Cefcapene inactivates chromosome-encoded class C β-lactamases

J Infect Chemother (2002) 8:207–210 DOI 10.1007/s10156-002-0177-7

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2002

ORIGINAL ARTICLE Jimena Alba · Yoshikazu Ishii · Moreno Galleni Jean-Marie Frère · Masahiko Ito · Keizo Yamaguchi

Cefcapene inactivates chromosome-encoded class C β-lactamases

Received: January 24, 2002 / Accepted: March 27, 2002

Abstract The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes of βlactamases was evaluated. For the class A β-lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter (kcat/Km) values were calculated (3100
1.1  107 M
1·s
1), because these enzymes have very high Km values for cefpodoxime and cefotaxime. As for class B β-lactamases L1, IMP-1, and CcrA, in general, similar kcat/ Km values were obtained. However, regarding class C βlactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds. Cefpodoxime acted as a good substrate for the class C β-lactamases, except for the enzyme from E. cloacae; its kcat and Km values were successfully calculated (kcat/Km, 1.8  105
1.2  107 M
1·s
1). On the other hand, cefcapene acted as a poor substrate or an inactivator for class C βlactamases; its k2/K value was successfully calculated (8.7  105
7.0  106 M
1·s
1). In addition, k3 values were determined for β-lactamases from P. aeruginosa (2.3  10
2·s
1) and C. freundii (2.1  10
1·s
1). Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed. These findings suggest the potential of cephem compounds as inhibitors of class C β-lactamases. Key words β-Lactamase · Cefcapene · Inactivation of class C β-lactamase

J. Alba · M. Ito Department of Microbiology, Yamanashi Medical University, Tamaho-cho, Yamanashi 490-3898, Japan J. Alba · Y. Ishii ( *) · K. Yamaguchi Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan Tel. 81-3-3762-4151; Fax 81-3-5493-5415 e-mail: [email protected] Y. Ishii · M. Galleni · J.-M. Frère Centre for Protein Engineering, Institute of Chemistry B6, University of Liège, Liège B-4000, Belgium

Introduction β-Lactamases represent a major mechanism of bacterial resistance to antibacterial β-lactams. Ambler1 and Bush et al.2 divided these enzymes into four classes. Class A, C, and D β-lactamases require Ser-70 as an essential amino acid to induce their activity. On the other hand, class B β-lactamases require zinc ions to enable their activity.3 In gram-negative bacteria, class A and D β-lactamases are usually encoded by plasmids, whereas class C β-lactamases are encoded by chromosomes. In recent years, some researchers have discovered plasmid-encoded MOX-1-,4 FOX-,5 LAT-,6 and CMY-type7 class C β-lactamases. Class B, C, and D β-lactamases are found not only in the bacteria of the family Enterobacteriaceae but also in glucosenonfermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter baumanii. Class A β-lactamases, on the other hand, have an even broader distribution, ranging from gram-negative bacteria to gram-positive bacteria, other than streptococci. Class A and D β-lactamases are usually inhibited by such β-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam,8,9 whereas class B and C βlactamases are difficult to inhibit with general β-lactamase inhibitors. The activity of class B β-lactamases is inhibited by ethylenediamine tetraacetic acid (EDTA), 2,6pyridinedicarboxylic acid,10 succinic acid, and mercaptocarboxylate;11 however, no inhibitors of class B enzyme producers are available for clinical use. Monobactams, carbapenems, and some penicillins inhibit class C βlactamases. Recently, some researchers have reported new inhibitors of class C β-lactamases.12–17 However, no reports are available on the inactivation of class C β-lactamases by oral antibacterial cephems. Here, we report on the potential of cefcapene, an oral antibacterial cephem, for the inactivation of class C β-lactamases.

