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 Cyclic Boronates Inhibit All Classes of β-Lactamases

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Samuel T. Cahill ◽  
Ricky Cain ◽  
David Y. Wang ◽  
Christopher T. Lohans ◽  
David W. Wareham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacterial Infections ◽  
Binding Mode ◽  
Gram Negative ◽  
Content Type ◽  
Class A ◽  
Class D ◽  
Dual Action ◽  
The Common ◽  
Continued Use

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

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.

Structural basis for DNA-mediated allosteric regulation facilitated by the AAA+module of Lon protease

2014 ◽  
Vol 70 (2) ◽  
pp. 218-230 ◽  
Author(s):  
Alan Yueh-Luen Lee ◽  
Yu-Da Chen ◽  
Yu-Yung Chang ◽  
Yu-Ching Lin ◽  
Chi-Fon Chang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Electrostatic Interactions ◽  
Allosteric Regulation ◽  
Binding Mode ◽  
Negative Regulation ◽  
Enzymatic Activities ◽  
Structural Basis ◽  
Lon Protease ◽  
Arginine Residues

Lon belongs to a unique group of AAA+proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease fromBrevibacillus thermoruber(Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain–domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

scholarly journals Bidimensional Polyiodide Netting Stabilized by a Cu(II) Macrocyclic Complex

Inorganics ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 12
Author(s):  
Matteo Savastano ◽  
Valeria Monini ◽  
Carla Bazzicalupi ◽  
Antonio Bianchi
Keyword(s):  
Crystal Structure ◽  
Crystal Engineering ◽  
Ternary Complex ◽  
Xrd Analysis ◽  
Binding Mode ◽  
Macrocyclic Complex ◽  
Redox Processes ◽  
Complex Stability ◽  
Formation Kinetic ◽  
The Stability

Iodine-dense polyiodide phases are interesting materials for a number of potential uses, including batteries and solid-state conductors. The incorporation of transition metal cations is considered a promising way to enhance the stability, tune the properties, and influence the architecture of polyiodides. However, several interesting metals, including Cu(II), may suffer redox processes, which generally make them not compatible with the I2/I− redox couple. Herein L, a simple derivative of cyclen, is proposed as a Cu(II) ligand capable of protecting the +2 oxidation state of the metal even in the presence of polyiodides. With a step by step approach, we report the crystal structure of free L; then we present spectrophotometric verification of Cu(II) complex stability, stoichiometry, and formation kinetic in DMF solution, together with Cu(II) binding mode elucidation via XRD analysis of [Cu(L)Cl]ClO4∙CH3CN crystals; afterwards, the stability of the CuL complex in the presence of I− is demonstrated in DMF solution, where the formation of a Cu:L:I− ternary complex, rather than reduction to Cu(I), is observed; lastly, polyiodide crystals are prepared, affording the [Cu(L)I]2I3I5 crystal structure. This layered structure is highly peculiar due to its chiral arrangement, opening further perspective for the crystal engineering of polyiodide phases.

scholarly journals Naphthoquinones as Covalent Reversible Inhibitors of Cysteine Proteases—Studies on Inhibition Mechanism and Kinetics

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2064 ◽  
Author(s):  
Philipp Klein ◽  
Fabian Barthels ◽  
Patrick Johe ◽  
Annika Wagner ◽  
Stefan Tenzer ◽  
...  
Keyword(s):  
Free Acid ◽  
Theoretical Calculations ◽  
Covalent Binding ◽  
Cysteine Proteases ◽  
Binding Mode ◽  
Benzyl Ester ◽  
Inhibition Mechanism ◽  
Reversible Inhibitors ◽  
Electron Withdrawing Group ◽  
Reversible Covalent

The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.

scholarly journals Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4

2016 ◽  
Vol 114 (3) ◽  
pp. 486-491 ◽  
Author(s):  
Irina F. Sevrioukova ◽  
Thomas L. Poulos
Keyword(s):  
Crystal Structure ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Information ◽  
Binding Mode ◽  
Structural Basis ◽  
Cytochrome P450 3A4 ◽  
Human Cytochrome ◽  
Cyp3a4 Substrate

Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 Å crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.

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 Structure of the complex of phosphorylated liver kinase B1 and 14-3-3ζ

2017 ◽  
Vol 73 (4) ◽  
pp. 196-201 ◽  
Author(s):  
Yongjian Lu ◽  
Sheng Ding ◽  
Ruiqing Zhou ◽  
Jianyong Wu
Keyword(s):  
Crystal Structure ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Binding Mode ◽  
Tumour Suppressor ◽  
Structural Basis ◽  
Dependent Manner ◽  
Multiple Cancers ◽  
Physiological Processes ◽  
Liver Kinase B1

The serine/threonine protein kinase liver kinase B1 (LKB1) is a tumour suppressor and plays important roles in development and metabolism. It phosphorylates AMPK and AMPK-related kinases to regulate multiple physiological processes. Mutations in LKB1 often occur in multiple cancers. LKB1 can be suppressed by 14-3-3 proteins in a phosphorylation-dependent manner. Previously, the structure of a 14-3-3ζ–LKB1 fusion protein has been reported, revealing a phosphorylation-independent binding mode of LKB1 to 14-3-3 proteins. Here, the crystal structure of phosphorylated LKB1 peptide in complex with 14-3-3ζ was solved, which provides a structural basis for the phosphorylation-dependent recognition of LKB1 by 14-3-3 proteins.

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