Competing off-loading mechanisms of meropenem from an l,d-transpeptidase reduce antibiotic effectiveness

Abstract

The carbapenem family of β-lactam antibiotics displays a remarkably broad spectrum of bactericidal activity, exemplified by meropenem's phase II clinical trial success in patients with pulmonary tuberculosis, a devastating disease for which β-lactam drugs historically have been notoriously ineffective. The discovery and validation of l,d-transpeptidases (Ldts) as critical drug targets of bacterial cell-wall biosynthesis, which are only potently inhibited by the carbapenem and penem structural classes, gave an enzymological basis for the effectiveness of the first antitubercular β-lactams. Decades of study have delineated mechanisms of β-lactam inhibition of their canonical targets, the penicillin-binding proteins; however, open questions remain regarding the mechanisms of Ldt inhibition that underlie programs in drug design, particularly the optimization of kinetic behavior and potency. We have investigated critical features of mycobacterial Ldt inhibition and demonstrate here that the covalent inhibitor meropenem undergoes both reversible reaction and nonhydrolytic off-loading reactions from the cysteine transpeptidase Ldt_Mt2 through a high-energy thioester adduct. Next-generation carbapenem optimization strategies should minimize adduct loss from unproductive mechanisms of Ldt adducts that reduce effective drug concentration.

Keywords: beta-lactam; beta-lactone; drug mode of action; l,d-transpeptidase; meropenem.

Fig. 1.

Key four-membered ring formations. In monocyclic β-lactams, the azetidinone ring is formed in A by a condensation domain while attached as thioesters to peptidyl carrier proteins in the case of nocardicin G (the nocardicins), or (B) a thioesterase as shown in sulfazecin (the monobactams). (C) Nitrocefin re-forms its β-lactam in the active site of Ldt_fm using its conjugated, anionic amine to substitute the enzyme thioester. (D) The obafluorin β-lactone ring is formed on a thioester. The meropenem-derived lactone (E) is formed by attack of the C6 hydroxyethyl group into the ester of class D, serine β-lactamases (SBLs). The condensation domain responsible for monocyclic β-lactam formation is depicted as a green circle, labeled C₅, as it is in the “module” responsible for incorporating the fifth amino acid in nocardicin G biosynthesis. The two peptidyl carrier proteins that deliver nocardicin biosynthetic intermediate to the condensation domain within module 5 are represented as cyan rectangles labeled PCP₄ and PCP_5. The thioesterases used to form the sulfazecin β-lactam and obafluorin β-lactone is a green circle labeled “TE.” The l,d-transpeptidase from E. faecium, Ldt_fm, is an orange circle. The C3′ position of nitrocefin is labeled with a black arrow. The class D β-lactamase is depicted as a green circle labeled “SBL.”

Fig. 2.

Intact protein UPLC–MS data demonstrate Ldt–meropenem adduct lability. (A) Replacement of meropenem on Ldt_Mt2 by faropenem can proceed through three mechanisms: 1) formation of a β-lactone product, 2) hydrolysis of the protein-drug thioester, and/or 3) reclosure of the meropenem β-lactam ring. (B) Ldt_Mt2-meropenem (decarboxylated, 38,425 Da, indicated with one blue circle; intact, 38,469 Da, indicated with two blue circles) was replaced by faropenem (38,172 Da, indicated with three blue circles) after prolonged incubation.

Fig. 3.

Adduct transfer from Ldt_Mt2 to DacB2. (A) The percent bound of Ldt_Mt2 and DacB2 are plotted versus time since admixture of Ldt_Mt2 and DacB2. Each point is a single measurement and curve is composed of straight lines, connecting contiguous points. (B) Spectra of DacB2 (Left) collected at time points after adding Ldt_Mt2–meropenem show apo-DacB2 (27,435 Da, single blue dot) gradually decrease as the two DacB2–meropenem species (27,774 Da, decarboxylated, two blue dots; 27,818 Da, intact, three blue dots) increase. Spectra of Ldt_Mt2 (Right) show apo-Ldt_Mt2 (single blue triangle, 38,086 Da) appear as Ldt_Mt2–meropenem (38,425 Da, decarboxylated, two triangles; 38,469 Da, intact, three triangles) is consumed.

Fig. 4.

Incubation of Ldt_Mt2 with meropenem results in a β-lactone product. (A) UPLC–MS total ion chromatograms demonstrate appearance of a lactone product with a later retention time than meropenem, as well as an additional hydrolysis peak, assigned as the less stable (2R) diastereomer of hydrolyzed meropenem. Compounds found under each peak are labeled with blue dots. (B) ¹H-NMR analysis of meropenem, Top traces, and a meropenem lactone product, Bottom traces. The β-lactone C8 hydrogen, shown in red, gives an apparent pentet with a coupling constant of J = 6.3 Hz. The sidechain amide methyl groups, in orange, appear in the region of δ2.9 to 3.1 for both meropenem and meropenem lactone. The methyl doublets are drawn in blue. The hydroxyethyl methyl group peak is seen at ∼1.32 ppm in meropenem and is downfield at ∼1.68 ppm in the meropenem lactone product, while the 1-β-methyl group moves upfield from ∼1.24 ppm in meropenem to ∼1.06 ppm in the meropenem lactone product.