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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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Stereoviewof the ADC-7/S02030 complex. (A) The 2Fo – Fc electron density map iscontoured at 1.0 σ and colored gray. (B) Hydrogen bonding interactionsobserved between ADC-7 and the boronic acid inhibitor S02030. Theactive site shown is the C monomer (the inhibitor in the B monomerbinds similarly). Hydrogen bonds and ionic interactions are shownas yellow dashed lines and represent distances between 2.6–3.3Å. Water molecules are indicated with red spheres. Carbon atomsof the active site residues are colored salmon, oxygens red, nitrogensblue, sulfurs yellow, and boron is light pink.
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fig3: Stereoviewof the ADC-7/S02030 complex. (A) The 2Fo – Fc electron density map iscontoured at 1.0 σ and colored gray. (B) Hydrogen bonding interactionsobserved between ADC-7 and the boronic acid inhibitor S02030. Theactive site shown is the C monomer (the inhibitor in the B monomerbinds similarly). Hydrogen bonds and ionic interactions are shownas yellow dashed lines and represent distances between 2.6–3.3Å. Water molecules are indicated with red spheres. Carbon atomsof the active site residues are colored salmon, oxygens red, nitrogensblue, sulfurs yellow, and boron is light pink.

Mentions: To better understand the structuralbasis for inhibition of ADC-7 by BATSIs, the X-ray crystal structureof the ADC-7/S02030 complex was determined to 2.03 Å resolution(Figure 3; Table 4).The complex was obtained by soaking the inhibitor into preformed ADC-7crystals. The space group was P21, withfour molecules in the asymmetric unit. Inspection of the initial Fo – Fc mapscontoured at 3σ showed unambiguous electron density for theinhibitor bound in the active site of all four molecules. The densitywas contiguous with the Oγ atom of Ser64, suggesting that theinhibitor was covalently attached to the catalytic serine residue.The inhibitor molecule was built into the density in all active sitesand further refined with Refmac5. 2Fo – Fc maps calculated from the refined model confirmedthe covalent attachment to Ser64. A tetrahedral geometry was observedaround the boron atom of the inhibitor. In addition to the four inhibitors,the final model of the complex contained 1,421 protein residues and668 water molecules. The final R and Rfree were 19.6% and 24.0%, respectively, and the coordinatesand structure factors have been deposited in the Protein Data Bankas 4U0X.


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)

Stereoviewof the ADC-7/S02030 complex. (A) The 2Fo – Fc electron density map iscontoured at 1.0 σ and colored gray. (B) Hydrogen bonding interactionsobserved between ADC-7 and the boronic acid inhibitor S02030. Theactive site shown is the C monomer (the inhibitor in the B monomerbinds similarly). Hydrogen bonds and ionic interactions are shownas yellow dashed lines and represent distances between 2.6–3.3Å. Water molecules are indicated with red spheres. Carbon atomsof the active site residues are colored salmon, oxygens red, nitrogensblue, sulfurs yellow, and boron is light pink.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Stereoviewof the ADC-7/S02030 complex. (A) The 2Fo – Fc electron density map iscontoured at 1.0 σ and colored gray. (B) Hydrogen bonding interactionsobserved between ADC-7 and the boronic acid inhibitor S02030. Theactive site shown is the C monomer (the inhibitor in the B monomerbinds similarly). Hydrogen bonds and ionic interactions are shownas yellow dashed lines and represent distances between 2.6–3.3Å. Water molecules are indicated with red spheres. Carbon atomsof the active site residues are colored salmon, oxygens red, nitrogensblue, sulfurs yellow, and boron is light pink.
Mentions: To better understand the structuralbasis for inhibition of ADC-7 by BATSIs, the X-ray crystal structureof the ADC-7/S02030 complex was determined to 2.03 Å resolution(Figure 3; Table 4).The complex was obtained by soaking the inhibitor into preformed ADC-7crystals. The space group was P21, withfour molecules in the asymmetric unit. Inspection of the initial Fo – Fc mapscontoured at 3σ showed unambiguous electron density for theinhibitor bound in the active site of all four molecules. The densitywas contiguous with the Oγ atom of Ser64, suggesting that theinhibitor was covalently attached to the catalytic serine residue.The inhibitor molecule was built into the density in all active sitesand further refined with Refmac5. 2Fo – Fc maps calculated from the refined model confirmedthe covalent attachment to Ser64. A tetrahedral geometry was observedaround the boron atom of the inhibitor. In addition to the four inhibitors,the final model of the complex contained 1,421 protein residues and668 water molecules. The final R and Rfree were 19.6% and 24.0%, respectively, and the coordinatesand structure factors have been deposited in the Protein Data Bankas 4U0X.

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