208

Results

Fig. 1. Structure of cefcapene and cefpodoxime

Materials and methods Enzymes and antibiotics used All β-lactamases were purified by the Centre for Protein Engineering, University of Liège (Liège, Belgium). As class A β-lactamases, TEM-1, TEM-18,18 SHV-1,19 Toho-1,20 and NMC-A21 were used. The β-lactamases of L1 from Stenotrophomonas (Xanthomonas) maltophilia,22 IMP-1,4 and CcrA from Bacteroides fragilis23 were used as class B enzymes. The β-lactamases of Enterobacter cloacae 908R, Escherichia coli K12, Citrobacter freundii OS60, and P. aeruginosa 18SH were used as class C β-lactamases.24,25 PSE-2 (OXA-10) was used as a representative class D enzyme.19 Cefcapene (∆ε262  8500 M
1·cm
1) was a gift from Shionogi (Osaka, Japan) (Fig. 1). Cefpodoxime (∆ε261  10 000 M
1·cm
1) was a gift from Sankyo (Tokyo, Japan) (Fig. 1). Nitrocefin (∆ε482  15 000 M
1·cm
1) was purchased from Unipath Oxoid (Basingstoke, UK). Kinetic study of β-lactamases The hydrolysis of cefcapene and cefpodoxime by βlactamases was followed by monitoring the variation in the absorbance of the β-lactam solution in 50 mM phosphate buffer (pH 7.0), except for the class B β-lactamases. For class B enzymes, 50 mM of (N-2-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.0) was used to determine kinetic parameters. All measurements were demonstrated on a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) connected to a personal computer. Reactions were performed in a total volume of 500 µl at 30°C. A final concentration of 20 µg/ml of bovine serum albumin was added to diluted solutions of β-lactamases in order to prevent enzyme denaturation. In the case of a good substrate, the steady-state kinetic parameters (Km and kcat) were determined by analyzing the complete hydrolysis time courses, as described by De Meester et al.,24 or by using the MichaelisMenten equation. These values were obtained with different concentrations (10 µM to 1 mM) of substrate. In the case of a poor substrate, nitrocefin was used as a reporter substrate.25

The kcat and Km values of class A, B, and D β-lactamases were determined for cefcapene and cefpodoxime (Table 1). Because TEM-1, TEM-18, SHV-1, and NMC-A had large Km values, only kcat/Km values for class A β-lactamases were obtained, except for Toho-1. The Km values of the class B β-lactamases L1, IMP-1, and CcrA against cefcapene were 4.9 µM, 3.2 µM, and 7.4 µM, respectively. On the other hand, cefpodoxime showed larger Km values compared with cefcapene. The kcat values of class B βlactamases against both substrates were 19 s
1 to 430 s
1. The kcat/Km values of PSE-2 (OXA-10), one of the class D β-lactamases, against cefcapene and cefpodoxime were 470 M
1·s
1 and 500 M
1·s
1. However, each substrate showed different kcat and Km values. Table 2 shows the kinetic parameters of cefcapene and cefpodoxime against class C β-lactamases. Cefpodoxime showed small k3 values with class C β-lactamases from E. coli, P. aeruginosa, and C. freundii. However, cefpodoxime did not show a small kcat/ Km value, because its Km values were also small. In the case of cefcapene, we could only obtain k3 values and a k2/ K value as kinetic parameters. The k3 value and k2/K value display the deacylation speed and the acyl-intermediate constant. The k3 values of cefcapene against class C βlactamases from P. aeruginosa and C. freundii were 0.02 s
1, and 0.21 s
1, respectively. The k2/K values of class C enzyme from E. cloacae, E. coli, P. aeruginosa, and C. freundii against cefcapene were small. All the enzymes in this study presented a reactivation phenomenon (data not shown); thus, the inactivation was only temporary.

Discussion The hydrolytic reaction of β-lactamases is explained in scheme 1 (below). E·S* represents an acyl enzyme intermediate formed between enzyme E and substrate S (antibacterial β-lactam); E·S represents a Henri-Michaelis complex; P represents the hydrolysis product. Steady-state parameters are derived from the model (equation) shown below. In addition, both kcat/Km and k2/K essentially denote acylation rates. Scheme 1: k1

k

k

2 3 ES Æ E ◊ S Æ E ◊ S* Æ E  P ¨ k
1

The characteristic steady-state parameters derived from the model are: kcat  k2 ◊ k3 k2  k3

and Km  k3 ◊ K k2  k3

209 Table 1. Kinetic parameters of class A, B, and D β-lactamases against cefcapene and cefpodoxime Enzyme

Cefcapene

Cefpodoxime

Km or Ki

kcat (s
1)

kcat/Km (M
1·s
1)

