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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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Overlay of the S02030 inhibitor conformations in all fourADC-7 active sites. The R1 side chain binds similarly in all fourconformations. Differences arise in the placement of the inhibitorR2 group (carboxytriazole) and its interaction with Arg340. In monomersB (purple) and C (salmon), the R2 group adopts similar conformationsand interacts with Arg340, which is observed in a single conformation.In monomers A (white) and D (orange), the conformations of the inhibitorare somewhat different, and alternate and/or multiple conformationsof Arg340 are also observed.
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fig4: Overlay of the S02030 inhibitor conformations in all fourADC-7 active sites. The R1 side chain binds similarly in all fourconformations. Differences arise in the placement of the inhibitorR2 group (carboxytriazole) and its interaction with Arg340. In monomersB (purple) and C (salmon), the R2 group adopts similar conformationsand interacts with Arg340, which is observed in a single conformation.In monomers A (white) and D (orange), the conformations of the inhibitorare somewhat different, and alternate and/or multiple conformationsof Arg340 are also observed.

Mentions: Superposition of each of the monomersreveals that S02030 binds in similar, but not identical, orientationsin each of the sites (Figure 4). The overallconformation of the inhibitors is the same with the R1 and R2 groupsforming favorable intramolecular van der Waals interactions. Othersimilarities include the tetrahedral geometry around the boron atomand the position of the R1 group in the active site that is analogousto where the R1 side chains of β-lactam substrates bind in otherclass C β-lactamases. The O1 hydroxyl group is positioned inthe oxyanion hole and makes a favorable hydrogen bond with the mainchain nitrogen of Ser315 (2.8 Å) and long hydrogen bonds to themain chain nitrogen of Ser64 and main chain oxygen of Ser315 (3.2–3.4Å). The R1 amide group interacts with the side chain nitrogensof Gln120 and Asn152 and the main chain oxygen of Ser315. The thiophenering in the R1 side chain is in an identical conformation in the Band D monomers. In the C monomer the ring is rotated approximately180° around the C12–C13 bond from the orientation observedin A and D monomers. Electron density supports the existence of twoalternate conformations for the ring in the A monomer. One is thesame as the conformation in monomers A and D, and the other differsby an approximate 120° rotation around C12–C13. Regardless,the thiophene ring is not involved in any significant interactionswith the enzyme.


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)

Overlay of the S02030 inhibitor conformations in all fourADC-7 active sites. The R1 side chain binds similarly in all fourconformations. Differences arise in the placement of the inhibitorR2 group (carboxytriazole) and its interaction with Arg340. In monomersB (purple) and C (salmon), the R2 group adopts similar conformationsand interacts with Arg340, which is observed in a single conformation.In monomers A (white) and D (orange), the conformations of the inhibitorare somewhat different, and alternate and/or multiple conformationsof Arg340 are also observed.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Overlay of the S02030 inhibitor conformations in all fourADC-7 active sites. The R1 side chain binds similarly in all fourconformations. Differences arise in the placement of the inhibitorR2 group (carboxytriazole) and its interaction with Arg340. In monomersB (purple) and C (salmon), the R2 group adopts similar conformationsand interacts with Arg340, which is observed in a single conformation.In monomers A (white) and D (orange), the conformations of the inhibitorare somewhat different, and alternate and/or multiple conformationsof Arg340 are also observed.
Mentions: Superposition of each of the monomersreveals that S02030 binds in similar, but not identical, orientationsin each of the sites (Figure 4). The overallconformation of the inhibitors is the same with the R1 and R2 groupsforming favorable intramolecular van der Waals interactions. Othersimilarities include the tetrahedral geometry around the boron atomand the position of the R1 group in the active site that is analogousto where the R1 side chains of β-lactam substrates bind in otherclass C β-lactamases. The O1 hydroxyl group is positioned inthe oxyanion hole and makes a favorable hydrogen bond with the mainchain nitrogen of Ser315 (2.8 Å) and long hydrogen bonds to themain chain nitrogen of Ser64 and main chain oxygen of Ser315 (3.2–3.4Å). The R1 amide group interacts with the side chain nitrogensof Gln120 and Asn152 and the main chain oxygen of Ser315. The thiophenering in the R1 side chain is in an identical conformation in the Band D monomers. In the C monomer the ring is rotated approximately180° around the C12–C13 bond from the orientation observedin A and D monomers. Electron density supports the existence of twoalternate conformations for the ring in the A monomer. One is thesame as the conformation in monomers A and D, and the other differsby an approximate 120° rotation around C12–C13. Regardless,the thiophene ring is not involved in any significant interactionswith the enzyme.

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