Inhibition of the SHV-1 β-Lactamase by Sulfones:  Crystallographic Observation of Two Reaction Intermediates with Tazobactam<sup>,</sup>

Kuzin, A.P.; Nukaga, Michiyoshi; Nukaga, Yasuko; Hujer, Andrea M.; Bonomo, Robert A.; Knox, James R.

doi:10.1021/bi0022745

Cited by 82 publications

(123 citation statements)

References 33 publications

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“…This finding supports the main reaction chemistry and that of the inhibitors involving the active site nucleophile Ser70, as identified with TEM-2 and clavulanate (4), TEM-1 with tazobactam (62), and PC1 with tazobactam (62) and as shown in the crystal structure and mass spectrometry analysis of SHV-1 with tazobactam and clavulanate (29,50,52,53). We did not find definitive evidence that there was a resulting fragment that represented the "bridging species" connecting Ser70 to Ser130, but we did find a 52 Ϯ 3 amu adduct (propynyl addition) when sulbactam and tazobactam inactivated CTX-M-9 via ESI-MS that may have served as such ( Fig.…”

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

confidence: 84%

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

Bethel

Taracila

Shyr

et al. 2011

Antimicrob Agents Chemother

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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 ...

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

confidence: 84%

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

Bethel

Taracila

Shyr

et al. 2011

Antimicrob Agents Chemother

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“…Hence, the reactivity of the azide and acetylene precursors in the gorge and the resulting affinity of the triazole formed could not have been predicted from structures of apo-AChE (10) or of complexes with close congeners to the precursors (10,11). To date, inhibitors containing substituted 1,2,3-triazoles have been observed only in plant glycolate oxidase inhibitors (25) and ␤-lactam antimicrobials (26). 10, 11, 22, 23, 27, and 28).…”

Section: Resultsmentioning

confidence: 99%

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Bourne

Kolb

Radić

et al. 2004

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

311

262

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The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1-and anti1-mouse AChE complexes at 2.45-to 2.65-Å resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.A cetylcholinesterase (AChE) rapidly terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter, acetylcholine, and inhibitors of AChE have been used for over a century in various therapeutic regimens (1, 2). The structure of the target enzyme reveals a narrow gorge Ϸ20 Å in depth with the catalytic triad of the active center at its base (3). Distinctive inhibitors bind to the active center or to a peripheral anionic site (PAS) located at the rim of the gorge near the enzyme surface (4-6). Previously, we generated a library of active site and PAS inhibitors with respective tacrine and phenanthridinium nuclei, each equipped with an azide or acetylene group at the end of a flexible methylene chain, to enable the reporting 1,3-dipolar cycloaddition to occur (Scheme 1) (7). AChE itself served as the reaction vessel, synthesizing its own inhibitor from these building blocks, in effect, by equilibriumcontrolled sampling of various possible pairs of reactants in its active center gorge until irreversible cycloaddition between azide and acetylene ensued at an intersecting point within the gorge, between the two anchoring positions. From 49 building block combinations, the enzyme selected the TZ2͞PA6 pair to form, with an enhanced reaction rate, a highly regioselective syn1 triazole as the sole product (Scheme 1). In contrast, chemical synthesis by t...

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“…The bases of action of the many other action-at-a-distance IRTs, such as TEM-38 (M69V/R275L) and TEM-39 (M69L/W165R/N276D) which, like TEM-30 and TEM-32, result in relatively resistant, relatively active IRTs, appear ever more interesting in light of the conserved accommodations of this simplest of IRT mutants, S130G. Simplified hydrolytic and inhibition pathways of TEM β-lactamase for tazobactam (1,11). Electron density of the characteristic regions of the S130G structure.…”

Section: Discussionmentioning

confidence: 99%

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,

et al. 2005

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Abstractβ-Lactamase confers resistance to penicillin-like antibiotics by hydrolyzing their β-lactam bond. To combat these enzymes, inhibitors covalently cross-linking the hydrolytic Ser70 to Ser130 were introduced. In turn, mutant β-lactamases have emerged with decreased susceptibility to these mechanism-based inhibitors. Substituting Ser130 with glycine in the inhibitor-resistant TEM (IRT) mutant TEM-76 (S130G) prevents the irreversible cross-linking step. Since the completely conserved Ser130 is thought to transfer a proton important for catalysis, its substitution might be hypothesized to result in a nonfunctional enzyme; this is clearly not the case. To investigate how TEM-76 remains active, its structure was determined by X-ray crystallography to 1.40 Å resolution. A new water molecule (Wat1023) is observed in the active site, with two configurations located 1.1 and 1.3 Å from the missing Ser130 Oγ; this water molecule likely replaces the Ser130 side-chain hydroxyl in substrate hydrolysis. Intriguingly, this same water molecule is seen in the IRT TEM-32 (M69I/ M182T), where Ser130 has moved significantly. TEM-76 shares other structural similarities with various IRTs; like TEM-30 (R244S) and TEM-84 (N276D), the water molecule activating clavulanate for cross-linking (Wat1614) is disordered (in TEM-30 it is actually absent). As expected, TEM-76 has decreased kinetic activity, likely due to the replacement of the Ser130 side-chain hydroxyl with a water molecule. In contrast to the recently determined structure of the S130G mutant in the related SHV-1 β-lactamase, in TEM-76 the key hydrolytic water (Wat1561) is still present. The conservation of similar accommodations among IRT mutants suggests that resistance arises from common mechanisms, despite the disparate locations of the various substitutions.The catalytic activity of the TEM family of class A β-lactamases is a major resistance mechanism against β-lactam antibiotics, such as penicillins ( Figure 1A); hydrolysis of the β-lactam ring of these antibiotics renders them ineffective against bacteria. To combat these enzymes, inhibitors against β-lactamase, such as clavulanate, tazobactam, and sulbactam ( Figure 1B-D), were developed. In turn, inhibitor-resistant TEM β-lactamases (IRTs) 1 have emerged in the clinic with decreased susceptibility to these mechanism-based inhibitors. These † This work was supported by NIH Grants GM63815 (to B.K.S.) and AI33170 (to S.M.) and by the Achievement Rewards for College Scientists Foundation and a National Science Foundation predoctoral fellowship (to V.L.T.). ‡ Atomic coordinates and structure factors have been deposited in the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics at Rutgers University (entry 1YT4). * Corresponding authors. B.K.S.: phone, 415-514-4126; fax, 415-502-1411; e-mail,shoichet@cgl.ucsf.edu. S.M.: phone, 574-631-2933; fax, 574-631-6652; e-mail,mobashery@nd.edu inhibitors function by acylating the enzyme's active site. After reacting with the catalytic Ser70, these β-lac...

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Inhibition of the SHV-1 β-Lactamase by Sulfones: Crystallographic Observation of Two Reaction Intermediates with Tazobactam^,

Cited by 82 publications

References 33 publications

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

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

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,

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Inhibition of the SHV-1 β-Lactamase by Sulfones: Crystallographic Observation of Two Reaction Intermediates with Tazobactam,

Cited by 82 publications

References 33 publications

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

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

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase,

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Inhibition of the SHV-1 β-Lactamase by Sulfones: Crystallographic Observation of Two Reaction Intermediates with Tazobactam^,

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,