 2. Activity of NB2001 with or without ␤-lactamase
ENZYME-CATALYZED ANTIMICROBIAL ACTIVATION
1265
TABLE 4. In vitro activities of NB2001 and reference antimicrobial
agents against recent clinical isolates of gram-positive
bacteria (obtained from MRL)
inhibitors against wild-type and ␤-lactamase-producing E. coli
MIC (␮g/ml)a of:
MIC (␮g/ml)b
Organism (no. of strains)a
and antimicrobial agent
Relevant
phenotype
NB2001
With 4 ␮g
Strain
Range
50%
90%
Cephalothin Triclosan
Alone
of CLA/ml
MS S. aureus (20)
NB2001
0.06–0.25
0.5–1
0.12
0.5
2
0.25
0.25
0.12
1
0.25
1
2
0.25
0.5
0.25
16
E. coli N
E. coli/TEM-1
E. coli 25922
BLAϪ
BLAϩ
BLAϪ
0.5
0.016
1
0.5
0.25
0.5
8
Ͼ64
16
0.03
0.03
0.13
Vancomycin
Linezolid
1–2
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Oxacillin
Յ0.12–0.5
0.06–2
Յ0.06–2
Յ0.25–16
a BLA, ␤-lactamase; CLA, lithium clavulanate. Lithium clavulanate (4 ␮g/ml)
had no effect on cells when tested alone.
Ampicillin
MR S. aureus (20)
NB2001
our lead compounds, NB2001, was prepared by the condensa-
tion of cephalothin with triclosan under Mitsunobu reaction
conditions (28), followed by deprotection (Fig. 1). The purity
of NB2001 was 98.2% as determined by HPLC.
0.06–1
0.5–1
0.12
0.5
2
0.5
1
Vancomycin
Linezolid
1–2
2
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Oxacillin
0.25–0.5
0.25–4
4–16
0.25
2
0.5
4
8
NB2001 hydrolysis by TEM-1 ␤-lactamase. NB2001 was ex-
amined as a substrate of TEM-1 ␤-lactamase and compared to
nitrocefin, a widely used chromogenic ␤-lactamase substrate with
the same C-7 side chain as NB2001. As shown in Table 1, the
kinetic parameters of NB2001 were comparable to nitrocefin.
Interestingly, nitrocephin hydrolysis was slower than that re-
ported in the literature, probably due to the fact that the TEM-1
enzyme was His tagged. In a further study, TEM-1 ␤-lactamase-
mediated hydrolysis of NB2001 was found to be associated with
the concomitant production of triclosan (Fig. 2).
Triclosan formation from NB2001 by TEM-1-producing E.
coli. Triclosan formation by ␤-lactamase was examined in an E.
coli model system established in our laboratory. We used the
cloned ␤-lactamase-producing strain, E. coli/TEM-1, and its
parental strain E. coli N, differing only in the expression of
TEM-1 ␤-lactamase. As shown in Fig. 3, 80% of NB2001 was
degraded, and an equimolar amount of triclosan was produced
in E. coli/TEM-1 after 5 min, whereas no triclosan was detected in
E. coli N after up to 15 min of incubation. Thus, triclosan pro-
duction from NB2001 is dependent on ␤-lactamase.
