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Antimicrobial Agents and Chemotherapy, February 2004, p. 477-483, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.477-483.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by ß-Lactamases

Geoffrey W. Stone,^* Qin Zhang, Rosario Castillo, V. Ramana Doppalapudi, Analia R. Bueno, Jean Y. Lee, Qing Li, Maria Sergeeva, Gody Khambatta, and Nafsika H. Georgopapadakou

NewBiotics, Inc., San Diego, California 92121

Received 12 May 2003/ Returned for modification 1 September 2003/ Accepted 2 November 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit ß-lactamases to release triclosan through hydrolysis of the ß-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A ß-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C ß-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k_cat/K_m). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the ß-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ubiquitous occurrence of ß-lactamases in bacteria and their association with clinical resistance to ß-lactams have allowed strong interest in these enzymes to be sustained (3, 26). ß-Lactamases are periplasmic in gram-negative bacteria, while they are exocellular in gram-positive bacteria. They are conveniently divided into four classes (classes A, B, C, and D) on the basis of amino acid sequence homologies (Ambler classification) (1, 15). Of these classes, classes A and C are of the greatest clinical significance. ß-Lactamase inhibitors, such as clavulanic acid and the penicillanic acid sulfones, mostly target class A enzymes; and their use in combination with older compounds has restored the broad-spectrum activities of older compounds, such as amoxicillin (18, 22, 25, 26). Inhibitors that target class C and class B ß-lactamases have yet to reach clinical development.

Exploiting common ß-lactamases to generate novel antibacterials is a strategy originally used by O'Callaghan et al. (24) and subsequently by Mobashery and Johnston (20) and other investigators (6, 10, 16). It has been part of NewBiotics' general enzyme-catalyzed therapeutic activation (ECTA) prodrug approach that harnesses unique enzymes in bacteria to achieve selective release of cytotoxic agents from substrate-like molecules (16; M. V. Sergeeva, G. H. Khambatta, B. E. Cathers, R. S. Castillo, V. R. Doppalapudi, H. H. Bendall, A. R. Bueno, J. Y. Lee, Q. Li, and N. H. Georgopapadakou, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. A-113, 2002). These antibacterials developed by the ECTA approach are designed so that neither they nor their products inactivate the enzymes that activate them.

We have synthesized a variety of cephalosporins with different C-7 side chains that affect enzymatic hydrolysis, cephalosporins with different C-3 linkers that affect chemical stability, and different toxophores that are released upon opening of the ß-lactam ring. Two of these compounds, NB2001 and NB2030, contain cephalothin (thienyl) and cefazolin (tetrazole) side chains at position C-7, respectively and the enoyl reductase inhibitor triclosan at the C-3 position of the cephem nucleus (Fig. 1). Triclosan is designed to act as a toxophore that is released upon ß-lactam hydrolysis in ß-lactamase-positive bacteria. Here we describe studies aimed at elucidating the mechanisms of action of NB2001 and NB2030 in Escherichia coli and Staphylococcus aureus. In E. coli, the overall mechanisms of action of these compounds were deduced from the cell morphologies observed after treatment of the cells with the compounds. Their levels of transport into bacterial cells, their levels of penicillin-binding protein (PBP) binding, their activities as ß-lactamase substrates, and the activity of the bound triclosan moiety relative to that of free triclosan against the enoyl reductase target were also determined.

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FIG. 1. Chemical structures of NB2001 and NB2030.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals. Unless otherwise noted, biochemicals were obtained from Sigma (St. Louis, Mo.) and culture media were obtained from Difco (Sparks, Md). Triclosan was kindly provided by KIC Chemicals (Armonk, N.Y.), anhydrous solvents were purchased from Aldrich Chemicals (Milwaukee, Wis.), and nitrocefin was from Calbiochem (San Diego, Calif.). Benzyl-[¹⁴C]penicillin (58 mCi/mmol) and -[1-¹⁴C]aminoisobutyric acid (50 mCi/mmol) were purchased from Amersham (Piscataway, N.J.) and ICN Pharmaceuticals (Irvine, Calif.), respectively.

