Kinetic and physical studies of <i>β</i>-lactamase inhibition by a novel penem, BRL 42715

Farmer, T. H.; Page, J. W. J.; Payne, D. J.; Knowles, David J. C.

doi:10.1042/bj3030825

Cited by 35 publications

(23 citation statements)

References 35 publications

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“…Although penem 1 and penem 2 are hydrolyzed by KPC-2 while acting as mechanism-based inactivators, they potentially offer a better alternative than the commercial inhibitors for inhibition of KPC-producing strains. We suspect that unraveling the chemistry that drives the hydrolysis of the commercially available inhibitors and penems 1 and 2 through a branched kinetic mechanism (20,21,28) may serve to offer new approaches to inhibiting carbapenemases.…”

Section: Resultsmentioning

confidence: 99%

Inhibitor Resistance in the KPC-2 β-Lactamase, a Preeminent Property of This Class A β-Lactamase

Papp-Wallace

Bethel

Distler

et al. 2010

Antimicrob Agents Chemother

171

190

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As resistance determinants, KPC ␤-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type ␤-lactamases are often identified as resistant to standard ␤-lactam-␤-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to ␤-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k cat /k inact ratios, where k inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k cat /k inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k inact /K m ‫؍‬ 6.5 ؎ 0.6 M ؊1 s ؊1 ). We conclude that KPC-2 is unique among class A ␤-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type ␤-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current ␤-lactamase inhibitors.In Klebsiella pneumoniae, ␤-lactam resistance is mediated predominantly by class A SHV, TEM, and CTX-M ␤-lactamases (7, 35). Single amino acid substitutions in the SHV and TEM ␤-lactamases can drastically alter the substrate profiles of the enzymes and confer resistance to extended-spectrum cephalosporins and ␤-lactamase inhibitors (5,12,34,36). ␤-Lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant ␤-lactamases) have significantly challenged the clinician's approach to the treatment of serious infectious diseases (36). Thus, the search for effective mechanism-based inhibitors of novel ␤-lactamases merits significant effort (8,9,32).First identified in K. pneumoniae, KPC class A ␤-lactamases threaten the use of all current ␤-lactam antibiotics (57). These ␤-lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram-negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the United States (3,10,11,16,17,25,37,45,49,53,59). Moreover, KPC ␤-lactamases are becoming geographically widespread (having been detecte...

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Section: Resultsmentioning

confidence: 99%

Inhibitor Resistance in the KPC-2 β-Lactamase, a Preeminent Property of This Class A β-Lactamase

Papp-Wallace

Bethel

Distler

et al. 2010

Antimicrob Agents Chemother

171

190

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“…Most ␤-lactamases appear to follow this linear pathway to inactivation by the penem compounds, i.e., formation of an acyl-enzyme and rearrangement to the stable dihydrothiazepine ring adduct (119,250). However, some class A enzymes, such as the ␤-lactamases from Staphylococcus albus (Staphylococcus epidermidis) and K. pneumoniae K1, exhibit more complex kinetics (46,250).…”

Section: Penemsmentioning

confidence: 99%

“…Further, amoxicillin-BRL 42715 combinations were more effective than amoxicillin-clavulanate for E. coli, K. pneumoniae, H. influenzae, S. aureus, and N. gonorrhoeae strains producing plasmid-mediated ␤-lactamases (mostly TEM and SHV expressors) and for K. oxytoca, Proteus vulgaris, and P. mirabilis strains producing chromosomal ␤-lactamases. Kinetic studies of BRL 42715 and NCTC 11561, TEM-1, and P99 showed rapid, irreversible inactivation with an inhibition stoichiometry of 1:1, or turnover numbers of 0 (119).…”

Section: Penemsmentioning

confidence: 99%

“…Accordingly, mass spectrometry studies demonstrated that the methylidene penem inhibition pathway does not involve fragmentation in the active site (119,250,406). When TEM-1, P99, and the class A ␤-lactamase from B. cereus I were inactivated by BRL 42715, spectra showed a mass increases equivalent to the molecular masses of the inhibitor plus the enzyme (119). The crystal structure of the class C ␤-lactamase E. cloacae 908R in complex with BRL 42715 (PDB 1Y54) confirmed the formation of a stable seven-membered thiazepine ring covalently attached to Ser64 (263).…”

Section: Penemsmentioning

confidence: 99%

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Three Decades of β-Lactamase Inhibitors

Drawz¹,

2010

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SUMMARYSince the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of seriousEnterobacteriaceaeand penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a “second generation” of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases.

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“…1, compound 4), a previously studied compound, showed that the Z-penem nitrobenzyl ester reacted with the base (sodium methoxide in methanol) results in the formation of a seven-member thiazepine with max values of 253 and 370 nm (9). It is known from crystal structure, kinetic, and computational studies that a novel seven-member 1,4-thiazepine ring is formed from the reaction of methyldiene penems and BRL 42715 with class A enzymes (e.g., TEM-1 and SHV-1) and class C enzymes (11,15,36,41,55,56). The chemical similarity of penems 1 and 2 to these compounds, the 1 H NMR spectra, and the results of MS and peptide analysis argue that a similar intermediate, the 1,4-thiazepine derivative, is formed (9,56).…”

Section: Resultsmentioning

confidence: 99%

Inhibition of OXA-1 β-Lactamase by Penems

Bethel

Distler²,

Ruszczycky

et al. 2008

Antimicrob Agents Chemother

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The partnering of a ␤-lactam with a ␤-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to ␤-lactam antibiotics mediated by serine ␤-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D ␤-lactamase that is resistant to inactivation by tazobactam. The K i of each penem inhibitor for OXA-1 was in the nM range (K i of penem 1, 45 ؎ 8 nM; K i of penem 2, 12 ؎ 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 ␤-lactamase were 0.13 ؎ 0.01 s ؊1 and 0.11 ؎ 0.01 s ؊1 , respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in <900 s and the recovery of OXA-1 ␤-lactamase activity was not detected at 24 h. Covalent adducts of penems 1 and 2 (changes in molecular masses, ؉306 ؎ 3 and ؉321 ؎ 3 Da, respectively) were identified by electrospray ionization mass spectrometry (ESI-MS). After tryptic digestion of OXA-1 inactivated by penems 1 and 2, ESI-MS and matrix-assisted laser desorption ionization-time-of-flight MS identified the adducts of 306 ؎ 3 and 321 ؎ 3 Da attached to the peptide containing the active-site Ser67. The base hydrolysis of penem 2, monitored by serial 1 H nuclear magnetic resonance analysis, suggested that penem 2 formed a linear imine species that underwent 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative. Susceptibility testing demonstrated that the penem inhibitors at 4 mg/liter effectively restored susceptibility to piperacillin. Penem ␤-lactamase inhibitors which demonstrate high affinities and which form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine ␤-lactamases present in gramnegative bacteria.

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Kinetic and physical studies of β-lactamase inhibition by a novel penem, BRL 42715

Cited by 35 publications

References 35 publications

Inhibitor Resistance in the KPC-2 β-Lactamase, a Preeminent Property of This Class A β-Lactamase

Inhibitor Resistance in the KPC-2 β-Lactamase, a Preeminent Property of This Class A β-Lactamase

Three Decades of β-Lactamase Inhibitors

Inhibition of OXA-1 β-Lactamase by Penems

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