Inhibition of the Class C β-Lactamase from Acinetobacter spp.: Insights into Effective Inhibitor Design

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Currently, CTX-M ␤-lactamases are among the most prevalent and most heterogeneous extended-spectrum ␤-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by ␤-lactamase inhibitors. However, it is unknown if the pathway to inhibition by ␤-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV ␤-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 ␤-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. CTX-M enzymes are becoming one of the most prevalent extended-spectrum ␤-lactamases (ESBLs) (3,8,9,(30)(31)(32) in the world. The widespread dissemination of CTX-M ␤-lactamases, especially Escherichia coli ST131 possessing CTX-M-15, has had a significant impact on the treatment of hospital-and community-acquired infections caused by E. coli and other enteric bacilli (6, 7, 13, 23, 36, 41, 44-49, 55, 59).As class A family ␤-lactamases, CTX-Ms are the most genetically heterogeneous (5 major divisions, CTX-M-1, -2, -8, -25, and -9-like groups) (1, 24, 35, 44-46, 58, 60). Most CTX-M enzymes expressed in E. coli provide a high level of resistance to the oxyimino-cephalosporins, cefotaxime and ceftriaxone, and variable levels of resistance to cefepime and cefpirome (3,43). Depending on the type of CTX-M expressed by the isolates, the MICs of ceftazidime are also increased (43). In addition, the MICs of combinations of clavulanate with amoxicillin or ticarcillin vary, and in some cases, low-level resistance has been observed (3).As a consequence of their clinical importance, the reaction mechanism of CTX-M ESBLs has been the subject of intense study (10-12, 14, 16, 42). However, the molecular properties and characteristics of CTX-M that determine the level of susceptibility and resistance to ␤-lactam-␤-lactamase inhibitor combinations and carbapenems are still unknown. Of the currently available inhibitors, tazobactam is the most active (50% inhibitory concentrations [IC 50 s] are 2 to 10 nM for tazobactam and 9 to 90 nM for clavulanate), and sulbactam is the least active (IC 50 s are 0.1 to 4.5 M) (3).In order to develop more effective and broader-spectrum ␤-lactamase inhibitors (18), detailed kinetic and biochemical measurements are needed to reveal the important intermediates in the inactivation of the CTX-M family. In TEM-1 and SHV-1, Arg244 is important in the mechanism of inactivation of carbapenems (imipenem and meropenem), clavulanic acid, sulbactam, and tazobactam (27,28,51,53,63). CTX-M-9 does not contain Arg244, and mutagenesis of a potential corresponding position, Arg276, does not firmly establish this amino acid as an "Arg244 equivalent" (42). Given the differences among class A enzymes, we wondered what the intermediates of inactivation by ...

Section: Vol 55 2011 Ctx-m-9 Inhibition By ␤-Lactamase Inhibitors 3469supporting

confidence: 58%

“…The turnover number (t n ) or the partitioning of the initial enzyme-inhibitor complex between hydrolysis and enzyme inactivation, k cat /k inact (partition ratio), was determined as previously reported (2,17,53).…”

Section: Methodsmentioning

confidence: 99%

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Bethel

Taracila

Shyr

et al. 2011

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“…Studies have shown analogs of ampicillin, cephalothin, and cefoperazone to be inhibitors of clinically relevant class A and C ␤-lactamases in the nM range (i.e., 9.3 nM, 420 nM, and 11 nM for K. pneumoniae SHV and for AmpCs from P. aeruginosa and Acinetobacter spp., respectively) (Fig. 3b) (53)(54)(55)(56). These compounds are still preclinical.…”

Section: Boronic Acid ␤-Lactamase Inhibitorsmentioning

confidence: 97%

New β-Lactamase Inhibitors: a Therapeutic Renaissance in an MDR World

Drawz

Papp-Wallace

Bonomo

2014

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266

179

As the incidence of Gram-negative bacterial infections for which few effective treatments remain increases, so does the contribution of drug-hydrolyzing ␤-lactamase enzymes to this serious clinical problem. This review highlights recent advances in ␤-lactamase inhibitors and focuses on agents with novel mechanisms of action against a wide range of enzymes. To this end, we review the ␤-lactamase inhibitors currently in clinical trials, select agents still in preclinical development, and older therapeutic approaches that are being revisited. Particular emphasis is placed on the activity of compounds at the forefront of the developmental pipeline, including the diazabicyclooctane inhibitors (avibactam and MK-7655) and the boronate RPX7009. With its novel reversible mechanism, avibactam stands to be the first new ␤-lactamase inhibitor brought into clinical use in the past 2 decades. Our discussion includes the importance of selecting the appropriate partner ␤-lactam and dosing regimens for these promising agents. This "renaissance" of ␤-lactamase inhibitors offers new hope in a world plagued by multidrug-resistant (MDR) Gram-negative bacteria.

“…The ceftazidime BATSI and cefoperazone BATSI analogues ( Fig. 1) were synthesized as previously described (7). Compounds 1 and 2 were obtained in two steps according to the scheme shown in Fig.…”

Section: Synthesis Of Boronic Acid Inhibitorsmentioning

confidence: 99%

“…Compounds 1 and 2 were obtained in two steps according to the scheme shown in Fig. 1B by using the appropriate amino acids (D-homotyrosine, synthesized as hydrobromide from D-aspartic acid [15,28], and commercially available D-tyrosine, respectively), commercially available 4-ethyl-2,3-dioxopiperazine-1-carbonyl chloride, and bis-(trimethylsilyl)-aminomethaneboronate, synthesized as previously described (7).…”

Section: Synthesis Of Boronic Acid Inhibitorsmentioning

confidence: 99%

Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

Wei

Sampson

Ori

et al. 2011

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Boronic acid transition state inhibitors (BATSIs) are potent class A and C ␤-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 ␤-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 ␤-lactamase with micromolar affinity that is considerably weaker than their inhibition of other ␤-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other ␤-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.Production of ␤-lactamases (EC 3.5.2.6) is one of the major mechanisms by which bacteria develop resistance to ␤-lactam antibiotics. In nature, four classes of ␤-lactamase enzymes exist (classes A to D). Classes A, C, and D are serine-based ␤-lactamases, while class B uses a metal ion (Zn 2ϩ ) to hydrolyze the lactam bond. TEM-1 and SHV-1 ␤-lactamases are among the most commonly observed class A ␤-lactamases found in Escherichia coli and Klebsiella pneumoniae. As such, this family of ␤-lactamases presents a significant clinical threat (3, 21).To counteract ␤-lactamases, mechanism-based inhibitors were developed to be administered in concert with ␤-lactam antibiotics (9). Presently, there are three commercially available ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) which are effective primarily against class A ␤-lactamases (Fig. 1). Unfortunately, bacteria possessing ␤-lactamase enzymes have adapted under continued drug pressure and some variants of class A ␤-lactamases are now "inhibitor resistant" (9). Moreover, class C and D enzymes are poorly inhibited by any of the three commercial inhibitors. Therefore, there is an urgent need to develop novel ␤-lactam antibiotics and new types of ␤-lactamase inhibitors to counteract the current crisis in antimicrobial resistance (9).Based...