Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

Yang, Hao; Aitha, Mahesh; Hetrick, Alyssa; Richmond, Timothy; Tierney, David L.; Crowder, Michael W.

doi:10.1021/bi300056y

Cited by 91 publications

(182 citation statements)

References 55 publications

(193 reference statements)

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“…The temperature was kept constant at 22 °C using a circulating water bath. Absorbance data were converted to concentration data using the following extinction coefficients: substrate (28).…”

Section: Fluorescencementioning

confidence: 99%

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Cheng

Thomas²,

et al. 2018

Journal of Biological Chemistry

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107

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Infections by carbapenem-resistant Enterobacteriaceae (CRE) are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically-used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a dinuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing comparing melting temperature and refolding efficiency and also by spectroscopy (UV-Vis, 1 H-NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining of minimum inhibitory concentrations of various antibiotics, pre-steady state and steadystate kinetics, inhibitor binding, and zincdependence of resistance and activity. We observed only minor differences among the fullloaded dizinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants http://www.jbc.org/ Downloaded from 2 exhibited improved thermostability (up to ~10 °C increased Tm) and improved zinc affinity (up to ~10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as monozinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/KM = 5 × 10 6 M -1 s -1 ). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of β-lactam antibiotics and zinc deprivation.Carbapenem-resistant Enterobacteriaceae (CRE) continue to be classified as an "urgent threat," the highest hazard level assigned by the Centers for Disease Control and Prevention(1). The five carbapenemases currently of primary public concern include Klebsiella pneumonia carbapanemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integrin encoded metallo-β-lactamase (VIM), imipenemase (IMP), and oxacillinase-48-like carbapenemase (OXA-48)(2). Three of these carbapenemases (NDM, VIM, and IMP) are metal-dependent β-lactamases that are not susceptible to any of the β-lactamase inhibitors incorporated into combination drugs used in the clinic. Of these three β-lactamases, NDM is the most widespread in U.S. patients, with infections bearing a blaNDM gene reported in 34 / 50 states (as of December 2017)(3).The genes encoding NDM continue to evolve, with discovery of more than 20 variants (NDM-1 through NDM-21 at the time of writing, 16 at the start of this project). Most of these mutations occur at sites distant from the active site, and the functions they confer are not immediately obvious. A comparison of NDM-1 through NDM-8 showed only minor differences in kcat/KM values (≤ 5-fold) for a panel of diverse β-lactam drugs(4). However, a considerable increase in thermostability was noted for many of the variants, suggesting the functional impact of NDM...

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Section: Fluorescencementioning

confidence: 99%

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Cheng

Thomas²,

et al. 2018

Journal of Biological Chemistry

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107

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“…4,21−23 Steady-state kinetic studies and inhibition experiments of the mercaptoacetic acid thioesters as inhibitors against MβLs were conducted on an Agilent UV8453 spectrometer. Hydrolysis of substrate (cefazolin for CcrA, NDM-1, and L1; imipenem for ImiS) was monitored at 262 and 300 nm, respectively.…”

Section: Acs Medicinal Chemistry Lettersmentioning

confidence: 99%

Amino Acid Thioester Derivatives: A Highly Promising Scaffold for the Development of Metallo-β-lactamase L1 Inhibitors

Liu

Shi

Kang

et al. 2015

ACS Med. Chem. Lett.

