0-Lactamase substrates

N-(o, m, or p-carboxy)phenyl azetidin-2-ones 19 (Fig. 11.12), which have two characteristic features of a [3-lactamase substrate, a [3-lactam ring and a carboxy... 

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

Active site directed P-lactam-derived inhibitors have a competitive component of inhibition, but once in the active site they form an acyl en2yme species which follows one or more of the pathways outlined in Figure 1. Compounds that foUow Route C and form a transiendy inhibited en2yme species and are subsequendy hydroly2ed to products have been termed inhibitory substrates or competitive substrates. Inhibitors that give irreversibly inactivated P-lactamase (Route A) are called suicide inactivators or irreversible inhibitors. The term progressive inhibitor has also been used. An excellent review has appeared on inhibitor interactions with P-lactamases (28). 

The activity of P-lactamase inhibitors is often expressed as an IC q value, which is defined as the concentration of inhibitor that causes 50% inhibition of en2yme activity for a given set of conditions. IC q values, which vary widely according to substrate, time of incubation, and other factors, are presented herein solely to give an indication of potency and en2yme inhibitor specificity. Values that decrease with preincubation are indicative of irreversible inhibitors. 

Numerous other penicillin sulfones have been reported to be P-lactamase inhibitors, as illustrated in Table 5. The effect of C-6 substituents has been extensively explored starting with 6-APA sulfone (25, R = NH2, R = H, R" = R " = CH ), which has modest activity. Mechanistic considerations led to preparation of sulfones of poor substrates, compounds such as methicillin, cloxaciUin, nafaciUin, and quinaciUin sulfone (25,... 

Antibiotic Resistance. Figure 1 According to Bush, Jacoby and Medeiros [2] four molecular classes of (3-lactamases can be discriminated based upon biochemical and molecular features. Classes 1, 2, and 4 included serine-proteases, while metallo enzymes are included in class 3. The substrate spectrum varies between different subclasses and the corresponding genes can be part of an R-plasmid leading to a wider distribution or are encoded chromosomally in cells of specific species. 

The class A enzymes have Mx values around 30,000. Their substrate specificities are quite variable and a large number of enzymes have emerged in response to the selective pressure exerted by the sometimes abusive utilization of antibiotics. Some of these new enzymes are variants of previously known enzymes, with only a limited number of mutations (1 4) but a significantly broadened substrate spectrum while others exhibit significantly different sequences. The first category is exemplified by the numerous TEM variants whose activity can be extended to third and fourth generation cephalosporins and the second by the NMCA and SME enzymes which, in contrast to all other SXXK (3-lactamases, hydrolyse carbapenems with high efficiency. Despite these specificity differences, the tertiary structures of all class A (3-lactamases are nearly superimposable. 

Inactivators of class A (3-lactamases (clavulanate, sulbactam, tazobactam) are themselves (3-lactams and act as suicide substrates. They can be used in... 

The class B metallo- 3-lactamases have emerged more recently as a clinical problem but they are particularly dangerous since many of them hydrolyse all know (3-lactams, with the exception of monobac-tams. In particular, they hydrolyse the suicide substrates mentioned above, as well as carbapenems that usually escape the activity of all the SXXK enzymes, with the exception of the NMCA group. 

P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

SxxK (3-lactamases are uncoupled SxxK acyl transferases that work as (3-lactam antibiotic hydrolases. They represent a mechanism of defence of great efficiency. On good (3-lactam substrates, their catalytic centres can turn over 1000 times per second. 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

The functionalized phenaceturates 16 (Fig. 11.10) are substrates of class A and C [3-lactamases, especially the class C enzymes, as observed with the parent unfunctionalized phenaceturates 15. They are also modest inhibitors of these enzymes and the serine DD-peptidase of Streptomyces R61. The inhibition of class C [3-lactamases is turnover dependent, as expected for a mechanism-based inhibitor. Inhibition is not very dependent on the nature of the leaving group, suggesting that the QM is generated in solution after the product phenol has been released from the active site. It therefore... 

All these 3,4-dihydro-2H-1 -benzopyran-2-ones 17 and 18 are substrates of class A and class C (3-lactamases. They are thus the first 8-lactones that are hydrolyzed by [3-lactamases. The kcat values for these substrates are generally smaller than those of the analogous acyclic phenaceturates suggesting that the tethered leaving group obstructs the attack of water on the acyl-enzyme. Despite the apparent advantage of the long-lived acyl-enzymes, the irreversible inhibition by the functionalized compounds is no better than that of acyclic molecules 16. Thus, even the tethered QM cannot efficiently trap a second nucleophile at the [3-lactamase active site, at least as placed as dictated by the structure of compounds 18.70... 

The benzocarbacephems 22 (Fig. 11.12), with (X = Cl or F) or without (X = H) a halogen leaving group, were synthesized by a copper-mediated intramolecular aromatic substitution as the main step.73 74 These tricyclic (3-lactams are also competitive inhibitors of (3-lactamases and not substrates. 

Xing, B., Khanamiryan, A. and Rao, J. (2005). Cell-permeable near-infrared fluorogenic substrates for imaging beta-lactamase activity. J. Am. Chem. Soc. 127, 4158-4159. 

Since many natural substrates ( -lactam antibiotics) of -lactamases contain pendant aromatic groups, it was postulated that a hydroxybenzyl deriva-... 

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