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OXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low Ki values for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant Ki improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low Kis for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [tn] = 0) and tns ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.
The complexity and diversity of β-lactamases (EC 220.127.116.11) are largely determined by the unique manner in which the various amino acids define the active sites of these enzymes. OXA β-lactamases are class D enzymes (Bush-Jacoby group 2d) that are structurally related to the class C penicillin binding proteins (12, 14, 42, 61). Of primary importance, OXA β-lactamases are found in some of the most difficult-to-treat nosocomial pathogens (Pseudomonas aeruginosa and Acinetobacter spp.) (11, 33, 60). In these pathogens, OXA β-lactamases may confer resistance to penicillins, cephalosporins, and carbapenems (2, 27-29, 45, 55). The blaOXA genes encoding resistance to β-lactams are located in the chromosome, on plasmids, or in integrons and may be inducible (25, 27, 30, 45, 51, 54).
Currently, there are more than 140 different OXA β-lactamases reported (www.lahey.org). OXA-1 is a penicillinase found in Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa and exists as a monomer (58). Among the most-studied OXA enzymes are OXA-10, a dimer, and its clinically important derivatives, OXA-14 and -17. The last three enzymes confer resistance to ceftazidime and are regarded as extended-spectrum β-lactamases (ESBLs) of the class D family (22-24). The common OXA enzymes that confer resistance to carbapenems include OXA-23, -24/40, -48, -51, and -58 (8, 26, 34, 40, 52, 55). In contrast to the situation in P. aeruginosa, OXA carbapenemases are mostly found in Acinetobacter baumannii (49).
The growing number of OXA β-lactamases found in nature generates considerable interest in both understanding the mechanistic basis of resistance to inactivation and developing effective inhibitors (6). When they are found in clinical isolates, OXA β-lactamases are poorly inhibited by the currently available β-lactam-β-lactamase inhibitor combinations (ampicillin-sulbactam, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, and piperacillin-tazobactam) (9, 13, 14, 45, 53). For the OXA-1 β-lactamase, the affinity of tazobactam is reduced, and the turnover of the inhibitor is significantly elevated (6). Moreover, little is known about the inactivation kinetics of other OXA β-lactamases with experimental inhibitors (1, 41, 44, 50).
In the quest for new inhibitors, Buynak and coworkers designed and synthesized C-2-substituted 6-alkylidene penicillin sulfones and C-3-substituted 7-alkylidene cephalosporin sulfones as mechanism-based inactivators of class A β-lactamases (3, 16, 17, 48). In general, these β-lactamase inhibitors derive their success from their high affinities for the active site and ability to form stable reaction intermediates (46, 48). The aim of this present work was to determine the relative efficacies of C-2- and C-3-substituted 6/7-alkylidene penicillin and cephalosporin sulfones as β-lactamase inhibitors of the OXA-1, -10, -14, -17, and -24/40 β-lactamases. In contrast to what has been determined in the inactivation of the class C CMY β-lactamase and the class A SHV and TEM β-lactamases by mechanism-based or “suicide” inhibitors (clavulanic acid, tazobactam, and sulbactam; Fig. Fig.1,1, compounds 1 to 3), we show that C-2- and C-3-substituted penicillin and cephalosporin sulfone inhibitors form a covalent adduct that undergoes a unique reaction chemistry and does not fragment (10, 57, 59). This behavior may prove to be an important characteristic of successful β-lactamase inhibitors of class D enzymes.
The chemical structures of penicillin G, ampicillin, oxacillin, cephaloridine, and nitrocefin are shown in Fig. Fig.11 (compounds 4 to 8, respectively). The chemical structures of the C-2- and C-3-substituted sulfone β-lactamase inhibitors tested in this study are also illustrated in Fig. Fig.11 (compounds 9 to 12). The synthesis and initial evaluation of compounds 9 to 12 were reported and reviewed by Buynak and coworkers (17-19).
The blaOXA-1 gene was cloned from plasmid RGN238 into vector pET 12a (+) (Novagen, Gibbstown, NJ), as described previously (58). Plasmid RGN238 containing blaOXA-1 was maintained in E. coli DH10B cells (Invitrogen, Carlsbad, CA). For protein purification, E. coli BL21(DE3) cells (Novagen) were transformed with plasmid pET 12a (+) blaOXA-1 by electroporation.