8
8
Ampicillin
Յ0.25–Ͼ16
Ͼ16
MS S. epidermidis (15)
NB2001
Vancomycin
Linezolid
0.015–0.12
0.5–2
0.5–2
Յ0.12–0.5
0.06–0.25
Յ0.06–0.12
Յ0.25–16
0.06
1
1
Յ0.12
0.12
Յ0.06
Յ0.25
0.06
2
2
0.25
0.25
0.12
1
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Oxacillin
Ampicillin
MR S. epidermis (15)
NB2001
Vancomycin
Linezolid
0.03–0.5
1–2
Յ0.25–1
Յ0.12–0.25
0.25–4
2–Ͼ32
Յ0.25–8
0.06
0.25
2
1
1
1
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Oxacillin
0.12
0.25
2
1
4
2
32
8
Ampicillin
PS S. pneumoniae (4)
NB2001
Vancomycin
Linezolid
0.12–0.25
0.12–0.25
0.5–1
Յ0.015–0.06
Յ0.12–0.5
Յ0.015–Յ0.015
Յ0.25–Յ0.25
0.12
0.25
0.5
Յ0.015
Յ0.12
Յ0.015
Յ0.25
0.25
0.25
1
Amoxicillin-clavulanate
Cefuroxime axetil
Ceftriaxone
0.06
0.5
Յ0.015
Յ0.25
Ampicillin
Dependence of antibacterial activity of NB2001 on ␤-lacta-
mase. This was initially demonstrated in the ␤-lactamase-pro-
ducing strain E. coli/TEM-1. As shown in Table 2, NB2001 was
PR S. pneumoniae (6)
NB2001
Vancomycin
Linezolid
1–8
0.25–0.25
0.5–1
2
8
0.25
1
0.25
0.5
4
TABLE 3. Antibacterial activities of NB2001 and
Amoxicillin-clavulanate
Cefuroxime axetil
Ceftriaxone
0.12–8
8
1–32
8
32
4
reference antibiotics against S. aureus
0.25–4
1
MIC (␮g/ml) of:
Ampicillin
Յ0.25–Ͼ16
4
16
S. aureus type and ␤-Lactamase
ATCC no.
statusa
NB2001 Vancomycin Oxacillin Triclosan
VS E. faecalis (10)
NB2001
Vancomycin
Linezolid
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Ampicillin
4–8
1–2
4
8
2
1
8
1
2
Methicillin resistant
700698
1
Neg
4
2
Ͼ32
32
0.063
Յ0.016
Յ0.016
Յ0.016
Յ0.016
Յ0.016
0.5–2
1–8
1
700789
Pos
Pos
Pos
Pos
Pos
0.031
2
4
700787
700788
33591
33593
Յ0.016
Յ0.016
Յ0.016
Յ0.016
8
2
0.25
0.5
Ͼ32
Ͼ32
8
0.5–1
1–2
0.5
1
VR E. faecalis (5)
NB2001
Vancomycin
Linezolid
Quinupristin-dalfopristin
Amoxicillin-clavulanate
Ampicillin
8
2–8
32–Ͼ32
1–1
8–Ͼ8
0.25–1
0.5–2
4
32
1
Ͼ8
0.5
1
8
Ͼ32
Methicillin sensitive
33594
1
Ͼ8
1
Pos
Pos
8
4
0.5
0.25
0.5
0.5
0.5
0.25
0.25
0.25
2
2
0.5
4
13301
2
43300
Neg/Pos Յ0.016
Յ0.016
Յ0.016
Յ0.016
2
33592
Pos
Pos
Յ0.016
Յ0.016
a MS, methicillin susceptible; MR, methicillin resistant; PS, penicillin suscep-
tible; PR, penicillin resistant; VS, vancomycin susceptible; VR, vancomycin re-
sistant; S. pneumoniae, Streptococcus pneumoniae; E. faecalis, Enterococcus fae-
calis.
14154
11632
Neg/Pos Յ0.016
0.25 Յ0.016
a Neg, negative; Pos, positive; Neg/Pos, strains may have very low level expres-
sion of ␤-lactamase.
b MIC assays were performed at MRL.

1266
LI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Susceptibility to NB2001 of ␤-lactamase-positive and -negative strains of M. catarrhalis and H. influenzae.
32-fold more potent against the E. coli/TEM-1 than against the
␤-lactamase-negative strain E. coli N. The increased suscepti-
bility of E. coli/TEM-1 to NB2001 was abolished in the pres-
ence of the ␤-lactamase inhibitor clavulanate (Table 2), sug-
gesting that it was dependent on ␤-lactamase.
resistance to commonly used antibiotics (22), was also suscep-
tible to NB2001 (MIC50 ϭ 1 ␮g/ml).
NB2001 was also active against gram-negative species (Table
5). Its MIC50s against ␤-lactamase-producing M. catarrhalis
and H. influenzae were at least fourfold superior to ciprofloxa-
cin. Against Enterobacter aerogenes, Enterobacter cloacae, E.
coli, and K. pneumoniae, NB2001 was more active than amoxi-
cillin-clavulanate, ceftazidime, or ceftriaxone and less active
than imipenem and ciprofloxacin. However, NB2001 showed
no activity against P. aeruginosa (Table 5).
