scholarly journals Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

2006 ◽  
Vol 50 (7) ◽  
pp. 2516-2521 ◽  
Author(s):  
Eric Sauvage ◽  
Eveline Fonzé ◽  
Birgit Quinting ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Broad Spectrum ◽  
Active Site ◽  
Bacterial Resistance ◽  
Specific Activity ◽  
Structural Basis ◽  
Mycobacterium Fortuitum ◽  
Bacterial Strains ◽  
Class A ◽  
Extended Spectrum

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

scholarly journals In Vitro Activity of LK-157, a Novel Tricyclic Carbapenem As Broad-Spectrum β-Lactamase Inhibitor

2008 ◽  
Vol 53 (2) ◽  
pp. 505-511 ◽  
Author(s):  
Susanne Paukner ◽  
Lars Hesse ◽  
Andrej Preželj ◽  
Tomaž Šolmajer ◽  
Uroš Urleb
Keyword(s):  
Clavulanic Acid ◽  
Broad Spectrum ◽  
Clinical Settings ◽  
In Vitro Activity ◽  
Bacterial Strains ◽  
Class A ◽  
Class C ◽  
Further Development ◽  
Lactamase Inhibitor

ABSTRACT LK-157 is a novel tricyclic carbapenem with potent activity against class A and class C β-lactamases. When tested against the purified TEM-1 and SHV-1 enzymes, LK-157 exhibited 50% inhibitory concentrations (IC50s) in the ranges of the clavulanic acid and tazobactam IC50s (55 nM and 151 nM, respectively). Moreover, LK-157 significantly inhibited AmpC β-lactamase (IC50, 62 nM), as LK-157 was >2,000-fold more potent than clavulanic acid and approximately 28-fold more active than tazobactam. The in vitro activities of LK-157 in combination with amoxicillin, piperacillin, ceftazidime, cefotaxime, ceftriaxone, cefepime, cefpirome, and aztreonam against an array of Ambler class A (TEM-, SHV-, CTX-M-, KPC-, PER-, BRO-, and PC-type)- and class C-producing bacterial strains derived from clinical settings were evaluated in synergism experiments and compared with those of clavulanic acid, tazobactam, and sulbactam. In vitro MICs against ESBL-producing strains (except CTX-M-containing strains) were reduced 2- to >256-fold, and those against AmpC-producing strains were reduced even up to >32-fold. The lowest MICs (≤0.025 to 1.6 μg/ml) were observed for the combination of cefepime and cefpirome with a constant LK-157 concentration of 4 μg/ml, thus raising an interest for further development. LK-157 proved to be a potent β-lactamase inhibitor, combining activity against class A and class C β-lactamases, which is an absolute necessity for use in the clinical setting due to the worldwide increasing prevalence of bacterial strains resistant to β-lactam antibiotics.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals Structural Basis for Imipenem Inhibition of Class C β-Lactamases

2002 ◽  
Vol 46 (12) ◽  
pp. 3978-3980 ◽  
Author(s):  
Beth M. Beadle ◽  
Brian K. Shoichet
Keyword(s):  
Crystal Structure ◽  
Carbonyl Oxygen ◽  
Structural Basis ◽  
Enzyme Complex ◽  
X Ray ◽  
Class A ◽  
Lactam Carbonyl ◽  
Class C ◽  
Expected Position

ABSTRACT To determine how imipenem inhibits the class C β-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 Å. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180° compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

scholarly journals Structural Basis and Binding Kinetics of Vaborbactam in Class A β-Lactamase Inhibition

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Orville A. Pemberton ◽  
Ruslan Tsivkovski ◽  
Maxim Totrov ◽  
Olga Lomovskaya ◽  
Yu Chen
Keyword(s):  
Crystal Structure ◽  
Covalent Bond ◽  
Binding Mode ◽  
Binding Kinetics ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Class A ◽  
Reversible Covalent ◽  
The Stability ◽  
M 14

ABSTRACT Class A β-lactamases are a major cause of β-lactam resistance in Gram-negative bacteria. The recently FDA-approved cyclic boronate vaborbactam is a reversible covalent inhibitor of class A β-lactamases, including CTX-M extended-spectrum β-lactamase and KPC carbapenemase, both frequently observed in the clinic. Intriguingly, vaborbactam displayed different binding kinetics and cell-based activity for these two enzymes, despite their similarity. A 1.0-Å crystal structure of CTX-M-14 demonstrated that two catalytic residues, K73 and E166, are positively charged and neutral, respectively. Meanwhile, a 1.25-Å crystal structure of KPC-2 revealed a more compact binding mode of vaborbactam versus CTX-M-14, as well as alternative conformations of W105. Together with kinetic analysis of W105 mutants, the structures demonstrate the influence of this residue and the unusual conformation of the β3 strand on the inactivation rate, as well as the stability of the reversible covalent bond with S70. Furthermore, studies of KPC-2 S130G mutant shed light on the different impacts of S130 in the binding of vaborbactam versus avibactam, another recently approved β-lactamase inhibitor. Taken together, these new data provide valuable insights into the inhibition mechanism of vaborbactam and future development of cyclic boronate inhibitors.

scholarly journals GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C β-Lactamase

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Jung-Hyun Na ◽  
Young Jun An ◽  
Sun-Shin Cha
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Phosphate Group ◽  
The Other ◽  
Extended Spectrum ◽  
Competitive Inhibitors ◽  
Class C

ABSTRACT Nucleotides were effective in inhibiting the class C β-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 μM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

scholarly journals Microbiological characterization of VNRX-5236: a broad spectrum β-lactamase inhibitor for rescue of the orally bioavailable cephalosporin ceftibuten as a carbapenem-sparing agent against strains of Enterobacterales expressing extended spectrum β-lactamases and serine carbapenemases.

