INTRODUCTION

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Since the introduction of benzylpenicillin in clinical trials about 60 years ago, the effectiveness of penicillins and related compounds (-lactam antibiotics) has been continuously challenged by the emergence of resistant pathogenic strains. Although bacteria have developed several strategies for escaping the lethal actions of -lactam antibiotics (13, 31, 42), the most common and often the most efficient mechanism is the synthesis of -lactamases (14, 20, 29, 48). These enzymes, which are usually secreted into the external medium by gram-positive species and into the periplasm by their gram-negative counterparts, very efficiently catalyze the irreversible hydrolysis of the amide bond of the -lactam ring, yielding biologically inactive products. Despite the large number (~300 [7]) of -lactamases described to date, these enzymes are divided into only four classes, classes A, B, C, and D, on the basis of their amino acid sequences (35). Enzymes of classes A, C, and D are active-site serine -lactamases, whereas class B enzymes are Zn²⁺ dependent.

Therefore, much effort has been devoted to the synthesis of molecules which would not be cleaved by -lactamases of pathogenic strains and which have suitable physicochemical and pharmacodynamic profiles. Absolute stability has, however, not been achieved with any one drug. An alternative strategy to the use of these so-called -lactamase-stable compounds rests on the use of two -lactams in synergy: one is an efficient -lactamase inactivator but a poor antibiotic, while the second is a good, but -lactamase-sensitive, antibiotic. The former is able to potentiate the action of the latter by protecting it from enzymatic hydrolysis (31, 43). Thus, -lactamase inactivators such as clavulanic acid, sulbactam, and tazobactam have been successfully used against bacteria that produce the ubiquitous and prevalent TEM-1 or TEM-2 and SHV-1 class A -lactamases (38). These "wonder" drugs display, however, little or no activity against class B and C enzymes. In addition, bacterial susceptibility to such combinations has recently been challenged by the spontaneous appearance of new -lactamases of the TEM family, which are resistant to the mechanism-based inactivators (9, 28, 46, 50).

Recently, by following a rational drug design approach, novel tricyclic carbapenem compounds (12) with potential inhibitory activity against serine -lactamases were synthesized. In this paper we describe the kinetics of the interaction between two of these compounds (Lek 156 and Lek 157; Fig. 1) and representative -lactamases of the four classes. Streptomyces sp. strains R61 and K15 D-alanyl-D-alanine peptidases (DD-peptidases), which are prototypes for penicillin-binding proteins (23), were also studied. The specificities of the interactions of Lek 156 and Lek 157 with active-site serine -lactamases and DD-peptidases, which constitute two closely related families of penicillin-recognizing enzymes (26), have been determined. The influence of the compounds on the MIC of ampicillin for Enterobacter cloacae P99 was also evaluated.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Structures of Lek 157 {sodium (4S,8S,9R)-4-methoxy-10- [(E)-ethylidene]-11-oxo-1-azatricyclo[7.2.0.03,8]undec-2-en-2-carboxylate} (A), Lek 156 {sodium (4R,8R,9R)-4-methoxy-10-[(E)-ethylidene]-11-oxo-1-azatricyclo[7.2.0.03,8]undec-2-en-2-carboxylate} (B), and Lek 1A [3-(1-carboxy-1-propenyl)-3',4,5,6-tetrahydro-3H-isoindole-1-carboxylate] (C), which is obtained from base- or enzyme-catalyzed hydrolysis of the first two compounds.

	MATERIALS AND METHODS

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Compounds. Lek 156 and Lek 157 (Fig. 1) were prepared by Lek (Ljubljana, Slovenia), as described by Copar et al. (12). Tazobactam was a gift from Wyeth-Ayerst Laboratories (West Chester, Pa.). Nitrocefin was purchased from Unipath (Basingstoke, United Kingdom). Ampicillin was purchased from Sigma Chemical Co. (St. Louis, Mo.). Phenylacetyl-D-alanyl-thio-lactate (S₂x) was a gift of Hoechst-Marion-Roussel (Romainville, France), and benzoyl-D-alanyl-thioglycolate (S₂d) was synthesized as described by Adam et al. (1).