By using recombinant plasmid pOXA-40 obtained from Nordmann and coworkers as a template (35), blaOXA-24/40 was amplified with primers containing 5′ NdeI and 3′ BamHI restriction sites. The primers were designed to amplify the blaOXA-24/40 gene, including the start and stop codons for the mature protein, without including the upstream leader sequence. The PCR product was subcloned directionally into pET 24a (+) (Novagen) and was electrotransformed into E. coli DH10B cells. Following verification of the sequence of the construct, the plasmid was transformed into E. coli BL21(DE3). This plasmid was used to express and purify the OXA-24/40 β-lactamase.
Preparation of the OXA-1 and OXA-24/40 β-lactamases was performed by inducing E. coli BL21(DE3) cells containing either the pET 12a (+) or the pET 24a (+) vector with the cloned blaOXA-1 and blaOXA-24/40 genes, respectively (6). Briefly, 500-ml culture flasks containing cells were induced at an optical density at 600 nm of 0.8 with isopropyl-β-d-galactopyranoside (IPTG) to a final concentration of 0.2 mM. The culture was harvested 3 to 6 h after IPTG induction, centrifuged, frozen at −20°C, and, on the next day, resuspended in 50 mM Tris HCl buffer, pH 7.4. β-Lactamase was liberated by using lysozyme and EDTA (7, 36). All cell manipulations for the isolation of OXA β-lactamases were done in 50 mM sodium phosphate (monobasic and dibasic) buffer, pH 7.2.
The OXA-1 and OXA-24/40 β-lactamases were purified by preparative isoelectric focusing (36). We assessed the purity of each preparation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Samples were resolved on a 5% stacking, 12% separating SDS-polyacrylamide gel and were stained with Coomassie brilliant blue R250 (Fisher, Pittsburgh, PA) to visualize the proteins. Further purification of the OXA-1 and OXA-24/40 β-lactamases was performed by size-exclusion chromatography with a Pharmacia ÄKTA Purifier System (GE Healthcare, Piscataway, NJ). We employed a Hi Load 16/60 Superdex 75 column (GE Healthcare) and performed elution with 10 mM phosphate-buffered saline (PBS; pH 7.4). The proteins were concentrated and dialyzed with 50 mM sodium phosphate buffer, pH 7.2. The protein concentrations were determined by a Bio-Rad protein assay with bovine serum albumin (Sigma, St. Louis, MO) as a standard.
The kinetic constants of the OXA β-lactamases were determined by continuous assays at room temperature with a model 8452 diode array spectrophotometer (Agilent, Palo Alto, CA) (32). Each experiment was performed in triplicate in 50 mM sodium phosphate buffer supplemented with 20 mM bicarbonate (31). Nitrocefin (NCF) was purchased from Becton Dickinson (Cockeysville, MD); and oxacillin, ampicillin, and cephaloridine were purchased from Sigma. Clavulanic acid and tazobactam were generous gifts from GlaxoSmithKline (Surrey, United Kingdom) and Wyeth Pharmaceuticals (Pearl River, NJ), respectively.
The values of the kinetic parameters Vmax and Km were determined by measuring the hydrolysis of NCF (Δ482 = 17,400 M−1 cm−1), oxacillin (Δ263 = 258 M−1 cm−1), ampicillin (Δ235 = −900 M−1 cm−1), and cephaloridine (Δ260 = −10,000 M−1 cm−1) and obtaining the nonlinear least-squares fit of the data to the Henri-Michaelis-Menten equation (equation 1) by using the program Enzfitter (Biosoft Corporation, Ferguson, MO):
where v is the initial rate of hydrolysis and [S] is the substrate concentration.
The reaction between the β-lactamase enzyme (E) and the mechanism-based inhibitors (I) studied in this paper can be represented by the follow equation:
In this model, E:I represents the formation of the preacylation complex and E-I represents the acyl-enzyme species. The acyl-enzyme (E-I) can proceed to hydrolysis (E + P1) or undergo rearrangement to a transiently inhibited species (E-IT). The E-IT intermediate may then return to E-I, proceed to hydrolysis (E + P2), or form an inactivated acyl-enzyme (E-I*). The rate constants (k's) describing each of these steps are represented in equation 2. P1 and P2 are reaction products.
