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Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

Powers RA, Swanson HC, Taracila MA, Florek NW, Romagnoli C, Caselli E, Prati F, Bonomo RA, Wallar BJ - Biochemistry (2014)

Bottom Line: Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase.In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams.The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Grand Valley State University , 1 Campus Drive, Allendale, Michigan 49401, United States.

ABSTRACT
β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

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Stereoview of the ADC-7 active site. (A) The2Fo – Fc electron density map is contoured at 1.0 σ and colored gray.(B) Hydrogen bonding interactions observed between active site residues,water molecules, and phosphate ion. Hydrogen bonds are shown as yellowdashed lines and represent distances between 2.4–3.2 Å.Water molecules are indicated with red spheres. Carbon atoms of theactive site residues are colored green, oxygens red, and nitrogensblue. The phosphorus of the phosphate is colored orange. This andall subsequent figures were made with PyMOL.59
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fig2: Stereoview of the ADC-7 active site. (A) The2Fo – Fc electron density map is contoured at 1.0 σ and colored gray.(B) Hydrogen bonding interactions observed between active site residues,water molecules, and phosphate ion. Hydrogen bonds are shown as yellowdashed lines and represent distances between 2.4–3.2 Å.Water molecules are indicated with red spheres. Carbon atoms of theactive site residues are colored green, oxygens red, and nitrogensblue. The phosphorus of the phosphate is colored orange. This andall subsequent figures were made with PyMOL.59

Mentions: The structure of apo ADC-7 was determined to 1.73 Å resolution(Figure 2; Table 4).ADC-7 crystallized in the P1 space group with eightmolecules in the asymmetric unit. A phosphate ion was observed inthe active sites of each of the eight monomers of the asymmetric unit(Figure 2). The quality of the final modelwas evaluated with MolProbity.50 In thismodel, 97.1% of all residues were in the favored region of the Ramachandranplot, and 99.9% were in the allowed region. The final R and Rfree were 19.6% and 24.0%, respectively,and the coordinates and structure factors have been deposited in theProtein Data Bank as 4U0T. The overall fold of ADC-7 is a mixed α/β structureand resembles that observed in other class C β-lactamases, includingthe recently determined structure of ADC-1.51


Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

Powers RA, Swanson HC, Taracila MA, Florek NW, Romagnoli C, Caselli E, Prati F, Bonomo RA, Wallar BJ - Biochemistry (2014)

Stereoview of the ADC-7 active site. (A) The2Fo – Fc electron density map is contoured at 1.0 σ and colored gray.(B) Hydrogen bonding interactions observed between active site residues,water molecules, and phosphate ion. Hydrogen bonds are shown as yellowdashed lines and represent distances between 2.4–3.2 Å.Water molecules are indicated with red spheres. Carbon atoms of theactive site residues are colored green, oxygens red, and nitrogensblue. The phosphorus of the phosphate is colored orange. This andall subsequent figures were made with PyMOL.59
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263437&req=5

fig2: Stereoview of the ADC-7 active site. (A) The2Fo – Fc electron density map is contoured at 1.0 σ and colored gray.(B) Hydrogen bonding interactions observed between active site residues,water molecules, and phosphate ion. Hydrogen bonds are shown as yellowdashed lines and represent distances between 2.4–3.2 Å.Water molecules are indicated with red spheres. Carbon atoms of theactive site residues are colored green, oxygens red, and nitrogensblue. The phosphorus of the phosphate is colored orange. This andall subsequent figures were made with PyMOL.59
Mentions: The structure of apo ADC-7 was determined to 1.73 Å resolution(Figure 2; Table 4).ADC-7 crystallized in the P1 space group with eightmolecules in the asymmetric unit. A phosphate ion was observed inthe active sites of each of the eight monomers of the asymmetric unit(Figure 2). The quality of the final modelwas evaluated with MolProbity.50 In thismodel, 97.1% of all residues were in the favored region of the Ramachandranplot, and 99.9% were in the allowed region. The final R and Rfree were 19.6% and 24.0%, respectively,and the coordinates and structure factors have been deposited in theProtein Data Bank as 4U0T. The overall fold of ADC-7 is a mixed α/β structureand resembles that observed in other class C β-lactamases, includingthe recently determined structure of ADC-1.51

Bottom Line: Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase.In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams.The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Grand Valley State University , 1 Campus Drive, Allendale, Michigan 49401, United States.

ABSTRACT
β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

Show MeSH