Structure-Based Design of β-Lactamase Inhibitors. 1. Synthesis and Evaluation of Bridged Monobactams

Abstract

Bridged monobactams are novel, potent, mechanism-based inhibitors of class C beta-lactamases, designed using X-ray crystal structures of the enzymes. They stabilize the acyl-enzyme intermediate by blocking access of water to the enzyme-inhibitor ester bond. Bridged monobactams are selective class C beta-lactamase inhibitors, with half-inhibition constants as low as 10 nM, and are less effective against class A and class B enzymes (half-inhibition constants > 100 microM) because of the different hydrolysis mechanisms in these classes of beta-lactamases. The stability of the acyl-enzyme complexes formed with class C beta-lactamases (half-lives up to 2 days were observed) enabled determination of their crystal structures. The conformation of the inhibitor moiety was close to that predicted by molecular modeling, confirming a simple reaction mechanism, unlike those of known beta-lactamase inhibitors such as clavulanic acid and penam sulfones, which involve secondary rearrangements. Synergy between the bridged monobactams and beta-lactamase-labile antibiotics could be observed when such combinations were tested against strains of Enterobacteriaceae that produce large amounts of class C beta-lactamases. The minimal inhibitory concentration of the antibiotic of more than 64 mg/L could be decreased to 0.25 mg/L in a 1:4 combination with the inhibitor.

Three Decades of β-Lactamase Inhibitors

“…The design of bridged monobactam derivatives was guided by the crystal structure of the monobactam aztreonam in complex with the class C ␤-lactamase from Citrobacter freundii (PDB 1FR1) (152,299). Studies of the structure demonstrated that before deacylation, aztreonam had to rotate around the C-3-C-4 bond to allow a hydrolytic water access to the ester bond.…”

“…Heinze-Krauss et al synthesized and evaluated of a panel of bridged monobactams that exhibited very favorable inhibition of the class C C. freundii and P. aeruginosa ␤-lactamases (IC 50 s of as low as 10 nM) but lower affinities for the class A TEM-3 (IC 50 s of Ͼ100 M) (152). At sufficiently high concentrations, class A ␤-lactamases were acylated rapidly, but the rate of deacylation was higher, leading to high hydrolysis rates and low active-site occupancy.…”

“…At sufficiently high concentrations, class A ␤-lactamases were acylated rapidly, but the rate of deacylation was higher, leading to high hydrolysis rates and low active-site occupancy. Comparison of the crystal structures of class A TEM-1, PC1, the ␤-lactamase from Bacillus licheniformis 749/C, and the class C ␤-lactamase from C. freundii revealed that in class A enzymes, hydrolysis occurs on the opposite side of the bridged monobactam ester by a water molecule activated by hydrogen bond networks not present in class C enzymes (152,166,191,267,299,392). Rotation about the C-3-C-4 bond is less critical to the inhibition mechanism in class A enzymes, and restricting this rotation does less to stabilize the acyl-enzyme.…”

“…Inhibitors that can delay deacylation for longer than 15 to 20 min (k off rate of Յ0.001 s Ϫ1 ) may outlast the bacterial generation time, allowing the partner ␤-lactam time to disrupt the cell wall synthesis necessary for cell division (396,410). For example, the bridged monobactams delay deacylation by limiting the C-3-C-4 rotation necessary for approach of the hydrolytic water in class C enzymes (152,238). Additionally, relatively minor changes introduced by the acylation of meropenem by SHV-1, including minor displacements to active-site residues (overall root mean square deviation [RMSD] of 0.29 Å) and a restructured hydrogen bonding network, lead to persistent complex formation (295).…”

A Dynamic Structure for the Acyl−Enzyme Species of the Antibiotic Aztreonam with the Citrobacter freundii β-Lactamase Revealed by Infrared Spectroscopy and Molecular Dynamics Simulations

“…An additional feature of structural difference is the presence of the sulphonyl group on the ring of aztreonam; the negative charge is proposed to interact with Lys-315 in the Michaelis complex, but not in the acylenzyme species. Additionally, the presence of the C-4 methyl group hinders rotation in the ring-opened form (30). Fig.…”