Wednesday, September 01, 2010

Identification of Beta-lactamase Inhibitor; a Strategy for Drug Development against Antibiotics Resistant Bacteria

Introduction:                                                                             By Bishnu Marasini

       The most threatening to human health is due to bacterial infection and intensive research has been addressing to treat these maladies since the known history. The infectious bacteria are microscopic, unicellular prokaryotes and different than mammalian eukaryotic cells. The outer layer of the bacterial cell consists of cell wall which is not found in mammalian cell and can be taken as target for development of bactericidal agents. The finding of clear zone of inhibition of bacterial culture around the growth of Penicillium notatum in the experiment of Alexander Fleming in 1928 was the positive indication to get rid of such threat.
       Nowadays almost all bacterial infection can be cured using antibiotics. The world consumes tons of antibiotics per year and half of them are beta-lactam type e.g. penicillin, amoxicillin, cephalosporin, cephalexin, cefixime, ceftriaxome, monobactam, carbapenem, methicillin etc. Beta-lactam antibiotics have broad spectrum activity, economical friendly on production, good safety profile, have clinical efficacy. It is also target specific for prokaryotic cells, little side effects except some allergic reaction. So, it has gained wide popularity.
       One of the components of bacterial cell wall is peptidoglycan which is cross linked polymer of repeatedly units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The final step in cell wall biosynthesis is transamidation reaction catalyzed by the enzyme cell wall transamidase (CWT) also called as penicillin binding protein (PBP). This enzyme helps to cross link the polymer of NAG and NAM. The highly strained and reactive beta-lactam ring of the antibiotics reacts and binds irreversibly with the serine hydroxyl group of PBP (shown in figure 1) which inactivates the enzyme and ultimately death of bacteria (Frère et al, 1984; Tipper and Strominger, 1965).

Figure1: Binding of β-lactam Ring with Penicillin Binding Protein
       However, beta-lactam antibiotics are becoming ineffective against pathogenic bacteria. The most common reason is due to the production of beta-lactamase enzyme (EC which catalyze the hydrolysis of the antibiotics i.e., formation of carboxyl group degrading beta-lactam ring (Shown in figure 2). Hydrolyzed antibiotics lose its activity or binding affinity towards the PBP hence no effect against bacteria. Hydrolysis of beta-lactam is rapid by beta-lactamase than binding of beta-lactam to PBP (Bush 1988)

Figure 2: Degradation of β-lactam ring by β-lactamase enzyme
       The beta-lactamse (penicillinase) was reported just few years after the first antibiotic discovered (Abraham and Chain, 1940). Although penicillin is the oldest antibiotic and most of the organisms acquired resistant, it is first therapeutic choice in some diseases like syphilis. Beta-lactamse enzyme is an extra cellular enzyme in Gram-positive bacteria and found in periplasmic membrane in Gram-negative bacteria (Bowden and Georgiou, 1990; Dyke and Richmond, 1967). More than 200 types of beta-lactamse have been found (Bush et al, 1995). The difference among them is only the catalytic efficacy and turn over rate range from 0.004 to 1,200 molecules per second by 1 molecule of enzyme. Among them two types i.e. penicillinase and cephalosporinase type has a potent influence on the profile of the beta-lactam resistant antibiotics. Class A beta-lactamse has high affinity towards penicillin G but low affinity towards cephalosporin while class C beta-lactamse has opposite. Class B beta-lactamse hydrolyze the antibiotics by binding with the co-factor zinc (Zn) and class A, C and D hydrolyze by binding through serine residue of it to beta-lactam ring (Sawai et al, 1981). Beta-lactamase became widespread via the mechanism of plasmid exchange/insert among the pathogens (Sykes and Richmond, 1970). The rapid spread and evolution of these enzymes have seriously threatened the present antimicrobial arsenal.
       Two strategies have been developed to combat the problem of resistant. The first approach has been the synthesis/production of beta-lactamase resistant antibiotics e.g. penicillinase resistant beta-lactam antibiotics, nafcillin, oxacillin, ceftriaxone, cefoxitime, aztreonam, imipenem etc. But after few exposure to pathogens these antibiotics also become susceptible to extended spectrum beta-lactamase (ESBL) produced by multi-drug resistant (MDR) pathogens.
       The second approach is to use beta-lactamase inhibitors coupled with beta-lactam antibiotics. These enzyme inhibitors function to permanently inactivate the beta-lactamase in the periplasmic space so that the partner antibiotics can reach its target, penicillin binding protein (PBP). Broad spectrum beta-lactam antibiotics plus beta-lactamase inhibitors combination have been found good safety records and clinical efficacies (Munoz et al, 1996). Augmentin, the production of GlaxoSmithKline which is composed of amoxicillin and clavulanate in 2:1; Timentin (ticarcillin and clavulanate); Sultamicillin (ampicillin and sulbactam) are examples of beta-lactamase inhibitors in combination with beta-lactam used clinically. Clavulanate exhibited clinical efficacy than others and used as standard inhibitor. However, clavulanate was not found so effective against class C beta-lactamase (cephalorinase type) (Bush K, 1989). It also exhibited side effects on the long term of use like liver function destruction, gastrointestinal toxicity etc. (Ioannidis et al, 2002). Also it contains beta-lactam ring it and may be susceptible to beta-lactamase enzyme in upcoming days as broad spectrum beta-lactam antibiotics which were resistant to beta-lactamase, now become susceptible.

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