Mechanism of Inactivation of β-Lactamases by Novel 6-Methylidene Penems Elucidated Using Electrospray Ionization Mass Spectrometry

Tabei, Keiko; Feng, Xun; Venkatesan, Aranapakam M.; Abé, Takao; Ushirogochi, Hideki; Mansour, Tarek S.; Siegel, Marshall M.

doi:10.1021/jm049903j

Cited by 28 publications

(15 citation statements)

References 29 publications

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“…Other classes of β-lactamase inhibitors undergo chemical rearrangements from their initial acyl-enzyme species, and such rearrangements are observable at high ratios of inhibitor to enzyme (17,18). To investigate this possibility, a high concentration of avibactam was incubated with TEM-1, and the off-rate was measured (Fig.…”

Section: Resultsmentioning

confidence: 99%

Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor

Ehmann

Jahić

Ross

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

457

450

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Avibactam is a β-lactamase inhibitor that is in clinical development, combined with β-lactam partners, for the treatment of bacterial infections comprising Gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a β-lactam core but maintains the capacity to covalently acylate its β-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the offrate for deacylation. The deacylation off-rate was 0.045 min −1 , which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant β-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors.antibacterial | drug discovery | enzymology T here is an urgent need for new antibacterial agents that are active against drug-resistant bacteria. In particular, some Gram-negative pathogens have accumulated enough resistance mechanisms to render them virtually untreatable by modern antibacterial chemotherapy (1, 2). A mainstay for treatment of Gram-negative infections is the β-lactam classes of drugs. The most common form of resistance to β-lactam antibiotics is the expression of various β-lactamase enzymes capable of hydrolyzing the β-lactam ring of β-lactam drugs, rendering them ineffective. As new β-lactams have been introduced into clinical use, a changing landscape of β-lactamases has been selected and disseminated. Presently, over 1,000 β-lactamases have been documented comprising several structural classes and a wide range of substrate promiscuities and catalytic efficiencies (3, 4).In efforts to restore the efficacy of β-lactam antibiotics, β-lactamases have also been targeted with a variety of inhibitors (5, 6). The three inhibitors approved for clinical use are clavulanic acid, tazobactam, and sulbactam, all of which contain a β-lactam core. A challenge for the development of broad-spectrum β-lactamase inhibitors is the mechanistic diversity in β-lactamase enzymes, with the largest distinction being between the enzyme classes that use a serine residue as the nucleophilic species and the metallo-β-lactamases, which directly activate water for hydrolysis (7). A shared mechanistic feature of the marketed β-lactam-based inhibitors is their reaction with the serine enzymes to form a covalent acylenzyme intermediate. On ring opening, the acyl-enzyme intermediate can undergo additional rearrangements or be released through hydrolysis to regenerate the active β-lactamase enzyme (8). Originally designed to combat class A serine β-lactamase enzymes such as TEM-1, the clinical use of β-lactam-based inhibitors has been diminished by the emergence of enzymes against which they are ineffective. Despite intense investigation by pharmaceutical companies, no new β-lactamas...

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Section: Resultsmentioning

confidence: 99%

Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor

Ehmann

Jahić

Ross

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

457

450

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“…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).…”

Section: Penemsmentioning

confidence: 99%

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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“…Thus, we wondered if penem inhibitors that possess an sp 2 -hybridized C 3 carboxylate (a property resembling a characteristic of carbapenems), a complex and reactive R1 side chain, and inactivation chemistry different from that of clavulanic acid could be exploited to inhibit KPC enzymes (41). The methylidene inhibitors penem 1 and penem 2 have dihydropyrazolo [1,5-c] [1,3]thiazole and dihydropyrazolo [5,1-c] [1,4] thiazine moieties, respectively (see Fig.…”

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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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Mechanism of Inactivation of β-Lactamases by Novel 6-Methylidene Penems Elucidated Using Electrospray Ionization Mass Spectrometry

Cited by 28 publications

References 29 publications

Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor

Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor

Three Decades of β-Lactamase Inhibitors

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

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