Inhibition of Lactamases

The mechanism of catalysis and the inhibition of -lactamases have been reviewed... 

Because they cause prolonged inactivation of certain fi-lactamases. class I inhibitors ate particularly useful in combination with extended-spectrum, lactamase-sen.sitive penicillins to treat infections caused by lactamase-producing bacteria. Three such inhibitors, clavulanic acid, sulbactam, and tazobactam. are currently marketed in the United States for this purpose. A class II inhibitor, the carbapenem derivative imipenem. has potent antibacterial activity in addition to its ability to cause transient inhibition of some /3-lactamases. Certain antibacterial cephalosporins with a leaving group at the C-3 position can cause transient inhibition of lactamases by forming stabilized acylenzyme intermiediates. These are discu.ssed more fiilly below in this chapter. 

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). 

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. 

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 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... 

In a recent report [67], the spirocyclopropyloxy structural motif was incorporated in sulbactam, and the compounds 12a and 12b had good activity against various -lactamases. The mechanism of inhibition of /3-lactamase by 12a or 12b is unique. After the initial acylation, the cyclopropyloxy group can promote the subsequent chemical events to form the aldehyde or the oxycar-benium moiety for further cross-linking with other active site residues of the enzyme (Scheme 5). 

Further, using a combination of X-ray crystallography and mass spectroscopy, Knox et al. [73] has firmly established a central role for Ser-130 in the inhibition of SHV-1 /1-lactamase (class A) by tazobactam. Many additional modifications (Table 3) were carried out on tazobactam with the aim of increasing inhibitory activity against AmpC enzymes, but none of these derivatives (e.g., 13c, 13d, and 13e) had any advantage over tazobactam [74— 77]. Renewed interest in the modification at the C-2 position of sulbactam was developed when scientists from Hoffmann-La Roche disclosed a series of 2/J-alkenyl penam sulfones that possess the ability to simultaneously inactivate both class A penicillinase as well as class C cephalosporinase. Compound... 

C. C. Chen, O. Herzberg, Inhibition of beta-Lactamase by Clavulanate. Trapped Intermediates in Cryocrystallographic Studies , J. Mol. Biol. 1992, 224, 1103-1113. 

C. J. Easton, J. R. Knowles, Inhibition of the RTEM /3-Lactamase from Escherichia coli. Interaction of the Enzyme with Derivatives of Olivanic Acid , Biochemistry 1982, 21, 2857-2862. 

Y. Yang, K. Janota, K. Tabei, N. Huang, M. M. Siegel, Y. I. Lin, B. A. Rasmussen, D. M. Shlaes, Mechanism of Inhibition of the Class A beta-Lactamases PCI and TEM-1 by Tazobactam. Observation of Reaction Products by Electrospray Ionization Mass Spectrometry , J. Biol. Chem. 2000, 275, 26674-26682. 

N. Li, J. Rahil, M. E. Wright, R. F. Pratt, Structure-Activity Studies of the Inhibition of Serine beta-Lactamases by Phosphonate Monoesters , Bioorg. Med. Chem. 1997, 5, 1783-1788. 

Efforts to overcome the actions of the p-lactamases have led to the development of such p-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam. They are called suicide inhibitors because they permanently bind when they inactivate p-lactamases. Among the p-lactamase inhibitors, only clavulanic acid is available for oral use. Chemical inhibition of p-lactamases, however, is not a permanent solution to antibiotic resistance, since some p-lactamases are resistant to clavulanic acid, tazobactam, or sulbactam. Enzymes resistant to clavulanic acid include the cephalosporinases produced by Citrobacter spp., Enterobacter spp., and Pseudomonas aeruginosa. 

Some boronic acid-based enzyme inhibitors undergo strong yet reversible covalent attachment to a nucleophile at the enzyme s active site, while others simply act as competitive inhibitors in their borate conjugate base form. Boronic acid-based inhibition of thrombin has been achieved <93MI109>, and that of P-lactamases has been particularly effective <95TL8399, 96M1688>. When compared to other covalent transition-state analog inhibitors of P-lactamases like phos-... 

Inhibition of enzymatic inactivation Enzymatic inactivation of 3-lactam antibiotics is a major mechanism of antibiotic resistance. Inhibition of 3-lactamase by 3-lactamase inhibitor drugs (eg, sulbactam) results in synergism. 

All -lactam antibiotics are bactericidal. They interfere with the synthesis of the bacterial wall by inhibiting the bacterial ftanspeptidase enzymes essential for the construction of peptidoglycan of the wall. Some -lactams may be inactivated by the -lactamases (penicillinases) produced by bacteria and, thus, the activity of both penicillins and cephalosporins can be determined by their ability to withstand the destructive action of -lactamases also produced by the organism for its optimal protection. Bacterial resistance caused by -lactamase production... 

Additional analyses will normally be required in order to identify and quantify individual residues within a group of antimicrobials to determine whether a positive result exceeds the MRL level. For suspect penicillin residues, identification can be made by repeating the microbiological assay in the presence of -lactamase. If the inhibition zone disappears by this addition, penicillin residues are present in the sample. If not, no conclusion can be drawn because several new-generation -lactams are less sensitive to inactivation by -lactamase. 

Several classes of (3-lactamases, often encoded in transmissible plasmids, have spread worldwide rapidly among bacteria, seriously decreasing the effectivenss of penicillins and other (3-lactam anti-biotics.t y Most (3-lactamases (classes A and C) contain an active site serine and are thought to have evolved from the dd transpeptidases, but the B typey has a catalytic Zn2+. The latter, as well as a recently discovered type A enzyme,2 hydrolyze imipenem, currently one of the antibiotics of last resort used to treat infections by penicillin-resistant bacteria. Some (3-lactam antibiotics are also powerful inhibitors of (3-lactamases.U/aa/bb These antibiotics may also have uses in inhibition of serine proteasesCC/dd such as elastase. Some antibiotic-resistant staphylococci produce an extra penicillin-binding protein that protects them from beta lactams.ee Because of antibiotic resistance the isolation of antibiotics from mixed populations of microbes from soil, swamps, and lakes continues. Renewed efforts are being... 

Inhibition of a staphylococcal /3-lactamase by certain dipeptides has been reported 102), but it is not clear whether the active site was directly involved. Other remotely related and unrelated compounds as well as metals have been implicated, but the results are largely inconclusive 2,9,20,44) Of the nonspecific inhibitors, the thiol reagents have been most extensively tested and usually found ineffective. This is not surprising in view of the available data on the amino acid composition of /3-lactamases (Table III) which show total absence of cysteine. However, interesting exceptions have been reported. Thiol reagents inhibit the Zn2+-dependent /3-lactamase II of B. cereus 87, 66) and the /3-lac-... 

The presence of a phenyl group at the C-4 position of the azetidinone ring favored a specific hydrophobic interaction with the active site of class A (3-lactamases. Instead, the stereochemistry of the C-4 position appeared to be not important for the inhibition [310]. Studies recently reported for the structure-function analyses of the sulfonate moiety have argued for the requirement of a hydrophobic functionality, but its size did not appear to be restrictive. The absence of any hydrophobic functionality at this position lowered the ability of the molecules to inhibit (3-lactamases [314]. 

GG Hammond, JL Huber, ML Greenlee, JB Laub, K Young, LL Silver, JM Balko-vec, KD Pryor, JK Wu, B Leiting, DL Pompliano, JH Toney. Inhibition of IMP-1 metallo-beta-lactamase and sensitization of IMP-1-producing bacteria by thioester derivatives. FEMS Microbiol Lett 179 289-296, 1999. 

---