We determined the Ki for the inhibitors using competition assays that employed the enzyme, the reporter substrate NCF, and each inhibitor. We measured the initial velocities (v0) in the presence of a constant concentration of enzyme (11 nM) and increasing concentrations of the inhibitor (range, 4 μM to 9 mM, depending on the inhibitor and the enzyme) that were competed against 50 μM NCF. In these competition assays, the Ki value approximates the Km value, and initial velocities can be represented by the following equation:
To determine Ki, the initial velocities immediately after mixing were plotted 1/velocity as a function of the inhibitor concentration and fitted to a linear equation, and the value of Ki was determined by dividing the y intercept by the slope of the line.
The first-order rate constant for enzyme and JDB/LN-1-255 complex inactivation, kinact, was obtained by monitoring the reaction time courses in the presence of the inhibitor and NCF until the reaction reached the final steady state. Fixed concentrations of enzyme (11 nM) and NCF (100 μM) and increasing concentrations of JDB/LN-1-255 (0.5 to 100 μM, depending on the enzyme) were used in each assay. The observed inactivation rate constant (kobs) was determined by using a nonlinear least-squares fit of the data obtained using Origin 7.5 (Northampton, MA):
where A is the absorbance, A0 is the initial absorbance, v0 (expressed as the variation of the absorbance per unit of time) is the initial velocity, vf is the final velocity, and t is time. Each kobs was plotted versus I and was fit to determine kinact as the maximum asymptote.
The turnover number, tn (i.e., partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation, or kcat/kinact), was derived by incubating increasing amounts of inhibitor with a fixed concentration of β-lactamase. After 24 h, an aliquot was removed from the mixture and the initial velocity was measured and compared with that for a control sample to which no inhibitor was added. The ratio of inhibitor to β-lactamase (I:E) that resulted in greater than 90% inactivation after 24 h was defined here as the tn (15).
The susceptibility of E. coli DH10B cells harboring plasmid RGN238 blaOXA-1 was determined by lysogeny broth agar dilution MICs. A steers replicator was used to deliver 10 μl of a diluted overnight culture containing 104 CFU (4, 5). Piperacillin was purchased from Sigma. Tazobactam and JDB/LN-1-255 were tested at a constant concentration of 4 μg/ml in combination with increasing concentrations of piperacillin.
For mass spectrometry studies, we incubated 20 μM each OXA enzyme for 15 min with and without JDB/LN-1-255 at an I:E ratio of 30:1. Each reaction was terminated by the addition of 0.1% trifluoroacetic acid, and the mixture was then immediately desalted and concentrated by using a C18 ZipTip (Millipore, Bedford, MA), according to the manufacturer's protocol.
The spectra of the OXA protein alone and the OXA and JDB/LN-1-255 proteins were generated on a Q-STAR XL quadrupole time-of-flight mass spectrometer (Applied Biosystems, Framingham, MA) equipped with a nanospray source. The experiments were performed by diluting the protein sample with 50% acetonitrile-0.1% trifluoroacetic acid to a concentration of 10 μM. This protein solution was then infused at a rate of 0.5 μl/min, and the data were collected for 2 min. The spectra were deconvoluted by using the Analyst program (Applied Biosystems). All measurements on the Q-STAR spectrometer have an error of ±4 m/z.
According to previously published methods, the UV difference (UVD) spectra were obtained for JDB/LN-1-255 reacted with OXA-1 β-lactamase at a 1,000:1 inhibitor-to-enzyme ratio (50 μM to 0.05 μM) for 30 min (48, 57). The spectra were measured at wavelengths ranging from 200 to 400 nm.
To create overlays of the β-lactamase structures, the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) entries for TEM-1 (PDB accession no. 1ZG4), OXA-1 (PDB accession no. 1M6K), and PC-1 (PDB accession no. 3BLM) were manipulated with the Sybyl molecular modeling program (Tripos Inc., St. Louis, MO). We also created models of OXA-1 with oxacillin, cephaloridine, and the inhibitor JDB/LN-1-255 docked in the active site. These studies utilized the FlexX docking protocol module (BioSolveIT) within the Sybyl program.