Of the 60 M. catarrhalis and H. influenzae isolates tested, 40
were ␤-lactamase-producing strains. The distribution of NB2001
MICs clearly showed greater activity against ␤-lactamase-pro-
ducing strains than ␤-lactamase-negative ones (Fig. 4).
Bactericidal activity of NB2001. We examined whether
NB2001 was bactericidal or bacteristatic in E. coli/TEM-1. At
concentrations up to eightfold its MIC, NB2001 showed no
bactericidal activity after 6 h of incubation. Similarly, its toxo-
phore triclosan was also clearly bacteristatic (Fig. 5).
Activity of NB2001 against S. aureus. Most ␤-lactam-resis-
tant isolates of S. aureus produce ␤-lactamase (16, 17). We
compared the in vitro antibacterial activity of NB2001 against
12 ATCC S. aureus strains to a panel of reference antimicrobial
agents, including vancomycin, the antibiotic of choice for clin-
ical applications. The MIC50 of NB2001 was Յ0.016 ␮g/ml,
which is at least 32-fold lower than vancomycin (MIC50 ϭ 0.5
␮g/ml) (Table 3). NB2001 exhibited higher activity against
most ␤-lactamase-producing S. aureus strains than the nonpro-
ducing ATCC 700698, although the ␤-lactamase nonproducer
was also sensitive to triclosan (MIC ϭ 0.063 ␮g/ml). Predict-
ably, NB2001 was not very active against two ␤-lactamase-
producing, but relatively triclosan-resistant strains ATCC
33594 and ATCC 13301 (Table 3).
In vitro antibacterial activity of NB2001 against other
pathogens. The antibacterial activity of NB2001 and compar-
ator antimicrobial agents was evaluated with 300 recent clinical
isolates, including S. aureus and other major human pathogens,
at MRL. Antimicrobial comparator agents were chosen from
appropriate drug classifications for the treatment of gram-
positive or gram-negative infections. Generally, NB2001 dem-
onstrated broad-spectrum activity against all species with the
exception of P. aeruginosa (Tables 4 and 5).
NB2001 inhibited all 70 strains of staphylococci tested
(including methicillin-resistant S. aureus and S. epidermidis)
at a concentration of 1 ␮g/ml or less, being equipotent to
quinupristin-dalfopristin and more active than vancomycin,
linezolid, amoxicillin-clavulanate, cefuroxime axetil, and ceftri-
axone (Table 4). It also demonstrated activity against Entero-
coccus faecalis, including vancomycin-resistant strains (MIC50
ϭ 4 ␮g/ml), although this was substantially less than other
against gram-positive bacteria, probably reflecting the low lev-
els of ␤-lactamase in that organism. Nevertheless, the data
highlight the potential of NB2001 against this clinically signif-
icant problem pathogen. Streptococcus pneumoniae, the major
pathogen for community-acquired pneumonia with increasing
In a separate experiment, we performed a synergy study
between cephalothin and triclosan in E. coli ATCC 25922 and
found that the two agents have additive effects (data not shown).
DISCUSSION
ECTA is a novel prodrug strategy, whose effectiveness de-
pends upon the activation of nontoxic compounds by bacteri-
um-specific enzymes. We chose as a target ECTA enzyme
␤-lactamase, a well-characterized and widely prevalent bacte-
rial enzyme expressed extracellularly or in the periplasmic
space. A key disease resistance mechanism, overexpression of
␤-lactamase, was thereby exploited for the formation of anti-
bacterial agents, and it became a marker of enhanced suscep-
tibility to NB2001. This compound was synthesized as a proof-
of-principle molecule for the ECTA technology.
