In these pathogens, OXA -lactamases may confer resistance to penicillins, cephalosporins, and carbapenems (2, 27-29, 45, 55). ceftazidime and are regarded as extended-spectrum -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 (49). The growing number of OXA -lactamases found in nature generates considerable interest in both understanding the mechanistic basis of resistance to inactivation and developing effective inhibitors (6). When they Licochalcone C are found in clinical isolates, OXA -lactamases are poorly inhibited by the currently available -lactam–lactamase inhibitor combinations (ampicillin-sulbactam, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, and piperacillin-tazobactam) (9, 13, 14, 45, 53). For the OXA-1 -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 -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 -lactamases (3, 16, 17, 48). In general, these -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 -lactamase inhibitors of the OXA-1, -10, -14, -17, and -24/40 -lactamases. In contrast to what has been determined in the inactivation of the class C CMY -lactamase and the class A SHV and TEM -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 Fgfr1 a unique reaction chemistry and does not fragment (10, 57, 59). This behavior may prove to be an important characteristic of successful -lactamase inhibitors of class D enzymes. Open in a separate window FIG. 1. Chemical structures of commercially available inhibitors: clavulanic acid, compound 1; tazobactam, compound 2; and sulbactam, compound 3. Substrates used in this study: penicillin G, compound 4; ampicillin, compound 5; oxacillin, compound 6, cephaloridine, compound 7; and nitrocefin, compound 8. Novel compounds used in this study: C-2-substituted 6-alkylidene penicillin sulfones, compounds 9 to 11; C-3-substituted 7-alkylidene cephalosporin Licochalcone C sulfone, compound 12. The accepted ring numbering system is shown for cephaloridine, and C-3 of the isoxazole substituent on oxacillin is labeled. MATERIALS AND METHODS Chemical synthesis. 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 -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). Bacterial strains and plasmids. The DH10B cells (Invitrogen, Carlsbad, CA). For protein purification, BL21(DE3) cells (Novagen) were transformed with plasmid pET 12a (+) DH10B cells. Following verification of the sequence of the construct, the plasmid was transformed into BL21(DE3). This plasmid was used to express and purify the OXA-24/40 -lactamase. Protein purification. Preparation of the OXA-1 and OXA-24/40 -lactamases was performed by inducing BL21(DE3) cells containing either the pET 12a (+) or the pET 24a (+) vector with the cloned were determined by measuring the hydrolysis of NCF (?482 = 17,400 M?1 cm?1), oxacillin (?263 = 258 M?1 cm?1), ampicillin (?235 = ?900 M?1 cm?1), Licochalcone C and cephaloridine (?260 = ?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): (1) where is the initial rate of hydrolysis and [represents the formation of the preacylation complex and represents the acyl-enzyme species. The acyl-enzyme (+ intermediate may then return to + for the inhibitors using competition assays that employed the enzyme, the reporter substrate NCF, and each inhibitor. We measured the initial velocities (value approximates the value, and initial velocities can be represented by the following equation: (3) To determine was determined by dividing the intercept by the slope of the line. The first-order rate constant for enzyme and JDB/LN-1-255 complex inactivation, is the absorbance, is Licochalcone C the final velocity, and is time. Each and was fit to determine (i.e., partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation, or (15)..