The β-lactam carboxylate is a key ligand recognition point which is held in place primarily by the side chain hydroxyl groups of Ser115 and Thr213, as well as by its proximity to Lys212. As the FlexX program automatically sets all (Ser and Thr) side chain C—C—O—H torsion angles at 180°, we manually adjusted the torsional angle of the hydroxyl side chains of the Ser and Thr residues in close contact with the β-lactam carboxylate. This allowed us to obtain the most ideal fit of the antibiotic, as judged by the proximity of the β-lactam carbonyl oxygen (of the docked antibiotic) into the oxyanion hole pocket formed by the backbone N—H's of Ser67 and Ala215. Accordingly, the torsional angle of Thr213 was set to 90° to provide a logical conformation in which the positive end of the O—H dipole faces the incoming carboxylate of the antibiotic. Ser67 is the active-site nucleophile, and its side chain oxygen is partially negatively charged. Thus, the C—C—O—H torsional angle of this residue was set at −30° to allow the positive end of the O—H dipole moment to face the negatively charged —CO2− of carboxylated Lys70 in a position for the proton to be abstracted. Furthermore, at this angle, the negative end of the Ser67 O—H dipole faces the β-lactam carbonyl carbon preparing for acylation. The C—C—OlH torsional angle of the remaining side chain hydoxylic residue, Ser115, was left at 180°, since this angle positions the proton toward the β-lactam carboxylate.
The OXA-1, -10, -14, -17, and -24/40 β-lactamases were purified to greater than 95% homogeneity. The high kcat of OXA-1 for oxacillin translated to an elevated catalytic efficiency (kcat/Km = 19 ± 1 μM−1 s−1; Table Table1),1), while for ampicillin, the catalytic efficiency was threefold lower. The kcat/Km of OXA-1 for NCF was 10 ± 3 μM−1 s−1 and was 48-fold lower for cephaloridine.
As the substrate profiles of OXA-10, -14, -17, and -24/40 were previously reported, we focused upon the behavior of NCF, as this cephalosporin served as our indicator substrate (22, 23, 31, 35). The catalytic efficiencies for NCF by the ESBL OXAs (OXA-10, -14, and -17) and OXA-24/40 ranged from 4.0 ± 0.1 to 9.6 ± 0.3 μM−1 s−1 (see footnote a of Table Table22).
Previous studies reported biphasic kinetic profiles for some OXA enzymes and particular substrates, and while this behavior is not completely understood, it appears to be related to the equilibrium of enzyme monomer/dimer species in solution as well as the concentration of bicarbonate (22, 31, 39, 47). Under the conditions tested, we did not observe biphasic kinetics with the OXA enzymes against any of the substrates tested. Our assays used a nanomolar concentration of enzyme that is likely well below the value of the dissociation constant for any dimer species and a relatively high concentration of supplemental bicarbonate (20 mM). These experimental conditions may offer an explanation for the lack of biphasic kinetic behavior.
Each of the C-2- and C-3-substituted sulfone compounds had a lower Ki for the OXA-1 β-lactamase than clavulanic acid and tazobactam (Table (Table2).2). The C-3 cephalosporin inhibitor DVR-II-214 had the highest Ki of the panel, 57 ± 5 μM, while the C-2 penicillin inhibitors showed Kis of <2 μM (Ki of JDB/LN-III-26, 1.60 ± 0.30 μM; the remaining data are shown in Table Table2).2). JDB/LN-1-255 demonstrated 830- and 270-fold lower Kis than tazobactam and clavulanic acid, respectively, for OXA-1. Determination of the 24-h tn values for JDB/LN-1-255 against OXA-1 revealed stoichiometric inactivation of the β-lactamase (tn = 0) (Table (Table3).3). These values are compared to tns of >100 for both clavulanic acid and tazobactam. While the first-order rate constant, kinact, of JDB/LN-1-255 for OXA-1 (0.094 ± 0.004 s−1) was not significantly different from the previously determined kinact for tazobactam (0.12 ± 0.01 s−1), the decreased Ki of this novel inhibitor yielded an 89-fold increase in inhibitor efficiency (kinact/Ki values = 0.134 ± 0.027 and 0.0015 ± 0.0003 μM−1 s−1, respectively) (6).