Enzymes. The various enzymes studied in the present work are listed in Table 1. All enzyme preparations were at least 95% pure.

In vitro susceptibility tests. The MICs of ampicillin were determined in the absence or in the presence of Lek 156 or Lek 157 at final concentrations of 10 or 30 µg/ml by a dilution technique with Mueller-Hinton broth and a bacterial inoculum of approximately 5 × 10⁵ CFU per tube, according to the guidelines of the National Committee for Clinical Laboratory Standards. The strain tested was E. cloacae P99, a strain which constitutively overproduces a class C -lactamase (19). The P99 and the Q908 enzymes are virtually identical (24, 25). A control experiment was also performed in the presence of 10 µg of tazobactam per ml.

Experimental conditions for kinetic studies. All kinetic measurements were performed at 30°C. The buffer for -lactamases of classes A and D was 50 mM sodium phosphate (pH 7.0), that for the class C -lactamase was 10 mM HEPES (pH 7.0), that for the class B -lactamase was 25 mM HEPES (pH 7.5) with 100 µM ZnSO₄; that for the DD-peptidase of Streptomyces sp. strain R61 was 10 mM sodium phosphate (pH 7.0) with 0.4 M NaCl, and that for the enzyme of Streptomyces sp. strain K15 was 25 mM sodium phosphate (pH 7.2) with 4 µM dithiothreitol. Dilutions of the -lactamases below a concentration of 0.1 mg/ml were made with buffer solutions containing 0.1 mg of bovine serum albumin per ml.

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Data analysis. Kinetic data were fitted by linear or nonlinear regression with the program GraFit (32).

Evaluation of the kinetic results. (i) Kinetic models. Both active-site serine -lactamases and DD-peptidases generally hydrolyze their substrates according to a simple three-step acylation-deacylation pathway (23, 35, 48), as follows: k₊₁    k₂      k₃ E +C  EC  EC*  E + P(s) (model 1a) k₁

(1)

(2)

(3)

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(ii) Determination of kinetic parameters. Progressive inactivation of the Streptomyces sp. strain K15 and R61 DD-peptidases was monitored discontinuously by measuring the residual activity either after increasing periods of time at fixed inhibitor concentrations or after a fixed period of time at different inhibitor concentrations (30), to give pseudo-first-order rate constants for inactivation (k_i). With -lactamases, inactivation can be monitored continuously with a reporter substrate (15). When inactivation is complete, a steady state (v_ss = 0) is eventually reached and the pseudo-first-order inactivation rate constant, k_i, can be computed (15). For all the models described above, with the exception of models 2a and 2c, the value of k_i is given by

(5)

where k_r takes the values of zero for models 1b and 2b, the value of k₃ for model 1a, and the value of k₅ for model 2a'. The correction factor  equal to 1 + [S]/K_m,S accounts for the protection of the enzyme by the reporter substrate (S).

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

	RESULTS

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Class A -lactamases: NmcA and TEM-1. Both Lek 156 and Lek 157 are readily hydrolyzed by the NmcA enzyme, and the UV spectra of the products (P) are the same whether they are obtained with sodium hydroxide, the NmcA and TEM-1 enzymes, or the Zn²⁺-containing Bacillus cereus II -lactamase as hydrolytic agents (Fig. 2). Hydrolysis of the two compounds was monitored at 310 nm to avoid protein absorption at lower wavelengths. A major absorbance decrease (_M, 1,850 and 1,350 M¹ cm¹ for Lek 156 and Lek 157, respectively) is followed by a very slow and partial recovery in intensity (_M  1,000 M¹ cm¹ for both compounds; Fig. 2A). This probably arises from the formation of the rearrangement product shown in Fig. 1C, in which the methoxy group of the cyclohexane ring has been eliminated. Indeed, mass spectrometry experiments (M. Vilar, B. Turk, and T. Solmajer, unpublished data) suggest that the major product in these hydrolysis experiments is the 3-(1-carboxy-1-propenyl)-3', 4, 5, 6-tetrahydro-3H-isoindole-1-carboxylate (Lek 1A) rearrangement product (Fig. 1C), which lacks the methoxy group. In addition, it is reasonable to assume that the normal -2 pyrroline intermediate covalently rearranges into the tautomeric and thermodynamically more stable -1 pyrroline (31).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Spectral changes observed during hydrolysis of 100 µM Lek 156. (A) Hydrolysis by 0.08 µM NmcA -lactamase. The heavy solid line corresponds to the starting compound; the dashed line refers to the hydrolysis product obtained after long-term incubation (~24 h). The thin lines are the spectra obtained after intermediate periods of incubation. (B) Difference spectra of the final hydrolysis product against the intact Lek 156 (dashed line) and between the adduct obtained with 60 µM TEM -lactamase and the starting compound (solid lines). A, difference in absorbance.