Km or Ki

kcat (s
1)

kcat/Km (M
1·s
1)

Class A TEM-1 TEM-18 SHV-1 Toho-1 NMC-A

– 460 µM – 46 µM –

– 0.1 – 100 –

9.6  105a 220 1.0  106a 2.2  106 3100a

– – – 25 µM –

– – – 110 –

8.5  104a 480a 1.1  107a 4.4  106 3800a

Class D PSE-2(OXA-10)

340 µM

0.2

590

1.2 mM

0.6

490

Class B L1 IMP-1 CcrA

4.9 µM 3.2 µM 7.4 µM

430 19 41

8.8  107 5.6  105 5.5  106

110 µM 37 µM 15 µM

300 51 79

2.7  106 1.4  106 5.4  106

a

First order kinetics

Table 2. Kinetic parameters of class C β-lactamases against cefcapene and cefpodoxime Enzyme

Class C Enterobacter cloacae 908R Escherichia coli K12 Pseudomonas aeruginosa Citrobacter freundii

Cefcapene

Cefpodoxime

k3 (s
1)

k2/K (M
1·s
1)

Km or Ki

kcat (s
1)

kcat/Km (M
1·s
1)

ND ND 2.3  10
2 2.1  10
1

7.0  8.7  1.4  6.7 

0.3 µM 1.7 µM 2.1 µM 0.1 µM

ND 0.3 1.3 1.2

1.8  105 6.2  105 1.2  107

106 105 106 106

k2/K was computed as kcat/Km ND, Not detected

where K  k
1  k2 k1

Furthermore, kcat/Km ( k2/K) characterized the acylation rate.26 The results obtained from the present study showed that cefcapene and cefpodoxime acquired stability to class A and D β-lactamases in nearly the same fashion. Specifically, cefcapene and cefpodoxime do not exhibit high affinity for class A or class D β-lactamases, except for Toho-1 (low Km values obtained). Cefcapene and cefpodoxime were therefore considered to be generally effective against class A and D β-lactamase-producers. However, both compounds were proven to be ineffective against class B β-lactamase producers. Cefcapene and cefpodoxime became stable to class C βlactamases in a fashion differing from that of their stability to class A and D β-lactamases. Cefpodoxime showed low kcat values for class C β-lactamases. Cefcapene acted as a poor substrate or an inactivator for class C β-lactamases. In fact, we failed to calculate the kcat or Km values of cefcapene for the class C β-lactamases used in our present study. Some reports have recently described inhibitors of class B and C β-lactamases, such as mercaptocarboxylate,11 salicyloyl cyclic phosphate,13 penam sulfones,14 boronic acids,15 sulfactams,16 oxamazins,16 and tetrahedral adducts,17 but these compounds are not cephems. In this article, we

suggest that cefcapene, a cephem compound, may specifically inhibit class C β-lactamases. However, cefcapene was found to have k3 values for the class C β-lactamases from P. aeruginosa (2.3  10
2·s
1) and C. freundii (2.1  10
1·s
1). In addition, all these enzymes showed transient inactivation when cefcapene was used. These findings show that the activity of cefcapene is insufficient to inactivate class C β-lactamases. Future study will clarify the mechanism of this phenomenon. In the present study, we investigated β-lactamases only from an enzymological viewpoint. Furthermore, the class C β-lactamases used were selected from among chromosomeencoded enzymes. Accordingly, it is considered necessary to conduct an enzymological investigation using plasmidencoded β-lactamases, and a drug-sensitivity study using other producers of class C β-lactamases. Our findings suggest that compounds with a cephem backbone may have a potential use as selective inhibitors of class C β-lactamases. Acknowledgments This study was supported in part by a grant from the Ministry of Health, Labour and Welfare of Japan during 2000–2001 (Scientific Research Foundation on Drug Resistant Bacteria), by a grant from the Japan Health Sciences Foundation, by a grant from Shionogi and Co., Ltd., and by Project Research Grants 12-20 and 1312 from the Toho University School of Medicine. J. Alba was supported by a scholarship from the Japanese Ministry of Education, Culture, Sports, Science and Technology. We are grateful to Professor A. Iwamoto, University of Tokyo, for his useful advice.

210

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