Organisms. Unless stated otherwise, experiments were performed with standard susceptible strains obtained from the American Type Culture Collection (ATCC; Manassas, Va.): E. coli ATCC 25922, S. aureus ATCC 29213, Enterobacter cloacae ATCC 13047, Enterococcus faecalis ATCC 29212, S. aureus ATCC 33591 (methicillin resistant), Pseudomonas aeruginosa ATCC 27853, Streptococcus pneumoniae ATCC 49619, and S. pneumoniae ATCC 700671. E. coli TE18 (22) was kindly provided by H. Nikaido, E. cloacae P99 (13, 17, 31) was provided by K. Bush, and S. aureus strains RN4220 and PC1 (14, 30, 32) were provided by J. Iandolo. E. coli (TEM-1) and its parent strain, E. coli N, have been described previously (16).

MIC determinations. Antibacterial activity was determined by the broth microdilution method in Mueller-Hinton broth (Difco) according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines (21).

Expression and assay of ß-lactamase in whole cells. ß-Lactamase expression in each strain was determined colorimetrically. A 1-ml mid-log-phase culture was grown under standard conditions for MIC determination. Nitrocefin was added at a final concentration of 25 µg/ml in dimethyl sulfoxide (DMSO; 0.25%). The cultures were scored positive if the broth color changed from yellow to red within 30 min at room temperature.

ß-Lactamase preparation and spectrophotometric assay. The AmpC and TEM-1 ß-lactamases were partially purified by previously published procedures (8). Briefly, AmpC (or TEM-1) was released from the E. coli periplasm by osmotic shock, and the supernatant (the pH of which was adjusted with 10 mM Tris-HCl [pH 7.0] for AmpC or 10 mM Tris-HCl [pH 7.5] for TEM-1) was run through an Amersham CM Sepharose column (AmpC) or an Amersham DEAE Sepharose column (TEM-1). ß-Lactamase was eluted with 100 mM Tris-HCl at pH 7.0 (AmpC) or pH 7.5 (TEM-1), and active fractions were identified by the nitrocefin assay. Positive fractions were pooled, concentrated to 0.5 mg/ml (AmpC) or 10 mg/ml (TEM-1) in a Centricon-10 filter (Millipore, Bedford, Mass.), mixed with 1 volume of 100% glycerol, and stored at -20°C.

The PC1 ß-lactamase was partially purified by the procedure of Kernodle et al. (14). Briefly, S. aureus PC1 was grown for 16 h at 37°C in 3 liters of modified CY broth (23) containing 0.5 µg of methicillin per ml, the cells were removed by centrifugation at 8,000 x g for 10 min, and the supernatant was mixed with 100 ml of P11 cellulose (Whatman) for 1 h at room temperature with shaking. The cellulose was then packed on a column, washed with 2 volumes of 0.02 M sodium citrate (pH 6.0), and eluted with 200 ml of 2.0 M ammonium sulfate-0.02 M sodium citrate (pH 6.0). Fractions were assayed with nitrocefin, and active samples were pooled and concentrated to 2 mg/ml in a Centricon-10 filter (Millipore), followed by the addition of 1 volume of 100% glycerol and storage at -20°C.

ß-Lactamase activity was assayed spectrophotometrically at 25°C in a Beckman DU-640 spectrophotometer with the Beckman DU series kinetics software package by a modification of previously published methods (8, 27, 32). Differential spectra after P99-mediated hydrolysis gave optimal wavelengths of 230 nm for benzylpenicillin and 262 nm for cephalothin, NB2001, and NB2030. Assay mixtures contained, in a 1-ml total volume, 10 mM Tris-HCl (pH 7.2), 2% DMSO, the ß-lactamase of interest, and substrate concentrations from 0.33 to 4 times the estimated K_m. The optimal enzyme concentration for each assay was determined by use of a saturation curve with estimated K_m values for cephalothin (8, 29, 32). For the assays with benzylpenicillin, DMSO was kept at a concentration of less than 0.5% due to its absorbance at 230 nm. Initial rates were plotted against the substrate concentration, and K_m and V_max were calculated by Marquardt nonlinear regression analysis (2).