On the basis of our kinetic data obtained with OXA-1, we decided to focus our attention on the C-2-substituted sulfones, JDB/ASR-II-292 and JDB/LN-1-255, for their activities against the OXA-10, -14, and -17 ESBLs. Table Table22 summarizes our data, which show that these investigational inhibitors demonstrated decreases in Kis for the OXA enzymes compared to the Kis of clavulanic acid and tazobactam, from 6 → 225-fold and 10 → 200-fold, respectively. Overall, the Kis of the sulfones were in the nM to low μM range. As shown in Table Table3,3, the kinact/Ki ratios of JDB/LN-1-255 for these ESBL OXAs varied from 0.018 ± 0.001 μM−1 s−1 for OXA-10 to 1.51 ± 0.34 μM−1 s−1 for OXA-17. This 84-fold difference is due primarily to the larger Ki of JDB/LN-1-255 for OXA-10. However, the tn of JDB/LN-1-255 for OXA-10, as well as for the other ESBL-type enzymes, remained low (tns = 6 to 8).
The C-2-substituted penicillin sulfones, JDB/ASR-II-292 and JDB/LN-I-255, demonstrated low Ki values for OXA-24/40, 2.4 ± 0.4 and 0.65 ± 0.05 μM, respectively. Of all the β-lactamases studied, only the kinact/Ki of JDB/LN-1-255 for OXA-17 was higher than that for OXA-24/40.
β-Lactamase inhibitors with low Kis and tns are clinically useful only if they can protect partner β-lactam antibiotics from inactivation by β-lactamase enzymes. On the basis of the kinetic profile of the C-2-substituted penicillin sulfone inhibitors, as well as previously published data demonstrating the efficacy of JDB/LN-1-255 against class A β-lactamases, we chose to compare the in vitro activity of JDB/LN-1-255 to that of tazobactam (48). We reasoned that the unique C-2 catechol moiety of JDB/LN-1-255 may facilitate the uptake of the inhibitor by bacterial siderophore (iron) channels (16, 48). Against E. coli DH10B cells harboring plasmid RGN238 blaOXA-1, susceptibility tests revealed that piperacillin combined with 4 μg/ml of JDB/LN-1-255 was more potent than piperacillin combined with 4 μg/ml of tazobactam (MICs, 128 μg/ml and 512 μg/ml, respectively).
To gain insight into the nature of the inhibition reactions and the intermediates that are generated, we performed ESI-MS analysis of each OXA β-lactamase after 15 min of incubation with JDB/LN-1-255 at an I:E of 30:1. ESI-MS of each OXA β-lactamase alone showed a mass peak corresponding to the predicted molecular mass of each apoenzyme within experimental error (±4 m/z) (mass data are summarized in Table Table4;4; representative spectra for OXA-1 and OXA-10 are shown in Fig. Fig.2).2). For the enzyme and inhibitor assays, we observed a species corresponding to the covalent addition of the β-lactamase inhibitor to OXA-1, -10, -14, -17, and -24/40. Only the spectra for OXA-10 plus JDB/LN-1-255 showed an additional peak corresponding to the molecular mass of the apoenzyme, suggesting that not all of the enzyme was covalently inhibited at the 15-min time point. This result is consistent with the kinetic data revealing the lower inhibitory efficiency of the JDB/LN-1-255 compound for OXA-10 compared to the inhibitory efficiencies for the other OXA β-lactamases.
UVD spectroscopy can be used to provide insight into the presence of intermediates formed during the reaction of an inhibitor and a β-lactamase (15, 20, 48, 57). In our study of JDB/LN-1-255 and OXA-1 reacted at an I:E of 1,000:1, we observed the formation of two chromophores at 258 to 265 nm and 307 to 315 nm. Each of these chromophores developed by 12 s and continued to increase in intensity up to 30 min (Fig. (Fig.3).3). In accordance with previous data for this inhibitor with the class A β-lactamases, we tentatively assigned the chromophore at 307 to 315 nm to the reaction intermediate containing a bicyclic aromatic ring system (see below). It is also possible that these UVD spectra represent other chemical changes to the enzyme-inhibitor complex.
