Kinetics of avibactam inhibition against Class A, C, and D β-lactamases

Abstract

Avibactam is a non-β-lactam β-lactamase inhibitor with a spectrum of activity that includes β-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.

Keywords: Antibiotics; Drug Discovery; Enzyme Inhibitors; Enzyme Kinetics; Mass Spectrometry (MS).

FIGURE 1.

Structures of avibactam (1), avibactam ¹³C-labeled in the urea (2), and avibactam ¹³C-labeled at five carbon positions and ¹⁵N labeled at the N₁ position (3).

FIGURE 2.

A, acylation (left) and deacylation (right) time courses for avibactam reacting with OXA-10. B, acyl enzyme exchange from acyl-OXA-10 to TEM-1. Top, ESI-MS spectrum of acyl-OXA-10 incubated with TEM-1 for 25 h without bicarbonate. The appearance of acyl-TEM-1 in this spectrum may result from free avibactam present in the acyl-OXA-10 sample. Bottom, ESI-MS spectrum of acyl-OXA-10 incubated with TEM-1 for 25 h in the presence of 10 mm sodium bicarbonate.

FIGURE 3.

Time course of deacylation from KPC-2 followed by ESI-MS. A, excerpted time points displaying conversion of acyl-KPC-2 to unacylated KPC-2 and appearance of acyl-98 and acyl-79 peaks. B, plot of the relative fractions of KPC-2 and the three acylated forms over 32 h of incubation at 37 °C.

FIGURE 4.

Structures of acyl enzyme intermediates and avibactam modifications based on the species seen in the deacylation time course. The mass of enzyme (A) is increased by 264 Da upon acylation by avibactam (B). Hydrolytic loss of SO₃ would produce the hydroxylamine and acyl-80 species (C) from which loss of water would produce the imine and acyl-98 species (D). Imine hydrolysis would produce the carbonyl and acyl-97 species (E). Hydrolysis of the carbamate linkage and subsequent decarboxylation would regenerate enzyme A and produce compound 4.

FIGURE 5.

¹³C chemical shifts observed upon incubation of [¹³C]urea avibactam (compound 2) with KPC-2 at 37 °C. The 0 h spectrum was taken before the addition of KPC-2. Chemical shifts from peaks at excerpted time points are shown in Table 5.

FIGURE 6.

Possible mechanisms for avibactam deacylation from KPC-2. The protein environment of acylated avibactam is based on the class A CTX-M-15 x-ray structure (PDB code 4HBU). A, the proposed ring-closing deacylation reaction to re-form intact avibactam in class A enzymes. B, proposed mechanism for sulfactam hydrolysis to produce the hydroxylamine avibactam fragment. C, proposed direct β-elimination of sulfate to produce the imine avibactam fragment.