Membrane preparation and PBP binding assay. E. coli ATCC 25922 and S. aureus ATCC 29213 were grown in brain heart infusion medium (Difco) to late log phase, harvested by centrifugation, and washed with 50 mM potassium phosphate buffer (pH 7.0) as described previously (9). For E. coli, the membranes were prepared by sonication of the cells at 4°C three times for 30 s each time (with 1-min cooling intervals between sonications) in 1 to 2 volumes of 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM magnesium chloride and 2 µg of DNase per ml. For S. aureus, sonication was preceded by 30 min of incubation at 37°C with lysostaphin (50 µg/ml) and lysozyme (100 µg/ml). The membranes were collected by centrifugation at 45,000 x g for 30 min, washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10 mM magnesium chloride and 2 µg of DNase per ml, and stored at -70°C if they were not used immediately. For PBP binding assays, the membranes were solubilized in 50 mM potassium phosphate buffer (pH 7.5) containing 1 M sodium chloride, 2% Triton X-100, and 1 mM ß-mercaptoethanol, as described previously (9). Samples containing 100 µg of solubilized membrane protein were incubated with increasing concentrations of test compound at 30°C for 10 min. After the addition of 1 µl (0.5 µCi) of benzyl-[¹⁴C]penicillin, the incubation was continued for another 10 min. Protein was precipitated with acetone and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide and 0.1% bisacrylamide for E. coli; 10% acrylamide and 0.27% bisacrylamide for S. aureus). The gels were subsequently incubated with En³Hance (New England Nuclear, North Billerica, Mass.), dried, and fluorographed at -70°C to visualize the radioactive bands.

Enoyl reductase cloning, purification, and assay. The fabI gene, which encodes enoyl reductase, of E. coli MG1655 (11) was amplified by PCR with primers EC5 (5'-CCTCTCCTCATATGGGTTTTCTTTCCGGTAAG-3') and EC3 (5'-CCTCGGATCCTTATTTCAGTTCGAGTTCGTTCAT-3'). The PCR product was digested with NdeI and BamHI and ligated into NdeI- and BamHI-digested expression plasmid pET28b(+). The new construct was transformed into BL21(DE3) cells, and the expressed enoyl reductase was purified with Ni²⁺ His-binding metal chelation resin (Novagen) and Novagen His-tagged purification reagents. Protein purity was estimated to be >90% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Purified enoyl reductase (final concentration, 24 nM) was added to an assay mixture (100 µM NADH, 100 mM sodium phosphate buffer [pH 7.0], 4% glycerol, various concentrations of crotonyl coenzyme A [crotonyl-CoA], and DMSO [final concentration, 1%]) in a final volume of 0.12 ml and incubated at 25°C. The rate of the reaction was measured by the time to the loss of the NADH absorbance peak at 340 nm. The K_m of crotonyl-CoA was determined to be 0.5 mM.

The 50% inhibitory concentrations (IC₅₀s) of triclosan, NB2001, and NB2030 were determined by incubating the assay mixture with 0.50 mM crotonyl-CoA and increasing concentrations of inhibitor. The rates of the reaction with various inhibitor concentrations were plotted, and IC₅₀s were determined by plotting percent activity (relative to that of the control without an inhibitor) against the inhibitor concentration. To determine the IC₅₀s of each inhibitor, data were fitted to the equation percent activity = 100/[(1 + [protein])/IC₅₀].

To determine the IC₅₀s in the presence of NAD⁺, enoyl reductase was preincubated with 100 µM NAD⁺, test compound, and the reaction mixture (without crotonyl-CoA) at 25°C for 30 min; and then the reaction was initiated by the addition of crotonyl-CoA.

Membrane integrity. Membrane integrity was measured by determination of the release of -[1-¹⁴C]aminoisobutyric acid, a radiolabeled nonmetabolizable amino acid (7). Briefly, the assay involves feeding of the labeled amino acid to the cells for 30 min, treatment of the cells with the test compound for 60 min, and measurement of the radioactivity remaining in the cells. The cationic detergent hexadecyltrimethyl ammonium bromide was used as a positive control treatment; when it was used at a concentration of 200 µg/ml (0.02%; wt/vol), it released 90% of the cell-associated radioactivity.

Cell morphology. E. coli ATCC 25922 was grown to mid-log phase in antibiotic medium 3 (Difco) by previously published procedures (5). Cells were diluted 1/100 in 1 ml of antibiotic medium 3 containing one of the test compounds at 0.2, 1, and 5 times the MIC. At 1 and 2 h after dilution, 5-µl samples were taken, spread onto glass slides, and air dried. The cells were fixed on the slide with poly-L-lysine and methanol and allowed to dry. Prior to fluorescence microscopy, 10 µl of 4',6-diamidino-2-phenylindole (DAPI) solution (5 µg/ml) in phosphate-buffered saline (PBS) was placed on the sample, and a glass coverslip was added. Cells were observed under x1,000 magnification. Nucleoids were viewed in a Nikon TE-200 fluorescence microscope equipped with a UV-2E/C DAPI filter (excitation, 340 to 380 nm) and photographed with a Diagnostic Instruments Spot digital camera (model 1.3.0) by using Spot software (version 3.0).