View this table: [in this window] [in a new window]   TABLE 2.   Kinetic parameters of -lactamases for Lek 156a

View this table: [in this window] [in a new window]   TABLE 3.   Kinetic parameters of -lactamases for Lek 157a

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Dixon plot of inhibition of nitrocefin turn over by the NmcA -lactamase in the presence of Lek 157. [C] is the concentration of Lek 157, and v is the initial rate of nitrocefin hydrolysis. A total of 5 to 10 µg of enzyme was added to 450 µl of 100 µM nitrocefin in 50 mM sodium phosphate (pH 7.0) in the presence of various concentrations of Lek 157 (0.4 to 21 µM). The slope of the line obtained by linear regression analysis of the data and under the assumption of a competitive phenomenon yielded Ki (Km) equal to 3.6 ± 0.2 µM.

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Class C -lactamase: E. cloacae 908R. The reaction observed with the E. cloacae 908R enzyme appears to be more simple. The two carbapenems form long-lived (t_1/2 > 1 h) inactivated complexes with the -lactamase, and titration measurements indicate that, in both cases, complete inactivation occurs at an equimolar (1:1) ratio. Thus, the interaction between Lek 156 or Lek 157 and the -lactamase from E. cloacae 908R can be interpreted on the basis of a linear pathway. The presence of a putative leaving group at the C-3' positions of these compounds suggests that the accumulated acyl-enzymes are the rearranged adduct (EC**) (model 2a'). The difference spectra (data not shown) between the inactivated species and the starting compounds are closely similar to those shown in Fig. 2B. Thus, the inactivated species obtained with the E. cloacae 908R -lactamase probably involves the rearranged carbapenem molecules.

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View larger version (10K): [in this window] [in a new window]   FIG. 4.   Interaction between the E. Cloacae 908R -lactamase and Lek 156. (A) Saturation phenomenon at high concentrations of the compound. The line is drawn from the fit of the data to equation 5 by using the following parameters: k2 equal to 0.37 s1 and K' equal to 16 µM (K' is equal to 86 µM). Note that above 50 µM, the ki values (>0.135 s1) become very high so that the mixing dead time constitutes more that 50% of the complete time course, which explains the large errors. (B) The same data described for panel A but with low Lek 156 concentrations ([C]<<K'), from which a k2/K' value of 17,500 M1 s1 can be calculated. (C) Competitive inhibition between Lek 156 and the reporter substrate (100 µM nitrocefin) after establishment of the steady state, from which a Km value of 0.02 µM is derived by linear regression of the data according to equation 6.

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View larger version (14K): [in this window] [in a new window]   FIG. 5.   Recovery of activity of the E. cloacae 908R -lactamase after transient inactivation by Lek 156. The enzyme was incubated with an equimolar concentration of inactivator that resulted in ~95% inactivation at time zero, and the samples were assayed against 100 µM nitrocefin after various periods of time. Fitting of the data to a single exponential function gives a kr value of 1.5 × 104 s1.

