Energetic, structural, and antimicrobial analyses of β-lactam side chain recognition by β-lactamases

Caselli, Emilia; Powers, R.A.; Blasczcak, Larry C; Wu, C. Y. E.; Prati, Fabio; Shoichet, Brian K.

doi:10.1016/s1074-5521(00)00052-1

Cited by 82 publications

(146 citation statements)

References 38 publications

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“…these residues, as observed in the complexes of AmpC with 6, 8, and 12 20,24,26 (Table 1; Figure 3C). In the complexes of AmpC with the -lactams 10 and 13, the carbacephem and oxacephem rings, respectively, interact with the leucines.…”

Section: Resultssupporting

confidence: 64%

“…GRID and X-SITE calculations were performed on the structures of apo AmpC (molecule 2 of PDB entry 1KE4) and complexed AmpC (molecule 2 of AmpC/8; PDB entry 1FSY). 24 The complexed and apo forms of AmpC were used to observe any differences that might arise in the predictions due to the starting model. Additionally, they represent two conformations of the flexible residue Gln120.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, the structure of AmpC has been determined in complex with inhibitors such as -lactams, 4,[20][21][22] transition state analogues, 23,24 and arylboronic acids, 6,25,26 the leads for which were known before the first structures were determined. 27 There are now 18 inhibitor complex structures with class C -lactamases that have been published.…”

Section: Introductionmentioning

confidence: 99%

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Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

2002

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-Lactamases are the most widespread resistance mechanism to -lactam antibiotics and are an increasing menace to public health. Several -lactamase structures have been determined, making this enzyme an attractive target for structure-based drug design. To facilitate inhibitor design for the class C -lactamase AmpC, binding site "hot spots" on the enzyme were identified using experimental and computational approaches. Experimentally, X-ray crystal structures of AmpC in complexes with four boronic acid inhibitors and a higher resolution (1.72 Å) native apo structure were determined. Along with previously determined structures of AmpC in complexes with five other boronic acid inhibitors and four -lactams, consensus binding sites were identified. Computationally, the programs GRID, MCSS, and X-SITE were used to predict potential binding site hot spots on AmpC. Several consensus binding sites were identified from the crystal structures. An amide recognition site was identified by the interaction between the carbonyl oxygen in the R1 side chain of -lactams and the atom Nδ2 of the conserved Asn152. Surprisingly, this site also recognizes the aryl rings of arylboronic acids, appearing to form quadrupole-dipole interactions with Asn152. The highly conserved "oxyanion" hole defines a site that recognizes both carbonyl and hydroxyl groups. A hydroxyl binding site was identified by the O2 hydroxyl in the boronic acids, which hydrogen bonds with Tyr150 and a conserved water. A hydrophobic site is formed by Leu119 and Leu293. A carboxylate binding site was identified by the ubiquitous C3(4) carboxylate of the -lactams, which interacts with Asn346 and Arg349. Four water sites were identified by ordered waters observed in most of the structures; these waters form extensive hydrogen-bonding networks with AmpC and occasionally the ligand. Predictions by the computational programs showed some correlation with the experimentally observed binding sites. Several sites were not predicted, but novel binding sites were suggested. Taken together, a map of binding site hot spots found on AmpC, along with information on the functionality recognized at each site, was constructed. This map may be useful for structure-based inhibitor design against AmpC.

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Section: Resultssupporting

confidence: 64%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

2002

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“…Boronic acids, as non-lactam transition-state analogs, are impervious to such mutants and are often transparent to other β-lactam resistance mechanisms, such as β-lactamase upregulation. Using β-lactam functional groups on a boronic acid scaffold, we rapidly optimized an initial class of boronic acids to midnanomolar affinity (6,7), and with further derivatization to 1 nM affinity against the class C β-lactamase AmpC (8). Notwithstanding their high affinity, these compounds had relatively modest activity in bacterial cell culture.…”

mentioning

confidence: 99%

Fragment-guided design of subnanomolar β-lactamase inhibitors active in vivo

Eidam¹,

Romagnoli

Dalmasso

et al. 2012

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

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Fragment-based design was used to guide derivatization of a lead series of β-lactamase inhibitors that had heretofore resisted optimization for in vivo activity. X-ray structures of fragments overlaid with the lead suggested new, unanticipated functionality and points of attachment. Synthesis of three derivatives improved affinity over 20-fold and improved efficacy in cell culture. Crystal structures were consistent with the fragment-based design, enabling further optimization to a K i of 50 pM, a 500-fold improvement that required the synthesis of only six derivatives. One of these, compound 5, was tested in mice. Whereas cefotaxime alone failed to cure mice infected with β-lactamase-expressing Escherichia coli, 65% were cleared of infection when treated with a cefotaxime:5 combination. Fragment complexes offer a path around design hurdles, even for advanced molecules; the series described here may provide leads to overcome β-lactamase-based resistance, a key clinical challenge.antibiotic resistance | antimicrobial | drug discovery | structure-based | boronic acid

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“…Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, protein dynamics, and inhibitor binding (35,(37)(38)(39)(40)(41)(42).…”

mentioning

confidence: 99%

Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

Garrity

Pauff

Crowder

2004

Journal of Biological Chemistry

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A structural feature shared by the metallo-␤-lactamases is a flexible loop of amino acids that extends over their active sites and that has been proposed to move during the catalytic cycle of the enzymes, clamping down on substrate. To probe the movement of this loop (residues 152-164), a site-directed mutant of metallo-␤-lactamase L1 was engineered that contained a Trp residue on the loop to serve as a fluorescent probe. It was necessary first, however, to evaluate the contribution of each native Trp residue to the fluorescence changes observed during the catalytic cycle of wild-type L1. Five site-directed mutants of L1 (W39F, W53F, W204F, W206F, and W269F) were prepared and characterized using metal analyses, CD spectroscopy, steady-state kinetics, stopped-flow fluorescence, and fluorescence titrations. All mutants retained the wild-type tertiary structure and bound Zn(II) at levels comparable with wild type and exhibited only slight (<10-fold) decreases in k cat values as compared with wild-type L1 for all substrates tested. Fluorescence studies revealed a single mutant, W39F, to be void of the fluorescence changes observed with wild-type L1 during substrate binding and catalysis. Using W39F as a template, a Trp residue was added to the flexile loop over the active site of L1, to generate the double mutant, W39F/D160W. This double mutant retained all the structural and kinetic characteristics of wild-type L1. Stopped-flow fluorescence and rapid-scanning UV-visible studies revealed the motion of the loop (k obs ‫؍‬ 27 ؎ 2 s ؊1 ) to be similar to the formation rate of a reaction intermediate (k obs ‫؍‬ 25 ؎ 2 s ؊1 ).

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Energetic, structural, and antimicrobial analyses of β-lactam side chain recognition by β-lactamases

Cited by 82 publications

References 38 publications

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

Fragment-guided design of subnanomolar β-lactamase inhibitors active in vivo

Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

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