Transport assay. Mid-log-phase cells were pelleted at 5,600 x g, washed in 50 mM PBS (pH 7.2), and resuspended in PBS at 40 mg (wet weight) of cells per ml. The compound to be tested was added at a final concentration of 10 µg/ml, and 0.5-ml samples were removed at various time points and placed in prechilled Eppendorf tubes. By a modification of a previously published method (19), the cell suspensions were centrifuged at 10,000 x g, and the resulting pellets were resuspended in 1 ml of ice-cold PBS. The cells were pelleted again, resuspended in 300 µl of 50% aqueous acetonitrile, and incubated at room temperature for 10 min. The precipitate was removed by centrifugation, and 150 µl of supernatant was analyzed in an HP1100 high-pressure liquid chromatograph (Hewlett-Packard, Palo Alto, Calif.) equipped with a C₁₈ Alltech Adsorbosphere HS 5-µm column (4.6 by 50 mm), as described previously (16). Intracellular concentrations were calculated by using published values for volume (19), in which 1 g (wet weight) of cells equals 0.55 ml of ATCC 25922 cytoplasm and 0.52 ml of ATCC 29213 cytoplasm.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro antibacterial activity. As expected, cephalothin and cefazolin showed decreased activities against all strains expressing ß-lactamases: E. coli TEM-1, E. coli TE18, E. cloacae P99, and methicillin-resistant S. aureus (Table 1). They did not show decreased activity against S. aureus PC1, although the strain was resistant to benzylpenicillin (MIC > 32 µg/ml). Triclosan was highly active against all organisms (MIC < 1 µg/ml) except P. aeruginosa, against which it was inactive (MIC > 128 µg/ml). The lamectacins NB2001 and NB2030 generally had reduced activities relative to that of triclosan, but they were up to 1,000-fold more active against ß-lactamase-positive strains than against ß-lactamase negative strains, suggesting the ß-lactamase-mediated release of triclosan. The exception was against ß-lactamase-producing strain S. aureus PC1, which was reproducibly as susceptible as or less susceptible than the non-ß-lactamase producer S. aureus ATCC 29213 to the two lamectacins.

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TABLE 1. In vitro activities of cephalothin, cefazolin, triclosan, and cephalosporins developed by the ECTA approach against ß-lactamase-positive and -negative strains of major pathogens

ß-Lactamase kinetics. For cephalothin, the K_m values of all four enzymes were consistent with those reported in the literature (4, 8, 29, 32) (Table 2). For NB2001 and NB2030, the K_m values were generally similar to those for cephalothin; for the TEM-1 enzyme, however, the K_m values were significantly lower. The anomaly might be related to enzyme inhibition at higher concentrations of NB2001 and NB2030. The relative catalytic efficiencies of NB2001 and NB2030 were generally within a sixfold range of those of cephalothin except in tests with strain PC1, for which they were significantly higher (Table 2).

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TABLE 2. Kinetic parameters for compounds developed by the ECTA approach and comparison with those for ß-lactams with different ß-lactamasesa

Binding to PBPs. NB2001 and NB2030 had significantly reduced affinities for PBPs 1a, 3, and 4 of E. coli compared to those of cephalothin (Table 3). The two compounds also had reduced affinities for PBPs 1, 2, and 3 of S. aureus.

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TABLE 3. PBP-binding profiles of NB2001, NB2030, and standard compounds with E. coli ATCC 29522 and S. aureus ATCC 29213 membranesa

Enoyl reductase inhibition. Triclosan inhibited purified E. coli enoyl reductase, with IC₅₀s similar to those reported previously (11) (Table 4). On the other hand, the IC₅₀s of NB2001 and NB2030 were 16 and 70 times higher than those of triclosan in the absence of preincubation with NAD⁺. The IC₅₀ of NB2030 was 160 times higher than that of triclosan when the preincubation step was included. The results can be accounted for by the amount of free triclosan detected by high-pressure liquid chromatography (HPLC) in the two compounds developed by the ECTA approach (Table 4).

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TABLE 4. Inhibition of enoyl reductase (Fabl) activity by triclosan, NB2001, and NB2030a

Effects on membrane integrity. Both NB2001 and NB2030 produced direct effects on the membranes of E. coli and S. aureus, as did triclosan, but at concentrations of 50 to 100 µg/ml, which are much higher than their MICs or the concentrations that affect enoyl reductase, suggesting that their antibacterial activities are due to enoyl reductase inhibition rather than direct effects on the membrane. The results are consistent with the bacteriostatic effects of triclosan and NB2001 reported previously (16). Interestingly, the two compounds were toxic to mammalian cells at concentrations (80 µg/ml) comparable to those that produce direct effects on the bacterial membrane (unpublished data).

