Faropenem reacts with serine and metallo-β-lactamases to give multiple products

Abstract

Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.

Keywords: Antimicrobial resistance; Carbapenems; Metallo-β-lactamases; Penems; Serine-β-lactamases; β-Lactams.

Figure 1. β-Lactam hydrolysis by serine and metallo-β-lactamase.

(A) SBL catalysis proceeds via an acyl-enzyme complex formed after being hydrolysed via a transient tetrahedral intermediate. In MBL catalysis, a zinc ion activated water molecule (that bridges the two Zn ions in the resting states of B1 and B3 MBLs), reacts with the β-lactam ring. Hydrolysis also proceeds via a transient tetrahedral intermediate and a hydrolysed intermediate stabilised through interactions with the Zn ions[6]. (B) Classes of clinically used β-lactam antibiotics.

Figure 2. Carbapenem hydrolysis by β-lactamases.

SBL and MBL catalysed carbapenem hydrolysis is proposed to result (predominantly) in the initial formation of an enamine (Δ²-pyrroline) that isomerises to give [(2R)- and (2S)-Δ¹-imine] products[10,13]. Various isomeric imine / enamine forms of reacted carbapenems have been observed crystallographically at transpeptidase/SBL/MBL active sites. In the case of the class D SBLs, the acyl-enzyme intermediate can also react to give lactone products.

Figure 3. Potential reaction products of β-lactamase catalysed faropenem hydrolysis.

Note the possibility of isomer formation, e.g. epimerisation at C-5.

Figure 4. Faropenem derived complexes in the β-lactamase active sites as defined by electron density maps.

Views from the β-lactamase active sites, with the F_o-F_c electron density (green mesh, contoured at 3σ) calculated from the final model after removal of the ligand. (A) VIM-2, in the I222 space group (green, PDB code 7A5Z). (B) VIM-2 in the C2 space group (light red, PDB code 7A60). (C) L1 (pink, PDB code 7A63) and (D) KPC-2 (cyan, PDB code 7A61). The deacylating water (DW) is shown as a red sphere. Note the different conformations of the β-lactam derived carboxylate and hydroxyethyl groups in L1 compared to VIM-2 and the presence of the zinc bridging water with L1 (Wat, red sphere), but not VIM-2. No density was observed for the hydroxyl of the fragmented THF ring so it was omitted from the final models of the VIM-2 and KPC-2 complexes.

Figure 5. Interactions of the faropenem-derived complexes with serine- and metallo-β-lactamase active sites.

Protein residues are in light grey; faropenem-derived atoms are coloured as in Figure 4. Zinc ions are shown as grey spheres and the catalytic water (Wat) as a red sphere. Metal coordination and hydrogen bonds are shown as black dashes, with the distances labelled in Å. (A) VIM-2 (I222 space group), PDB 7A5Z; (B) VIM-2 (C2 space group) PDB 7A60; (C) L1 PDB 7A63; (D) KPC-2 PDB 7A61. Note the different conformations of the hydroxyethyl group in L1 (C) compared to VIM-2, leading to a lack of interaction with Zn1. KPC-2 (D) forms an acyl-enzyme complex with the faropenem derived complex.

Figure 6. ¹H NMR (AVIII 700 MHz) spectra displaying methyl group resonances of the faropenem derived products.

Enamines 1 and 2 ((5S) and (5R) enamine THF ring-closed products, orange), THF ring-opened (5R) and (5S) imines 3 and 4 (yellow) and THF ring closed imine 5 (pink) degradation products were formed after incubation of faropenem (green) with OXA-10 (5 μM, 16 h), OXA-23 (5 μM, 15 min), OXA-48 (5 μM, 55 min), VIM-1 (280 nM, 5 min), VIM-2 (280 nM, 5 min), NDM-1 (5 μM, 5 min), KPC-2 (125 nM, 72 min), L1 (125 nM, 5 min). All enzymes were incubated with 5 mM faropenem apart from OXA-23, which was incubated with 2 mM faropenem. Reactions were in 50 mM sodium phosphate, pH 7.5, 10% (v/v) D₂O. Note, 5 is only observed with VIM-1 and VIM-2. Colour coding corresponds to the structures shown and associated resonances. Individual spectra are provided in Figures S13-19. Characterisation data for compounds 2 and 5 are available in Figure 21 and Figures S22-27 respectively.

Figure 7. Summary of solution and crystallographic observations for faropenem reactions with SBLs/MBLs.

*Denotes crystallographically observed enzyme bound complexes. Note that 5 and 6 were only observed in the cases of solution studies with VIM-1 and VIM-2.