Class D -lactamase: OXA-10. As with the TEM-1 -lactamase, OXA-10 reacts according to a branched pathway with both compounds. Although complete enzyme inactivation is achieved at much lower [C]₀/[E]₀ ratios (k₃/k₄ equal to 4 and 2 for Lek 156 and Lek 157, respectively), the inactivated complexes (EC**) are not completely stable (t_1/2, 12 and 20 min for Lek 156 and Lek 157, respectively). Hence, we have analyzed the data according to model 2a.

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View larger version (11K): [in this window] [in a new window]   FIG. 6.   Interaction between the OXA-10 -lactamase and Lek 157. Variation of ki with [C]. Fitting of the data to equation 13 leads to A equal to (20 ± 6) × 103 s1 and B equal to 0.085 ± 0.01 µM (B equal to 0.66 µM).

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Class B -lactamase: the Zn²⁺-containing enzyme from B. cereus (BcII). The kinetic parameter values (Tables 2 and 3) show that the two carbapenems are hydrolyzed very efficiently by the BcII enzyme. In the absence of any indication of a more complex situation, the simple Henri-Michaelis model was used, and initial rate measurements at various substrate concentrations allowed the individual k_cat and K_m values to be determined. Kinetics similar to those of the NmcA enzyme are observed by monitoring the change in absorbance at 310 nm.

DD-Peptidases of Streptomyces sp. strains R61 and K15. The interactions between the two DD-peptidases and Lek 156 and Lek 157 can be analyzed on the basis of a simple linear pathway (model 1a). Due to the very slow (t_1/2 > 4 h) deacylation process, however, the rearrangement of the opened -lactam compounds probably occurs at the level of the acyl-enzyme (EC*  EC**), in which case model 2a' provides a better description of the phenomenon.

In vitro susceptibility tests. Lek 156 and Lek 157 had no effect at concentrations up to 284 µg/ml. The MICs (Table 5) of ampicillin were significantly reduced by the presence of 30 µg of Lek 157 per ml or 10 µg of tazobactam per ml. The presence of Lek 156 also decreased the MIC of ampicillin, but with a lesser efficiency. However, it should be noted that Lek 156 was unstable even in pure phosphate buffer (pH 7). Under these conditions, a UV and visible spectrum indicated that more than 50% of the compounds was degraded after 24 h at 20°C.

	DISCUSSION

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Both tricyclic carbapenem molecules tested in the present study, Lek 156 and Lek 157, are very efficient inactivators of the E. cloacae 908R class C -lactamase. With this enzyme, the reaction pathway appears to be linear (model 2a') due to negligible hydrolysis of the first covalent adduct (k₃ << k₄) and a very low k₅ value (1.5 × 10⁴ s¹ or less). At saturating concentrations, the enzyme is rapidly inactivated (k₂/K'  2 × 10⁴ to 4 × 10⁴ M¹ s¹), and the resulting species, EC**, although not completely stable, displays very low turnover values (t_1/2  1 to 5 h). Preliminary experiments also indicate that both compounds efficiently inactivate various extended-spectrum class C -lactamases. It is interesting that BRL 42715 (6, 17, 40), which is also a good inactivator of class C enzymes, shares structural characteristics with the compounds studied in the present work.

In agreement with these observations, Lek 157 significantly reduced the MIC of ampicillin for E. cloacae P99, which overproduces a class C -lactamase. The lesser decrease in the MIC observed with Lek 156 probably results from the intrinsic instability of this compound.

The NmcA -lactamase (class A) has been chosen for its unusual catalytic properties (34). It is a very broad spectrum enzyme, hydrolyzing efficiently not only classical penams and cephems but also a wide range of -lactam molecules usually considered resistant to class A -lactamases. In particular, NmcA hydrolyzes imipenem (and related carbapenem antibiotics) with k_cat and K_m values of ~200 s¹ and ~0.6 mM (k_cat/K_m = 2 × 10⁵ M¹ s¹), respectively (34, 45; Florence Mahy, Jean-Marie Frère, and Moreno Galleni, unpublished data). In the present case, both carbapenems are substrates of the NmcA enzyme, but the k_cat (~2 to 3 s¹) and K_m (~0.5 to 4 µM) values are about 3 orders of magnitude lower than those obtained with imipenem.