The structure of NB2001 is similar to that of cephalothin and
nitrocefin. It is composed of a cephem core with a 2-thienyla-
cetylamino side chain at the 7-position, which is an important
determinant of ␤-lactamase binding. In addition, NB2001 con-
tains a prodrug form of the antimicrobial agent triclosan, which
is converted to its fully active form by ␤-lactamase. Triclosan is

VOL. 46, 2002
ENZYME-CATALYZED ANTIMICROBIAL ACTIVATION
1267
TABLE 5. In vitro activities of NB2001 and reference antimicrobial agents against gram-negative bacteria
MIC (␮g/ml)b
MIC (␮g/ml)b
Organism (no. of strains)a
and antimicrobial agent
Organism (no. of strains)a
and antimicrobial agent
Range
50%
90%
Range
50%
90%
BP M. catarrhalis (20)
NB2001
Ampicillin
32–Ͼ32
0.25–2
Ͼ32
Ͼ32
Յ0.004–0.015
Յ0.015–0.5
Յ0.03–0.12
0.008–0.03
Յ1–Յ1
Յ0.004
Յ0.015
Յ0.03
0.015
Յ1
Յ0.004
0.25
0.06
0.015
Յ1
0.25
2
Gentamicin
0.5
1
Ceftriaxone
E. cloacae (30)
NB2001
Imipenem
0.5–Ͼ32
0.12–Ͼ32
0.03–Ͼ64
0.25–2
2
Ͼ32
32
8
Ͼ32
Ͼ64
2
2
Ͼ32
0.5
Ͼ32
4
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
Ceftazidime
Ceftriaxone
0.06–0.5
0.12
Imipenem
0.5
Յ0.12–2
0.5
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
Յ0.002–Ͼ4
4–Ͼ32
0.03
Ͼ32
0.06
Ͼ32
0.5
BN M. catarrhalis (20)
NB2001
Ceftriaxone
Imipenem
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
0.015–0.06
0.03
0.03
Յ0.015
Յ0.03
0.015
Յ1
0.12
Յ0.12
0.03–32
Յ0.015–Յ0.015
Յ0.03–Յ0.03
0.008–0.03
Յ1–Յ1
Յ0.015
Յ0.03
0.015
Յ1
2–Ͼ32
Gentamicin
0.25–Ͼ16
E. coli (30)
NB2001
Ceftazidime
Ceftriaxone
Imipenem
1–16
0.06–Ͼ32
Յ0.015–Ͼ64
0.06–0.5
0.004–Ͼ4
2–32
Յ0.015–Ͼ32
1–Ͼ32
0.25–Ͼ16
4
8
32
8
0.25
Ͼ4
32
Ͼ32
Ͼ32
2
0.03–0.12
Յ0.12
0.06
Յ0.12
0.25
0.06
0.12
0.008
4
BP H. influenzae (20)
NB2001
0.12–2
Յ0.03–0.12
Յ0.015–0.03
0.25–0.5
0.004–0.015
Յ1–4
0.5
0.06
Յ0.015
0.5
0.008
Յ1
1
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
Ceftazidime
0.06
Յ0.015
0.5
Ceftriaxone
0.06
32
0.5
Imipenem
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
0.015
4
16
Gentamicin
K. pneumoniae (30)
NB2001
0.06–16
8–Ͼ32
0.25
Ͼ32
1–Ͼ32
Յ0.03–Ͼ32
Յ0.015–Ͼ64
0.06–1
0.008–Ͼ4
Յ1–Ͼ32
0.06–Ͼ32
16–Ͼ32
0.25–Ͼ16
8
32
4
0.12
0.12
8
0.5
Ͼ32
4
16
Ͼ32
Ͼ64
1
Ͼ4
32
Ͼ32
Ͼ32
Ͼ16
Ͼ32
Ceftazidime
BN H. influenzae (10)
NB2001
Ceftriaxone
2–4
Յ0.03–0.12
Յ0.015–Յ0.015
0.06–0.5
0.008–0.015
Յ1–Յ1
2
4
Imipenem
Ceftazidime
0.06
Յ0.015
0.25
0.008
Յ1
0.06
Յ0.015
0.5
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
Ceftriaxone
Imipenem
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
0.008
Յ1
1
0.25
Gentamicin
0.12–1
Յ0.12–0.25
0.5
Յ0.12
P. aeruginosa (10)
NB2001
Ͼ32–Ͼ32
1–Ͼ32
2–Ͼ64
0.25–Ͼ16
0.12–4
4–Ͼ32
Ͼ32
Ͼ32
4
64
2
1
Ͼ32
8
Ͼ32
2
Ͼ32
Ͼ32
Ͼ64
16
2
Ͼ32
16
E. aerogenes (30)
NB2001
Ceftazidime
0.5–16
0.12–Ͼ32
0.03–64
0.25–2
0.008–Ͼ4
32–Ͼ32
0.03–32
4
8
Ͼ32
32
2
1
Ͼ32
0.5
Ceftriaxone
Ceftazidime
32
Imipenem
Ceftriaxone
16
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
Ampicillin
Imipenem
1
Ciprofloxacin
Amoxicillin-clavulanate
Sulfamethoxazole
0.015
32
4–Ͼ32
1–Ͼ16
Ͼ32
16
0.12
Gentamicin
a BP, ␤-lactamase positive; BN, ␤-lactamase negative; E. cloacae, Enterobacter cloacae, E. aerogenes, Enterobacter aerogenes.
