Binding of monobactams to penicillin-binding proteins of Escherichia coli and Staphylococcus aureus: relation to antibacterial activity.

A series of novel monocyclic beta-lactam antibiotics having side chains related to penicillin, piperacillin, azlocillin, and cefotaxime were examined with respect to binding to essential penicillin-binding proteins (PBPs) in Escherichia coli and Staphylococcus aureus. In the penicillin series, there was poor binding to all essential PBPs of E. coli (greater than 100 micrograms/ml) but good binding to PBPs 1, 2, and 3 of S. aureus (approximately 1 microgram/ml). In the piperacillin and azlocillin series, there was good binding to PBP 3 of E. coli (0.1 microgram/ml) and PBPs 1, 2, and 3 of S. aureus (approximately 1 microgram/ml). In the cefotaxime series, there was generally good binding to PBP 3 of E. coli (0.1 micrograms/ml) but poor binding to PBPs 1, 2, and 3 of S. aureus (greater than or equal to 100 micrograms/ml). With a few exceptions in the cefotaxime series, antibacterial activity paralleled essential PBP binding. Binding studies with radioactively labeled compounds revealed no additional essential monobactam-binding proteins in the two organisms. The studies suggest that monobactams are intrinsically active against both gram-positive and gram-negative bacteria; the activity spectrum of a given monobactam is determined by the binding to essential PBPs, which in turn is determined by the nature of the substituents on the beta-lactam nucleus.

but poor binding to PBPs 1, 2, and 3 of S. aureus (-100 ,ug/ml). With a few exceptions in the cefotaxime series, antibacterial activity paralleled essential PBP binding. Binding studies with radioactively labeled compounds revealed no additional essential monobactam-binding proteins in the two organisms. The studies suggest that monobactams are intrinsically active against both grampositive and gram-negative bacteria; the activity spectrum of a given monobactam is-determined by the binding to essential PBPs, which in turn is determined by the nature of the substituents on the l3-lactam nucleus.
Monobactams are monocyclic P-lactam antibiotics characterized by the 2-oxoazetidine-1sulfonic acid moiety (Table 1) recently isolated from gram-negative bacteria (14,26). The naturally occurring compounds have generally weak antibacterial activity, but synthetic derivatives are potent antibiotics (3), with stability to 1lactamases equal to or better than that of thirdgeneration cephalosporins (6). A synthetic monobactam, azthreonam, is currently being developed for clinical use (25).
Penicillins and cephalosporins are believed to kill bacteria by binding covalently to specific membrane proteins involved in peptidoglycan biosynthesis (1,23). These penicillin-binding proteins (PBPs) have been extensively studied in Escherichia coli, for which essential PBPs have been identified and their functions elucidated (22,24). E. coli PBPs appear to be representative of enterobacteria and pseudomonads. Other bacteria have different PBP patterns (amounts, molecular weights, and 1-lactambinding profiles) and different essential PBPs. In Staphylococcus aureus, for example, four PBPs have been detected, PBPs 2 (molecular weight, 80,000 [80K]), 3 (75K), and possibly 1 (87K) being essential (9,13  water, cooled to 0OC, and filtered. The-filtrate was concentrated, and applied to a 20-ml HP-20 AG column. Elution with water, followed by evaporation in vacuo, gave 10 to 11 mg of pure [14CISQ 81,377, as judged by analytical silica gel thin-layer chromatography in 1butanol-acetic acid-water (3:1:1 by vol) or electrophoresis at pH 7.2, followed by visualization with UV light, Rydon reagent, or autoradiography. The overall yield was 20%o, based on [methyl-'4Cjiodide.
Bacteria and culturing conditions. E. coli SC 8294 and S. aureus SC 2399 were from the Squibb Culture Collection; E. coli DC2 and Streptomyces sp. strain R61 were, respectively, gifts from M. Richmond, University of Bristol, and J. M. Ghuysen, University of Liege. All organisms were grown as previously described (10). PBP binding assay. Solubilized (2% Triton X-100) VOL. 23,1983 membranes of sonicated E. coli and S. aureus were incubated (-100 gig of protein) at 30°C with the appropriate ,-lactam for 10 min in a total volume of 50 gWl. Then 10 nmol of [14C]penicillin G was added, and the incubation was continued for 10 min. PBPs were visualized after SDS-polyacrylamide gel electrophoresis and fluorography (10). P-Lactam-binding protein assay. Solubilized membranes (-100 gig of protein) were incubated with the appropriate ,B-lactam for 10 min as described above, except that the f-lactam was radiolabeled ([125i] ,B-Lactam-binding proteins were detected as described above.
Release of bound 3-lactams. Solubilized membranes (-100 ,ug of protein) were incubated at 30°C with 10 nmol of [14C]penicillin G or [14C]SQ 26,324 for 10 min in a total volume of 50 glJ. Penicillinase (4,000 U) was added to destroy the unbound P-lactam, and the incubation was continued for 10, 20, or 50 min. Hydroxylamine-induced release was examined by adding the appropriate amount of neutral hydroxylamine (final concentration, 0.2, 0.4, and 0.8 M) after 20 min of incubation in the penicillinase and incubating the mixture for 30 min. Residual (14C11-lactam binding was detected after SDS-polyacrylamide gel electrophoresis and fluorography. DD-Carboxypeptldase assay. Partially purified Streptomyces sp. strain R61 DD-carboxypeptidase (8) was incubated at 30°C with the appropriate 1-lactam for 10 min in a total volume of 20 gil. [14C]diacetyl-L-Lys-D-Ala-D-Ala (2 nmol) was added, and the incubation was continued for 30 min. The hydrolysis product, ['4C]diacety1-L-Lys-D-Ala, was separated by highvoltage paper electrophoresis and quantitated by liquid scintillation counting (27).
Peptidoglycan transpeptdase ay. Ether-treated E. coli cells were incubated with the appropriate cofactors and ,-lactam for 10 min in a total volume of 40 gil (12). Then 0.25 nmol of UDP-N-['4C]acetylglucosamine and 6 nmol of UDP-N-acetylmuramyl pentapeptide were added, and the incubation was continued for 20 min. The SDS-insoluble peptidoglycan was collected and quantitated as previously described (12).