After consideration of our substrate and inhibitor profiles, we chose to focus our molecular modeling on OXA-1, a monomer. To this end, we selected a good substrate (oxacillin), a poor substrate (cephaloridine), and JDB/LN-1-255, the overall good inhibitor. Using our kinetic data to rationalize our models, we propose a mechanism by which JDB/LN-1-255 inhibits this enzyme.
To start, we studied the structure of OXA-1 in comparison to the structures of the class A enzymes TEM-1 and the staphylococcal β-lactamase PC1. Overlaying OXA-1 with TEM-1 and PC1 elucidated significant structural differences which may be related to the substrate profiles of classes A and D. Noticeably, the turn between the B9 and B10 β strands is significantly longer in the case of the OXA-1 enzyme than for both TEM-1 and PC1. This results in an effective widening of the active site, since, as the turn begins in the class A β-lactamases, the (somewhat displaced) side chains of residues Glu240 (TEM-1) and especially Ile239 (PC1) become angled back toward the active-site serine and significantly restrict the substrate binding site (Fig. 4a and b). In contrast, the closest corresponding residue of OXA-1 (Phe217) is oriented away from the site, thus providing additional space for the recognition of sterically bulky substrates, such as oxacillin.
The entry and binding of oxacillin (compound 6) in the active site of OXA-1 are depicted in Fig. Fig.5a.5a. The carbonyl oxygen of the β-lactam is positioned in the oxyanion hole, and both oxygens of the sp3-hybridized C-3 carboxylate interact with Ser115 and Thr213. In this orientation, the bulky hydrophobic phenyl group at C-3 of the isoxazole substituent creates favorable interactions with the side chains of Trp102 and Met99. Such interactions of the residues on this loop would not be possible with penicillin G (compound 4), nor are the loops of the TEM-1 and PC1 class A β-lactamases properly positioned to interact with this substrate, thus potentially explaining the higher hydrolytic efficiency of oxacillin than of the penicillins for OXA-1. In contrast, the carboxylate of cephaloridine (compound 7) does not appear to achieve optimal interactions with both Ser115 and Thr213 of OXA-1 (Fig. (Fig.5b)5b) due to the relatively flat geometry of cephaloridine imposed by the sp2-hybridized C-4 of the cephem. The docked cephaloridine molecule suggests that additional hydrophobic residues at C-3 (as is the case with NCF, a better substrate of OXA-1) might favorably interact with a number of residues in this part of the site.
The modeling of these OXA substrates helped us to better understand the affinity contributions of JDB/LN-1-255 modeled in the active site of OXA-1 (Fig. (Fig.6).6). In our model, the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213. This also allows optimal room for the C-2 substituent to be placed in the entry port between Gln113 and Leu259. Potential hydrophobic interactions between the pyridyl ring with hydrophobic residues Val116 and Leu161 may also contribute to the inhibitor's overall affinity.
In the study described here, we compared the activities of members of a novel class of β-lactamase inhibitors, C-2- and C-3-substituted penicillin and cephalosporin sulfones, to the activities of the class A mechanism-based inactivators clavulanic acid and tazobactam against select OXA β-lactamases. Class D β-lactamases possess significant differences in their primary sequences and structures compared to those of the other serine β-lactamases of classes A and C. Since these OXA β-lactamases are found in highly resistant bacteria encountered in the hospital setting, the need to understand the kinetic behavior of these enzymes is an important first step in designing effective class D β-lactamase inhibitors. The ultimate goal of this work was to find inhibitors that can inactivate all three types of OXA β-lactamases: penicillinases (OXA-1), ESBLs (OXA-10, -14, -17), and carbapenemases (OXA-24/40).
Our kinetic data show that the C-2-substituted penicillin sulfones JDB/LN-1-255 (compound 9), JDB/LN-III-26 (compound 10), and JDB/ASR-II-292 (compound 11) inhibit the OXA-1 β-lactamase with low Ki values. These novel compounds are more effective than the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214, compound 12) and have significantly lower Kis than clavulanic acid and tazobactam when they are tested against OXA-1. Susceptibility testing shows that JDB/LN-1-255 in combination with piperacillin is superior to the piperacillin-tazobactam combination for lowering the MIC for E. coli DH10B cells expressing the OXA-1 β-lactamase.
