P lactamases and

Substitution of penicillins by 6a-methoxy was found to be compatible with an a-acidic side chain in terms of antibacterial activity, but less beneficial when the side chain contained an a-acyl or a-ureido substituent. However, analogues of the ureido penicillin VX-VC-43 (Table 2) containing a 6a-methoxy substituent (10) were found to combine good stabiUty to P-lactamase and relatively high antibacterial activity (37). Following an extensive program to identify other 6a-substituents that would stabilize the acyl and ureido series of penicillins, the 6a-formamido series (11) represented by formidacillin (BRL 36650) (Table 2) was developed (38). 

AmpC P-Lactamase. A map of hot spots was constructed from the X-ray structure of AmpC P-lactamase and a university version of the program DOCK was used to search for noncovalent inhibitors in 229,810 compounds of the ACD database. Of 56 tested compounds three had values <650pM, for example, compound 41 Ki = 26pM Fig. 16.6) [117]. The experimental X-ray structure of its complex with AmpC P-lactamase closely resembles the predicted binding mode. 

Bush, K. Jacoby, G. A. Medeiros, A. A. A functional classification scheme for p-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 1995, 39,1211-33. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Widespread clinical acceptance continues to be accorded to the cephalosporins, and the field is extremely active as firms search for the ultimate contender. Among the characteristics desired is retention of the useful features of the older members (relatively broad spectrum, less antigenicity than the penicillins, relative insensitivity toward p-lactamases, and convenience of administration) while adding better oral activity and broader antimicrobial activity (particularly potency against Pseudomonas, anaerobes, meningococci, cephalosporinase-carrying organisms, and the like). To a considerable extent these objectives have been met, but the price to the patient has been dramatically increased. 

Although no conclusive evidence has been found yet for this assumption, it is postulated that the p-lactam opening reactions promoted by CN- or N3 -containing additives proceed via an acyl azide or an acyl cyanide intermediate. The mode of action of some p-lactamases and the fact that no p-lactam ring opening usually takes place in the absence of such additives support this hypothesis. 

JM Ghuysen. Serine P-lactamases and penicillin-binding proteins. Annu Rev Microbiol 45 37-67, 1991. 

It is known that p-lactamase catalyzes the rapid hydrolysis of the p-lactam ring of penicillins and cepharosporines. The hydrolytic activity of these enzymes eliminates the bacteriocidal action of many p-lactam antibiotics and makes the organism resistant to these molecules. For this reason, the p-lactamase inhibitors have long been regarded as promising targets from a medicinal viewpoint. A comparison between the kinetic characteristics of p-lactamase and penicillin-sensitive enzymes (carboxy-peptidase and transpeptidase) is of interest in this respect. p-Lactamases very efficiently hydrolyze p-lactam in contrast to penicillin-sensitive enzymes [high /e4 in Eq. (9)]. 

The compound (which had a dihydrothiazine ring fused to the P-lactam core) showed resistance to P-lactamases and was less toxic than benzylpenicillin. The discovery that the basic building block, namely 7-aminocephalosporanic acid (7-ACA), could be synthesised, led to the preparation of numerous cephalosporin derivatives eg cephalothin, cephaloglycin (orally active), cefaclor and cefuroxime (Figure 7). 

The activity of these semis)mthetic penicillins extends beyond the Gram-positive and Gram-negative cocci which are susceptible to benzylpenicillin, and includes many Gram-negative bacilli. They do not resist P-lactamases and their usefulness has reduced markedly in recent years because of the increased prevalence of organisms that produce these enzymes. 

The class A P-lactamases are a subset of the active-site serine P-lactamases. TEM-1 P-lactamase is a class A enzyme encoded by the ft/ajEM-l gene that is present on the transposons Tn2 and Tn3 (Datta et al, 1965). Epidemiological studies have shown that TEM-1 is the most common plasmid-mediated P-lactamase and is therefore a major determinant of bacterial resistance to P-lactam antibiotics (Wiedemann et al, 1989). Compounding the problem of resistance is the discovery that TEM-1 mutant variants with altered substrate specificity have been identified in natural isolates (Jacoby and Medieros, 1991). These variant enzymes contain from one to three amino acid substitutions that enable the enzyme to hydrolyze the newer extended-spectrum cephalosporin antibiotics such as cefotaxime and ceftazidime (Jacoby and Medieros, 1991). Thus, the selective pressure of antibiotic therapy le s to die creation of new enzymes with expanded hydrolytic capabilities. 

Because of the significant role of TEM-1 P-lactamase and its mutant derivatives in antibiotic resistance, it is of interest to understand how the amino acid sequence of the enzyme establishes its structure and activity. We have determined the tolerance of each residue in TEM-1 P-lactamase to amino acid substitutions to identify those residues that make critical contributions to the structure and activity of the enzyme. The tolerance of each residue was determined by randomizing three to six contiguous codons to create a random library containing all possible amino acid substitutions for the region randomized (Palzicill and Botstein, 1992). Functional random mutants were then selected from the libraries and sequenced to identify permissible substitutions at each position. The sequences for each set of mutants allowed the importance of... 

A large number of class A P-lactamases have now been identified and sequenced and an alignment of 20 class A P-lactamases has been published (Ambler et al., 1991). These aligned sequences permit a comparison between the conserved amino acid residue positions among class A p-lactamases and the conserved positions among the functional random mutants in TEM P-lactamase (Fig. 2). In general, there is agreement between the tolerance of a residue in TEM P-lactamase to amino acid substitutions and the amount a position is substituted in... 

Penicillanic acid derivatives are synthetically produced P-lactamase inhibitors. Penicillanic acid sulphone (Fig. 10.6D) protects ampicillin from hydrolysis by staphylococcal P-lactamase and some, but not all, of the P-lactamases produced by Gram-negative bacteria, but is less potent than... 

It must also be noted that a combination of two P-lactams does not necessarily produce a synergistic effect. Some antibiotics are excellent inducers of P-lactamase, and consequently a reduced response (antagonism) may be produced. 

SUltamieillin [ban, inn, usan] is a semisynthetic (penicillin) ANTIBIOTIC, a prodrug of ampicillin and sulbactam, joined by a double ester, where the latter is an enzyme inhibitor that is resistant to P-lactamase and inhibits this penicillindegrading enzyme. It can be used clinically as an ANTIBACTERIAL to treat certain infections. 

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