2021 ◽  
Author(s):  
Cassandra L. Chatwin ◽  
Jodie C. Hamrick ◽  
Robert E. L. Trout ◽  
Cullen L. Myers ◽  
Susan M. Cusick ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Clinical Isolates ◽  
Class A ◽  
Extended Spectrum ◽  
Oral Agents ◽  
Class C ◽  
Orally Bioavailable ◽  
Lactamase Inhibitor

There is an urgent need for oral agents to combat resistant gram-negative pathogens. Here we describe the characterization of VNRX-5236, a broad-spectrum boronic acid β-lactamase inhibitor (BLI) and its orally bioavailable etzadroxil prodrug, VNRX-7145. VNRX-7145 is being developed in combination with ceftibuten, an oral cephalosporin, to combat strains of Enterobacterales expressing extended spectrum β-lactamases (ESBLs) and serine carbapenemases. VNRX-5236 is a reversible covalent inhibitor of serine β-lactamases, with inactivation efficiencies on the order of 104 M−1. sec−1, and prolonged active site residence times (t1/2, 5 to 46 min). The spectrum of inhibition includes Ambler class A ESBLs, class C cephalosporinases, and class A and D carbapenemases (KPC and OXA-48, respectively). Rescue of ceftibuten by VNRX-5236 (fixed at 4 μg/mL) in isogenic strains of E. coli expressing class A, C or D β-lactamases demonstrated an expanded spectrum of activity relative to oral comparators including investigational penems, sulopenem and tebipenem. VNRX-5236 rescued ceftibuten activity in clinical isolates of Enterobacterales expressing ESBLs (MIC90 = 0.25 μg/mL), KPCs (MIC90 = 1 μg/mL), class C cephalosporinases (MIC90 = 1 μg/mL) and OXA-48-type carbapenemases (MIC90 = 1 μg/mL). Frequency of resistance studies demonstrated a low propensity for recovery of resistant variants at 4× the MIC of the ceftibuten/VNRX-5236 combination. In vivo, whereas ceftibuten alone was ineffective (ED50, >128 mg/kg), ceftibuten/VNRX-7145 administered orally protected mice from lethal septicemia caused by K. pneumoniae producing KPC carbapenemase (ED50, 12.9 mg/kg). The data demonstrate potent, broad-spectrum rescue of ceftibuten activity by VNRX-5236 in clinical isolates of cephalosporin-resistant and carbapenem-resistant Enterobacterales.

scholarly journals In Vitro Activities of the Potent, Broad-Spectrum Carbapenem MK-0826 (L-749,345) against Broad-Spectrum β-Lactamase-and Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae and Escherichia coli Clinical Isolates

1999 ◽  
Vol 43 (5) ◽  
pp. 1170-1176 ◽  
Author(s):  
Joyce Kohler ◽  
Karen L. Dorso ◽  
Katherine Young ◽  
Gail G. Hammond ◽  
Hugh Rosen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Broad Spectrum ◽  
Bacterial Resistance ◽  
Clinical Isolates ◽  
Inoculum Level ◽  
E Coli ◽  
Extended Spectrum ◽  
Group 1

ABSTRACT An important mechanism of bacterial resistance to β-lactam antibiotics is inactivation by β-lactam-hydrolyzing enzymes (β-lactamases). The evolution of the extended-spectrum β-lactamases (ESBLs) is associated with extensive use of β-lactam antibiotics, particularly cephalosporins, and is a serious threat to therapeutic efficacy. ESBLs and broad-spectrum β-lactamases (BDSBLs) are plasmid-mediated class A enzymes produced by gram-negative pathogens, principallyEscherichia coli and Klebsiella pneumoniae. MK-0826 was highly potent against all ESBL- and BDSBL-producingK. pneumoniae and E. coli clinical isolates tested (MIC range, 0.008 to 0.12 μg/ml). In E. coli, this activity was associated with high-affinity binding to penicillin-binding proteins 2 and 3. When the inoculum level was increased 10-fold, increasing the amount of β-lactamase present, the MK-0826 MIC range increased to 0.008 to 1 μg/ml. By comparison, similar observations were made with meropenem while imipenem MICs were usually less affected. Not surprisingly, MIC increases with noncarbapenem β-lactams were generally substantially greater, resulting in resistance in many cases. E. coli strains that produce chromosomal (Bush group 1) β-lactamase served as controls. All three carbapenems were subject to an inoculum effect with the majority of the BDSBL- and ESBL-producers but not the Bush group 1 strains, implying some effect of the plasmid-borne enzymes on potency. Importantly, MK-0826 MICs remained at or below 1 μg/ml under all test conditions.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C β-Lactamase from Escherichia coli

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 899 ◽  
Author(s):  
Pauline A. Lang ◽  
Anete Parkova ◽  
Thomas M. Leissing ◽  
Karina Calvopiña ◽  
Ricky Cain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
High Energy ◽  
Structural Basis ◽  
Side Chain ◽  
Class A ◽  
Dual Action ◽  
Class C ◽  
Tract Infections ◽  
Site Binding

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

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