b MIC assays were performed at MRL.
a broad-spectrum agent that acts via inhibition of the enoyl-
acyl carrier protein reductase (FabI) (7, 8, 12). Because of its
high potency in bacteria and its low toxicity in humans, tri-
closan itself is widely used in formulations as diverse as anti-
septic soap, toothpaste, cosmetics, and household products (2).
NB2001 was designed to have a dual mode of action:
cephalosporin activity against ␤-lactamase-negative organ-
isms and triclosan-associated activity against ␤-lactamase-
producing strains. As we have shown, NB2001 is a good
substrate for ␤-lactamase TEM-1. The idea of multiple
mechanisms of action for NB2001 is supported by the low
frequency (Ͻ10Ϫ8) of bacteria resistant to NB2001 (unpub-
lished data).
tamases present in amounts undetectable by the nitrocefin test
and/or acylation of target penicillin-binding proteins.
NB2001 had potent activity against S. aureus, a major human
pathogen (19, 30) of increasing significance in nosocomial
drug-resistant infections (6, 20, 27, 31). The compound was
also active against a number of clinically important gram-pos-
itive and -negative pathogens. Its high potency against problem
pathogens such as methicillin-resistant staphylococci (S. aureus
and S. epidermidis) and vancomycin-resistant Enterococcus fae-
calis underscores its potential for clinical use.
P. aeruginosa is known to be resistant to a variety of antibi-
otics. The primary reason for the multidrug resistance of P.
aeruginosa is its outer membrane (4) in combination with mul-
tidrug efflux pumps (13, 23). We have confirmed both of these
reasons to account for the low activity of NB2001 against P.
aeruginosa (unpublished data).
␤-Lactamase ECTA compounds have enhanced activity against
␤-lactamase-producing strains but also good activity against ␤-lac-
tamase-negative strains. For example, in E. coli N NB2001 has
higher activity relative to cephalothin (Table 2). The higher ac-
tivity might be due to triclosan production via hydrolysis by ␤-lac-
Antibiotics used in the treatment of most common bacterial
infections attack only a few distinct targets in the pathogen

1268
LI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. Time-kill curve of NB2001 and triclosan against ␤-lactamase-producing E. coli. The compounds were added to log-phase cultures of
E. coli/TEM-1 and incubated at 37°C. Culture aliquots were removed every hour up to 6 h, and the number of cells (CFU) was determined as
described in Materials and Methods.
contributing factor to ␤-lactam resistance. Antimicrob. Agents Chemother.
(24), such as cell wall synthesis, protein synthesis, or DNA
gyrase. ECTA represents a novel approach in that bacterium-
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shows, the ECTA approach is an effective way of harnessing
drug resistance caused by enzyme overexpression.
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15. Livermore, D. M., M. Akova, P. J. Wu, and Y. J. Yang. 1989. Clavulanate and
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ACKNOWLEDGMENTS
We gratefully acknowledge Nafsika Georgopapadakou for helpful
discussions and valuable aid during the revision of the manuscript.
Thanks are also extended to Analia Bueno for technical contributions
in the evaluation of NB2001.
19. Muder, R. R., C. Brennen, M. M. Wagener, R. M. Vickers, J. D. Rihs, G. A.
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