RESULTS
The monobactams studied possess side chains analogous to those found in penicillins and cephalosporins. On that basis, they can be conveniently divided into four groups (Table 1): penicillin-cephalothin, piperacillin-cefoperazone, azlocillin-mezlocillin, and cefotaxime-ceftazidime. Monobactams in the latter three groups are generally active against both E. coli and S. aureus, whereas those in the penicillin group are active mainly against S. aureus.
Binding to PBP 3 of E. coli is generally less affected by structural changes on the P-lactam nucleus than is binding to the other PBPs (or the ,-lactamases). For example, an amoxicillin derivative did not bind to PBPs lb and 2 and was not hydrolyzed by 1-lactamases (19) but retained the ability of the parent compounds to bind to PBP 3. (N. H. Georgopapadakou and F. Y. Liu, unpublished results). Thus, monobactams, by virtue of their high affinity for PBP 3 (a PBP relatively tolerant to structural changes), permit optimization of structure for both resistance to f-lactamase and outer membrane permeability. The latter involves predictable parameters, such as hydrophobicity and charge (17), whereas the former is mostly empirical. It   to be present in five copies per cell) which is possibly involved in septation (2). Monobactams also bind to the nonessential PBPs la and 4 of E. coli, which are generally sensitive to ,B-lactam antibiotics. However, binding to PBP 3 occurs at still lower monobactam concentrations and occasionally in the absence of binding to either nonessential PBP, as with SQ 81,755. Thus, monobactams appear to bind to PBP 3 of E. coli far more specifically than the bicyclic 1-lactam antibiotics.
Monobactams bind poorly to PBP 5/6 of E. coli and PBP 4 of S. aureus, the 3a-methoxylated compounds being exceptions. Monobactams do not bind to PBP lb of E. coli and accordingly do not induce lysis.
In conclusion, monobactam activity against gram-negative or -positive bacteria or both is most likely a function of binding to essential PBPs. In the case of E. coli and S. aureus, activity is due to binding to PBP 3 and PBPs 1, 2, and 3, respectively. The essential PBP profile of a given monobactam is in turn determined by the nature of the 313-side chain, as well as other substituents on the 3-lactam nucleus.