In contrast to the NmcA enzyme, both compounds inactivate the TEM-1 -lactamase (t_1/2  15 to 30 min for complete inactivation at high [C]₀/[E]₀ values) according to a branched pathway mechanism (k₃ > k₊₄). Most interestingly, the rearranged adducts (EC**) are found to be very stable (t_1/2 > 4 h). A similar mechanism is observed with the class D enzyme (OXA-10). In this case, however, the ratio between the number of productive turnovers and reactions that lead to enzyme inactivation (i.e., the partition ratio k₃/k₄) is quite low, and the enzyme is completely inactivated on a shorter time scale (t_1/2  2 to 4 min). Furthermore, the inactivated species (EC**), is not fully stable, and hydrolysis of the rearranged adduct (EC**) is observed (k₅ 0; t_1/2  10 to 20 min).

Like all other known carbapenems, which are usually only very poorly hydrolyzed by the active-site serine -lactamases (37), Lek 156 and Lek 157 are readily hydrolyzed by the Zn²⁺-containing class B enzyme (18). With the two compounds, both the k_cat/K_m (5 × 10⁶ M¹ s¹) and the k_cat (700 s¹) values are remarkably high, which confirms the high degree of catalytic efficiency of this enzyme (18).

With the representative enzymes considered in the present work, the spectral properties of the final hydrolysis products are identical and correspond to Lek 1A (Fig. 1C), which is also obtained after sodium hydroxide hydrolysis. It is probable that a very slow rearrangement of the primary hydrolysis product, i.e., elimination of the methoxy group of the cyclohexane ring and tautomerization to the -2 pyrroline, occurs after its release from the enzyme. These two events are characterized by significant changes in the molecular extinction coefficient value of the molecules at 310 nm, i.e., ca. 1,500 M¹ cm¹ and ca. + 1,000 M¹ cm¹ for the -lactamase-catalyzed opening of the -lactam ring and the spontaneous rearrangement of the cleaved -lactam, respectively. It appears, however, that the rearrangement takes place much faster when the opened -lactam is trapped as a stable acyl-enzyme species, as is the case with the TEM-1, E. cloacae 908R, and OXA-10 enzymes.

The results indicate that both compounds are very poor acylating agents (k₂/K' = 0.5 to 50 M¹ s¹) for the two model DD-peptidases. With imipenem, a semisynthetic carbapenem antibiotic, the k₂/K' values have been estimated to be 1,000 M¹ s¹ (for the Streptomyces sp. strain R61 enzyme [30]) and 100 to 200 M¹ s¹ (for the Streptomyces sp. strain K15 enzyme [33]). Imipenem is, however, a broad-spectrum antibiotic which very efficiently inactivates some of the essential penicillin-binding proteins of the pathogenic strains and is therefore often used as a last resort for patients in intensive care units. Thus, the intrinsic antibacterial activities of the new Lek carbapenem antibiotics, which have not yet been tested in vivo, cannot be predicted on the basis of the behaviors of the two DD-peptidases tested in the present study.

The present survey of the interaction between different -lactamases and two novel enantiomeric carbapenem molecules confirms the very different behaviors of the enzymes. Although both compounds are readily hydrolyzed by the so-called carbapenem-hydrolyzing -lactamases of class A (NmcA) and class B (BcII), they are efficient inactivators of the "classical" active-site serine -lactamases. This is a quite unusual, specific, and interesting property, since most other mechanism-based inactivators of active-site serine enzymes (e.g., clavulanic acid and sulbactam) generally exhibit rather poor activity against class C enzymes. These enzymes may be associated with resistance profiles that include virtually all -lactam antibiotics (7, 35), and the recent discovery of many new plasmid-mediated forms of genes encoding class C enzymes has now been recognized as a serious threat. Our kinetic data therefore suggest that further development of the tricyclic carbapenem compounds could be of clinical interest.