Morphology. As expected, addition of cephalothin at 0.2 or 1 time the MIC resulted in marked elongation and multinucleation of E. coli ATCC 25922 cells compared to the morphology of the control cells (Fig. 2). In contrast, both NB2001 and NB2030 produced bacterial cell morphology changes identical to those produced by the control treatment; at subinhibitory or inhibitory concentrations they had no effect on septation (Fig. 2). Morphology similar to that of the control was also seen by treatment with triclosan and cerulenin, another fatty acid synthesis inhibitor (data not shown). A longer exposure time and exposure to a suprainhibitory concentration of NB2001, NB2030, triclosan, or cerulenin also produced a cell morphology indistinguishable from that of the control (data not shown).

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FIG. 2. Morphologies of E. coli ATCC 25922 cells incubated in the presence of cephalothin, NB2001, and NB2030 and stained with DAPI.

Transport. Triclosan was readily transported into S. aureus and E. coli cells, reaching similar intracellular concentrations in both organisms (Fig. 3A). In contrast, the levels of NB2001 uptake into E. coli cells were reduced compared to those into S. aureus cells, while NB2030 was detected in E. coli cell extracts but not in S. aureus cell extracts after exposure to the compound (Fig. 3B and C). Intracellular levels of NB2001 and NB2030 may also have been affected by chemical or enzymatic degradation of the compounds during the assay (Fig. 3D). Free triclosan was detected in the transport assay samples with NB2030 and S. aureus and to a lesser extent in the samples with NB2001 and E. coli.

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FIG. 3. Transport of triclosan (A), NB2001 (B), and NB2030 (C) into E. coli () and S. aureus () cells. All intracellular concentrations were determined by HPLC analysis of cell lysates. Samples obtained at 0 min were removed from the original buffer containing 10 µg of compound per ml within approximately 1 to 2 min. The HPLC profiles of cell extracts from the NB2001 and NB2030 uptake experiments gave peaks consistent with the elution profiles of pure compound and those of control samples containing a mixture of cell extract and either NB2001 or NB2030. (D) Transport of triclosan released from NB2001 (, E. coli; , S. aureus) and NB2030 (•, E. coli; , S. aureus). Values for triclosan uptake were obtained from the same experiment whose results are shown in panels B and C; in this case peaks eluting in a manner consistent with the elution of triclosan were quantified.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experiments presented here suggest that the two ß-lactam-containing antibacterials, NB2001 and NB2030, developed by the ECTA approach deliver their toxophores via ß-lactamases since a trend of enhanced activity against organisms expressing class A or class C ß-lactamases was shown for the compounds. For example, there was a 62-fold increase in the activity of NB2001 between E. coli ATCC 25922 and TE18 and a 16-fold increase in the activity of NB2001 between E. coli ATCC 25922 and the strain overexpressing TEM-1. The results are somewhat complicated by differences in the triclosan susceptibilities of the different strains.

Nevertheless, in a comparison of isogenic strains, the non-ß-lactamase-producing strain N and strain N expressing TEM-1, there were 500- to 1,000-fold differences in the MICs of NB2001 and NB2030 and the MIC of triclosan. This suggests that ß-lactamase is responsible for the increased activity against this strain and that there is minimal (0.1%) triclosan contamination and/or non-ß-lactamase-mediated triclosan release. The large difference in MICs was reproducible throughout the period that the experiments described in this report were carried out, except for the biochemical experiments involving enoyl reductase (Table 4), which were performed a year later. In those experiments, NB2001 and NB2030 were analyzed directly for triclosan contamination, and the levels of contamination were found to be 4 and 1%, respectively.

NB2001 also showed at least an eightfold higher level of activity against ß-lactamase-overexpressing strain E. cloacae P99 than against E. cloacae ATCC 13047. Again, inherent differences in triclosan susceptibilities complicate interpretation of the results. Surprisingly, NB2001 was less active against ß-lactamase-overexpressing S. aureus strain PC1 than against S. aureus ATCC 29213. This may be due to differences in drug uptake between the two strains or enzymatic degradation of NB2001 by a non-ß-lactamase-mediated mechanism during MIC determination, as was observed for NB2030 in our S. aureus ATCC 29213 transport assays. The results of kinetic experiments support the lack of toxophore release by the PC1 ß-lactamase and show that NB2001 is a poor substrate for the PC1 enzyme relative to benzylpenicillin (Table 2). This is also an issue in the nitrocefin test, since nitrocefin has the same side chain as cephalothin.

