Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate

Abstract

Members of the beta-lactam class of antibiotics, which inhibit the bacterial d,d-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A beta-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum beta-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved beta-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.

Figure 1. Catalytic mechanism of Class A β-lactamases

Dual participation of Lys73 and Glu166 for activation of Ser70 (A), formation of the covalent acyl-enzyme (B), attack of the ester bond by the activated water molecule (C).

Figure 2. Inhibition profiles of M. tuberculosis BlaC.

Clavulanate (A), tazobactam (B) and sulbactam (C) structures are shown on the right. Times courses are shown in left panels. After fitting to equation 2, values of k_iso are plotted against inhibitor concentration on the right panels. For sulbactam, initial velocities were determined at 4μM (■), 3μM (△), 2μM (▼), 1μM (○) and 0μM (●) sulbactam concentrations.

Figure 3. FTICR Mass spectra of BlaC acyl adducts with clavulanate

Only the 25+ charge states are shown. Free enzyme is shown in top panel, with a experimental mass of 28784.475. Three minutes after the addition of clavulanate (middle panel), three enzyme forms are formed: peak A=28854.70, Δm= +70.22; peak B=28920.75, Δm= +136.27; and peak C=28939.52, Δm= +154.40. The bottom panel shows that all three peaks remains after one hour incubation.

Figure 4. Recovery of BlaC activity after incubation with β-lactamase inhibitors

Enzyme (20 μM) was incubated with 100 μM sulbactam (▲), 100 μM tazobactam (■) or 100 μM clavulanate (●), and activity was determined at the indicated times.

Figure 5. Proposed mechanism of BlaC inhibition by clavulanate

After acylation of the clavulanate molecule, rearrangement and decarboxylation lead to the imine (C), which undergoes loss of a water molecule (B), or hydrolysis to form the propionaldehyde (A).