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In light of the biomedical significance of metallo-β-lactamases (MβLs), ten new mercaptoacetic acid thioester amino acid derivatives were synthesized and characterized. Biological activity assays indicated that all these synthesized compounds are very potent inhibitors of L1, exhibiting an IC 50 value range of 0.018−2.9 μM and a K i value range of 0.11−0.95 μM using cefazolin as substrate. Partial thioesters also showed effective inhibitory activities against NDM-1 and ImiS with an IC 50 value range of 12−96 and 3.6−65 μM, respectively. Also, all these thioesters increased susceptibility of E. coli cells expressing L1 to cefazolin, indicated by a 2−4-fold reduction in MIC of the antibiotic. Docking studies revealed potential binding modes of the two most potent L1 inhibitors to the active site in which the carboxylate group interacts with both Zn(II) ions and Ser221. This work introduces a highly promising scaffold for the development of metallo-β-lactamase L1 inhibitors. KEYWORDS: Antibiotic resistance, metallo-β-lactamase, subclass B3, L1, inhibitor, mercaptoacetic acid thioester β-Lactam antibiotics, which include penicillins, cephalosporins, and carbapenems, remain the most important and frequently used antimicrobial agents, constituting more than 50% of the antibiotics prescribed worldwide.1 However, the overuse of antibiotics in the clinical setting as well as animal production has resulted in resistance 2 conferred, among other resistance mechanisms, by the production of β-lactamases. More than a thousand β-lactamases have been isolated, and these enzymes have been categorized into classes A to D, depending on their amino acid sequence homologies.1 Enzymes from classes A, C, and D comprise the serine β-lactamases. They use a catalytic mechanism that is characterized by the nucleophilic attack of an active-site serine on the β-lactam carbonyl, ultimately causing cleavage of the β-lactam ring. Class B enzymes, or metallo-β-lactamases (MβLs), require either one or two Zn(II) ions per enzyme molecule for full catalytic activity, and these enzymes inactivate all clinically important β-lactams except aztreonam. According to amino acid sequence homologies MβLs are divided into subclasses B1−B3, based on amino acid sequence homologies.3 MβLs from subclasses B1 and B3 can inactivate nearly all β-lactam antibiotics. Contrarily, B2 enzymes possess a narrow substrate preference for carbapenems. There are no known clinical inhibitors of the MβLs to date.

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“…10 New Delhi metallo-β-lactmase-1 (NDM-1), a B1 subclass enzyme first discovered in 2008, 11 has become a global threat because it confers resistance to almost all β-lactam antibiotics and due to its rapid spread of the plasmid-encoded NDM-1 gene. 12,13 The inhibitors of serine-β-lactamases (classes A, C, and D), such as tazobactam and sulbactam, have been used in combination with penicillins to treat infections with resistant bacteria. Novel effective MβL inhibitors for such combination therapies are urgently needed.…”

mentioning

confidence: 99%

Triazolylthioacetamide: A Valid Scaffold for the Development of New Delhi Metallo-β-Lactmase-1 (NDM-1) Inhibitors

Zhai

Zhang

Kang

et al. 2016

ACS Med. Chem. Lett.

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ABSTRACT:The metallo-β-lactamases (MβLs) cleave the β-lactam ring of β-lactam antibiotics, conferring resistance against these drugs to bacteria. Twenty-four triazolylthioacetamides were prepared and evaluated as inhibitors of representatives of the three subclasses of MβLs. All these compounds exhibited specific inhibitory activity against NDM-1 with an IC 50 value range of 0.15−1.90 μM, but no activity against CcrA, ImiS, and L1 at inhibitor concentrations of up to 10 μM. Compounds 4d and 6c are partially mixed inhibitors with K i values of 0.49 and 0.63 μM using cefazolin as the substrate. Structure−activity relationship studies reveal that replacement of hydrogen on the aromatic ring by chlorine, heteroatoms, or alkyl groups can affect bioactivity, while leaving the aromatic ring of the triazolylthiols unmodified maintains the inhibitory potency. Docking studies reveal that the typical potent inhibitors of NDM-1, 4d and 6c, form stable interactions in the active site of NDM-1, with the triazole bridging Zn1 and Zn2, and the amide interacting with Lys 211 (Lys224).

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Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

Cited by 91 publications

References 55 publications

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Amino Acid Thioester Derivatives: A Highly Promising Scaffold for the Development of Metallo-β-lactamase L1 Inhibitors

Triazolylthioacetamide: A Valid Scaffold for the Development of New Delhi Metallo-β-Lactmase-1 (NDM-1) Inhibitors

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