Against the ESBL OXA-10, -14, and -17 β-lactamases, JDB/ASR-II-292 and JDB/LN-1-255 demonstrated low Kis, which ranged from 0.20 ± 0.04 to 8.0 ± 0.2 μM and which are significantly improved compared to those of tazobactam and clavulanic acid. JDB/ASR-II-292 and JDB/LN-1-255 showed the lowest Kis for OXA-17 and the highest Ki values for OXA-10. The amino acid substitutions that characterize OXA-14 and -17 compared to the sequence of OXA-10 (Gly167Asp and Asn76Ser, respectively) suggest a role for the Ω-loop region in the inactivation mechanism of these inhibitors. The Ω loop is known to follow different paths in OXA-10 than in OXA-1, and these deviations may also influence the cephalosporin substrate profiles of the β-lactamases (39, 58). Similarly, the substitutions in OXA-14 and -17 may affect their affinities for the penicillin sulfone inhibitors; but despite these variations, the range of Ki values for the three ESBLs and OXA-1 offers substantial gains over the Kis of the available mechanism-based inhibitors. The low Kis of these novel sulfones stand out from the data previously reported for OXA-10 inhibition by 6-hydroxyalkylpenicillanates (Kis = 240 to 300 μM) (43).
Furthermore, JDB/ASR-II-292 and JDB/LN-1-255 maintained their potencies against the carbapenemase OXA-24/40. This remarkable property is noteworthy as the substrate profile of OXA carbapenemases is different from penicillinases and/or ESBLs. The crystal structure of OXA-48, a carbapenemase of increasing clinical importance, indicates that this enzyme possesses unique active-site shape and charge features compared to those of the OXA-24/40 β-lactamases (26, 56). This comparison suggests the evolution of multiple mechanisms that confer carbapenem resistance in class D β-lactamases, underscoring the need for inhibitors which can maintain activity across diverse active sites (26). Studies directed against OXA-48 and other OXA carbapenemases that are evolving in the clinic are planned.
Of the compounds tested against the OXA enzymes, JDB/LN-1-255 achieved the lowest Ki values and demonstrated activity in vivo (lowered MICs). Our data show that JDB/LN-1-255, compared to tazobactam, demonstrates significant improvements in turnover and inhibitor efficiency (kinact/Ki) for OXA-1. JDB/LN-1-255 extends these low tns to the ESBL- and carbapenemase-type β-lactamases. Furthermore, the efficiency of inhibition of OXA-17 and -24/40 by JDB/LN-1-255 is improved compared to that of OXA-1.
ESI-MS of each of the OXA β-lactamases tested with JDB/LN-1-255 revealed a covalent adduct corresponding to the addition of the inhibitor to the β-lactamase. This observation is consistent with previous MS and X-ray crystallography data showing the formation of a stable acyl-enzyme product consisting of unfragmented JDB/LN-1-255 covalently bound to SHV class A β-lactamases (48). On the basis of the findings of past studies with JDB/LN-1-255 and class A β-lactamases, we posit that this compound forms a stable acyl-enzyme intermediate with OXA enzymes that does not fragment along the inhibition pathway or reaction coordinate (48).
Crystallography reveals that the SHV-1 β-lactamase and JDB/LN-1-255 form a bicyclic aromatic intermediate that stabilizes the acyl-enzyme by a large conjugated π system. We propose that reaction of OXA β-lactamases with JDB/LN-1-255 leads to a similar intermediate in which the acyl-enzyme ester carbonyl is stabilized by both the bicyclic aromatic system and resonance interactions with the nitrogen atoms on the inhibitor. UVD spectroscopy of OXA-1 with JDB/LN-1-255 revealed the formation of reaction intermediates with characteristic absorption spectra. The appearance of the chromophore at 307 to 315 nm could be consistent with the formation of an aromatic intermediate. A similar UVD profile was observed with JDB/LN-1-255 and SHV-1 (48).