Finally, contamination of NB2001 and NB2030 with free triclosan or ß-lactamase-independent hydrolysis may account for the increased activities of NB2001 and NB2030 against S. pneumoniae ATCC 700671 and E. faecalis ATCC 29212 relative to that of the parent compound, cephalothin. While the results of the experiments with E. coli N support the dependence of the activities of NB2001 and NB2030 on ß-lactamase release, further experiments are needed to determine whether this effect is also the case with clinical isolates. However, it is known from HPLC time course experiments of NB2001 hydrolysis (16) and NB2030 hydrolysis (data not shown) that compound degradation and triclosan release are dependent on the presence of ß-lactamase.

Assays to determine the enzyme kinetics of NB2001 and NB2030 reveal that they are good substrates for both TEM-1 and AmpC ß-lactamases. Surprisingly, the former enzyme hydrolyzes NB2030 with increased efficiency relative to its efficiency of hydrolysis of NB2001 and cephalothin, while the latter enzyme hydrolyzes NB2030 with a correspondingly decreased efficiency. Significantly, while cephalothin is a poor substrate for PC1 (k_cat/K_m less than 0.01% of the value for benzylpenicillin), NB2001 and NB2030 have k_cat/K_m values of 6.4 and 61% of the value for benzylpenicillin, respectively.

Our results also suggest that, as ß-lactams, NB2001 and NB2030 are less active than cephalothin against E. coli and S. aureus. The level of binding to the essential PBP 3 of E. coli is significantly (10- to more than 50-fold) reduced, and the compounds are unable to induce filamentation. According to previous work (9, 28), PBPs 1b, 2, and 3 of E. coli and PBPs 2 and 3 of S. aureus are essential and are targets of ß-lactam antibiotics. The two lamectacins bound poorly to all essential PBPs in the two organisms.

The antibacterial activities of NB2001 and NB2030 appear to reflect the activity of the triclosan toxophore more than they reflect the activity of the ß-lactam moiety. Consistent with their triclosan-dependent antibacterial activities, treatment of E. coli cells with NB2001 and NB2030 at their MICs resulted in cells with normal morphologies. It is therefore possible that bacterial resistance to NB2001 or NB2030 could occur through the loss of ß-lactamase genes, which would then increase the susceptibility of the cell to ß-lactams. Although triclosan has additional activity at higher concentrations due to direct effects on the cell membrane and the activity is retained in NB2001 and NB2030, this activity is associated with mammalian cytotoxicity and thus is not clinically useful.

The experiments with enoyl reductase suggest that intact NB2001 and NB2030 do not inhibit the enzyme, consistent with computer modeling of its active site (M. Hixon, unpublished data). The three-dimensional structure of enoyl reductase containing a bound NAD⁺ molecule and a triclosan molecule (12) suggests that steric hindrance would not allow NB2001 and NB2030 to bind within the active site of the molecule. While our enoyl reductase assay results were complicated by the residual triclosan in the preparation, the reduction in enzyme inhibition that was observed is consistent with the presence of contaminating triclosan as opposed to inhibition by NB2001 or NB2030.

In summary, the ECTA approach presented here allows clinical pathogens that express class A or class C ß-lactamases to be targeted. Despite the antibacterial activity potentially stemming from the ß-lactam nucleus, evidence from in vitro and in vivo studies with NB2001 and NB2030 points to toxophore release as the major contributor to antibacterial activity. Neither compound possessed sufficient activity in animal models of infection to enter clinical development, most likely because serum binding caused more than 1,000-fold shifts in the MICs. However other cephalosporin conjugates that use triclosan analogs with decreased levels of serum binding (Q. Li, V. R. Doppalapudi, R. S. Castillo, A. R. Bueno, J. Y. Lee, G. W. Stone, Q. Zhang, S.-F. Chen, H.-P. Hong, S.-F. Lin, Y.-Y. Lu, J. Macdonald, and N. H. Georgopapadakou., Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-2142, 2003) might eventually yield candidates for clinical development.

 
* Corresponding author. Present address: Department of Medicine 0169, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0679. Phone: (858) 552-8585, ext. 7203. E-mail: gstone{at}ucsd.edu.

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Antimicrobial Agents and Chemotherapy, February 2004, p. 477-483, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.477-483.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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