P -Lactamases

An increasingly critical problem for mankind is the alarming ability of pathogenic bacteria to acquire resistance to antibiotics, which has been triggered as a result of the excessive applieation of these compounds by health care systems. Amongst the strategies employed by the bacteria to render these antibiotics inefficient is the expression of enzymes that hydrolyze, and thus inactivate, the 

NATASA MITIC, GERHARD SCHENK AND GRAEME R. HANSON 

Since the vast majority of enzymes that belong to the metallo-p-lactamase family are usually purified as di-zinc proteins [583], only a limited number of studies have been reported at present that describe EPR-spectroscopic parameters of 

In contrast to the cytosolic isoform, the metal ion eontent of mitochondrial GOX II contains almost exclusively iron and zinc [585], Nonetheless, the EPR spectrum of the mitochondrial isoform resembles that of cytosolic GOX II. Consequently, like its cytosolic counterpart, mitochondrial GOX II consists of a mixture of metal centers, including antiferromagnetically coupled Fe Fe (5 = 1/2), Fe Zn (S= 5/2), EPR-silent Fe Fe, and diamagnetic Zn Zn centers [585], 

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In essence, the cephalosporin acts as a carrier (63) for the quinolone. The quinolone is replaced in the bacterial ceU after the action of P-lactamase on the cephalosporin portion of the molecule. This codmg combination represents a relatively new class of antibacterial agents which appear to offer advantages over the separated components (64). A good introductory discussion of these exciting agents can be found (65) (see also Antibiotics P-lactams ... 

Carbapenems and penems, Cephalo sp orins, P-Lactamase inhibitors, Monobactams,... 

The sulfated compounds MM 13902 (3, n = (5) and MM 17880 (4) are also broad-spectmm agents, but not as potent as thienamycia and all lack any significant activity against Pseudomonas (73). Many carbapenems are excellent inhibitors of isolated P-lactamases, particularly the olivanic acid sulfoxide MM 4550 (3, n = 1) (3). The possible mechanism of action of the carbapenems as inhibitors of P-lactamases has been discussed in some detail (74). Other carbapenems such as PS-5 (5) (75), the carpetimycins (76), asparenomycins (77), and pluracidomycins (8) are all highly active as antibiotics or P-lactamase inhibitors. The parent nucleus itself (1, X = CH2) is intrinsically active, but chemically unstable (9). 

Beecham P-lactamase iiihibitoi BRL 42715 [102209-75-6] (89, R = Na), C IlgN O SNa (105). Lithium diphenylamide, a weaker base, was used to generate the anion of (88) which on sequential treatment with l-methyl-l,2,3-ttia2ole-4-carbaldehyde and acetic anhydride gives a mixture of diastereomers of the bromoacetate (90). Reductive elimination then provided the (Z)-penem (89, R = d5 Q [ OC15 -p) as major product which on Lewis acid mediated deprotection gave BRL 42715 (89, R = Na). 

C QHyN O SNa, as a potentially useful P-lactamase inhibitor capable of potentiating the activity of a number of clinically important P-lactam antibiotics against resistant strains (153). 

Resistance. Resistance to the cephalosporins may result from the alteration of target pencillin-binding sites (PBPs), decreased permeabdity of the bacterial ced wad and outer membrane, or by inactivation via enzyme mediated hydrolysis of the lactam ring (80,81,138—140). This resistance can be either natural or acquired. Although resistance is often attributed speciftcady to one of these factors, in reaUty it reflects the interplay of several factors. In most instances, however, resistance results from the production of a P-lactamase enzyme, which opens the P-lactam ring as depicted in Figure 2. 

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). 

Because of the highly permeable nature of the ced wad of gram-positive organisms, they produce P-lactamases which are not only found throughout the ced wad, but also in the extracedular environment. Hence the extracedular P-lactamases can act on the antibiotic before the ced is entered. 

Gram-negative organisms produce ced-bound P-lactamases which reside in the periplasmic space. Thus, for gram-negative bacteria, the antibiotic must penetrate the outer ced membrane/wad before coming in contact with a P-lactamase (80,139,140). 

The antibacterial spectmm of moxalactam (Table 9) is similar in breadth and potency to that of cefotaxime (36). Hence, moxalactam (48) is classified with the third-generation cephalosporins. In general 1-oxacephalosporins are considerably more susceptible to P-lactamases than their sulfur counterparts... 

However, moxalactam is highly resistant to a broad spectrum of P-lactamases owiag to the effect of the 7a-methoxy group (194,195). 

The antibacterial effectiveness of penicillins cephalospotins and other P-lactam antibiotics depends upon selective acylation and consequentiy, iaactivation, of transpeptidases involved ia bacterial ceU wall synthesis. This acylating ability is a result of the reactivity of the P-lactam ring (1). Bacteria that are resistant to P-lactam antibiotics often produce enzymes called P-lactamases that inactivate the antibiotics by cataly2ing the hydrolytic opening of the P-lactam ring to give products (2) devoid of antibacterial activity. 

A book (1) and several general reviews (2—4) on P-lactamases have been pubUshed. Based on sequence data, it has been suggested that P-lactamases evolved from the enzymes involved in bacterial cell wall synthesis (5—7). 

One approach to combating antibiotic resistance caused by P-lactamase is to inhibit the enzyme (see Enzyme inhibition). Effective combinations of enzyme inhibitors with P-lactam antibiotics such as penicillins or cephalosporins, result in a synergistic response, lowering the minimal inhibitory concentration (MIC) by a factor of four or more for each component. However, inhibition of P-lactamases alone is not sufficient. Pharmacokinetics, stability, ability to penetrate bacteria, cost, and other factors are also important in determining whether an inhibitor is suitable for therapeutic use. Almost any class of P-lactam is capable of producing P-lactamase inhibitors. Several reviews have been pubUshed on P-lactamase inhibitors, detection, and properties (8—15). 

Organism P-Lactamase producers, % Chromosomal Enzyme origin % Plasmid Richmond-Sykes classification... 

Fig. 1. Scheme for the interaction of P-lactamase inhibitor s and P-lactamases where the en2yme is presented by... 

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. 

Fig. 2. Tiansiendy inliibited species foi cephalospoiins bound to P-lactamase. R, an aminotbiazole oxime.

Clavulanic acid has only weak antibacterial activity, but is a potent irreversible inhibitor for many clinically important P-lactamases (10—14,57,58) including penases, and Richmond-Sykes types 11, 111, IV, V, VI ([Bacteroides). Type I Cephases are poorly inhibited. Clavulanic acid synergizes the activity of many penicillins and cephalosporins against resistant strains. The chemistry (59—63), microbiology (64,65), stmcture activity relationships (10,13,60—62,66), biosynthesis (67—69), and mechanism of action (6,26,27,67) have been reviewed. 

Table 2. P-Lactamase Inhibitory Activity for Clavulanic Acid and Analogues ...

Garbapenem P-Lactamase Inhibitors. Carbapenems are another class of natural product P-lactamase inhibitors discovered about the same time as clavulanic acid. Over forty naturally occurring carbapenems have been identified many are potent P-lactamase inhibitors. Garbapenem is the trivial name for the l-a2abicyclo[3.2.0]hept-2-ene ring system (21) shown in Table 3. The synthesis (74), biosynthesis (75), and P-lactamase inhibitory properties (13,14,66) of carbapenems have been reviewed. Carbapenems are often more potent than clavulanic acid and include type I Cephases in the spectmm of inhibition. Table 3 Hsts the available P-lactamase inhibition data. Synergy is frequendy difficult to demonstrate because the compounds are often potent antibacterials. 

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