In the case of irreversible inhibitors of serine β-lactamases, efficacy is determined by a combination of factors, including inhibitor recognition, the speed and efficiency of acylation, and the hydrolytic stability of the acyl-enzyme (32, 48, 57). Class D β-lactamases are not usually inhibited by commercial inhibitors such as clavulanic acid or tazobactam, while class A enzymes are generally susceptible (14). Clavulanic acid and tazobactam were shown to be mechanism-based enzyme inactivators that require either isomerization of an intermediate imine (represented by E-IT in Equation 2) to a resonance-stabilized β-aminoacrylate enamine or interception of this reactive intermediate by a second nucleophilic amino acid side chain, such as Ser130 (leading to the formation of the species represented by E-I* in equation 2) (Fig. (Fig.7a).7a). In either case, the inhibitory process requires appropriately placed enzymatic machinery (e.g., a base to promote the isomerization or an appropriately positioned nucleophilic residue). Glu166 of the class A β-lactamases promotes the imine-to-enamine isomerization of clavulanic acid (37, 38). In class D enzymes, the carboxylated active site, Lys70, may serve as the general base by activating both the nucleophilic serine for acylation and the hydrolytic water for deacylation. However, in reference to the mechanism-based inhibitors, the carboxylated lysine of the class D enzymes may not be properly positioned or may otherwise be unable to promote the second isomerization to the stabilized intermediate enamine (31, 43, 44).
In contrast, the 6-(pyridylmethylidene)penicillin sulfone JDB/LN-1-255 possesses an intramolecular nucleophile in the form of the pyridine nitrogen. Previous work with SHV β-lactamases suggests that the formation of the proposed inactivating species, an aromatic indolizine ring system (represented by E-I* in Equation 2), involves the intramolecular capture of the imine (represented by E-IT in equation 2), followed by the final loss of a proton (48). This proton is lost from C-5 (not from C-6, as is the case for clavulanic acid and sulbactam), which is directly adjacent to the newly basic nitrogen (formerly the β-lactam nitrogen). This mechanism of intramolecular rearrangements leading to the JDB/LN-1-255 bicyclic intermediate is probably common to SHV class A and OXA class D β-lactamases (21, 48). However, the details of β-lactam acylation and deacylation differ between these two enzyme classes. Thus, we propose a general mechanism of OXA inactivation by JDB/LN-1-255, as outlined in Fig. Fig.7b7b (43, 58).
Taken together, our kinetics, spectroscopic, and susceptibility data and molecular representations serve as guides for important active site-inhibitor interactions with class D β-lactamases. From the findings of these studies, we highlight four important features of JDB/LN-1-255: (i) the ability of the conserved β-lactam carboxylate to make multiple interactions with active-site residues, dictated in part by the stereochemistry and rotational freedom of the substituent (e.g., sp2- versus sp3-hybridization); (ii) high-affinity interactions between the C-6 group and residues that have evolved for recognition of the preferred substrate of the enzymes, oxacillin; (iii) a C-2 group which may enhance cell entry, as has been supported by this and other MIC studies (48); and (iv) a novel inhibition mechanism that is less reliant on appropriately placed enzymatic machinery than the currently available suicide inhibitors are.
In summary, the JDB/LN-1-255 inhibitor is likely to be a broad-spectrum class D β-lactamase inhibitor with activity against the pencillinase-, ESBL-, and carbapenemase-type OXA enzymes. This C-2-substituted 6-alkylidene penicillin sulfone and its novel inactivation chemistry may offer significant advantages compared to the currently available inhibitors.
We thank F. Danel and M. G. P. Page for providing the OXA-10, -14, and -17 proteins and P. Nordmann for plasmid pOXA-24/40. We also thank Krisztina Papp-Wallace and Magdalena Taracila for helpful comments and discussions and the Case Center for Proteomics for assistance with mass spectrometry.
J.D.B. is supported by grant N-0871 from the Robert A. Welch Foundation. This work was supported in part by the U.S. Department of Veterans Affairs Merit Review Program (to R.A.B. and M.J.S.) and National Institutes of Health grant 1RO1 A1063517-01. R.A.B. is also supported by the Veterans Integrated Service Network (VISN) 10 Geriatric Research, Education, and Clinical Center (GRECC). S.M.D. was supported in part by NIH grant T32 GM07250 and the Case Medical Scientist Training Program.
Published ahead of print on 19 January 2010.