Synergistic effects of sulopenem in combination with cefuroxime or durlobactam against Mycobacterium abscessus

Abstract

Mycobacterium abscessus (Mab) affects patients with immunosuppression or underlying structural lung diseases such as cystic fibrosis (CF). Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual β-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with β-lactamase inhibitors [BLIs-avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration (MIC) values for 54 clinical Mab isolates and ATCC19977 (MIC₅₀ and MIC₉₀ ≤0.25 µg/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding ~1.5 Log₁₀ CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in an additional reduction of CFUs (~3 Log₁₀ for sulopenem-CXM and AVI and ~4 Log₁₀ for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (Ldts; Ldt_Mab1-Ldt_Mab4), penicillin-binding-protein B (PBP B), and D,D-carboxypeptidase (DDC). Acyl complexes, identified via mass spectrometry analysis, demonstrated the binding of sulopenem with Ldt_Mab2-Ldt_Mab4, DDC, and PBP B and CXM with Ldt_Mab2 and PBP B. Molecular docking and mass spectrometry data suggest the formation of a covalent adduct between sulopenem and Ldt_Mab2 after the nucleophilic attack of the cysteine residue at the β-lactam carbonyl carbon, leading to the cleavage of the β-lactam ring and the establishment of a thioester bond linking the Ldt_Mab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLIs. These findings potentially broaden the selection of oral therapeutic agents to combat Mab.

Importance: Treating infections from Mycobacterium abscessus (Mab), particularly those resistant to common antibiotics like macrolides, is notoriously difficult, akin to a never-ending struggle for healthcare providers. The rate of treatment failure is even higher than that seen with multidrug-resistant tuberculosis. The role of combination β-lactams in inhibiting L,D-transpeptidation, the major peptidoglycan crosslink reaction in Mab, is an area of intense investigation, and clinicians have utilized this approach in the treatment of macrolide-resistant Mab, with reports showing clinical success. In our study, we found that cefuroxime and sulopenem, when used together, display a significant synergistic effect. If this promising result seen in lab settings, translates well into real-world clinical effectiveness, it could revolutionize current treatment methods. This combination could either replace the need for more complex intravenous medications or serve as a "step down" to an oral medication regimen. Such a shift would be much easier for patients to manage, enhancing their comfort and likelihood of sticking to the treatment plan, which could lead to better outcomes in tackling these tough infections. Our research delved into how these drugs inhibit cell wall synthesis, examined time-kill data and binding studies, and provided a scientific basis for the observed synergy in cell-based assays.

Keywords: Mycobacterium abscessus; dual β-lactams; oral carbapenem; sulopenem.

FIG 1

Sulopenem chemical representation.

FIG 2

Minimal inhibitory concentration distribution: comparison for sulopenem (SUL), cefuroxime (CXM), and combined sulopenem with 4 µg/mL cefuroxime against 54 Mab clinical strains and the ATTCC 19977 strain (See Table 1 for MIC data).

FIG 3

Time-kill curves of sulopenem (A) in monotherapy, cefuroxime (CXM) in monotherapy (B), the combination of sulopenem and CXM (C and D), sulopenem in the presence of avibactam (AVI) (E), and the combination of sulopenem and sulbactam with or without DUR (F) against ATCC 19977. To counteract thermal degradation, 10% of sulopenem and 20% of cefuroxime were supplemented every 24 h.

FIG 4

Capturing covalent adduct formation between sulopenem and Ldt_Mab2 (A) and Ldt_Mab3 (B) using timed electrospray ionization mass spectrometry. After 5 minute incubation of sulopenem with Ldt_Mab2, the adduct formed is 86 Da (A). When incubated with LdtMab₃, a 349 Da (sulopenem Mw) adduct is preserved after 2.5 hours (B).

FIG 5

Interaction between Bla_Mab, L,D-transpeptidases (Ldt_Mab1–Ldt_Mab4), D,D-carboxypeptidase, and PBP B with β-lactams (sulopenem and cefuroxime) and β-lactamase inhibitors (durlobactam and avibactam).

FIG 6

Proposed mechanism of action between sulopenem and Ldt_Mab transpeptidases. The covalent adduct formation between sulopenem and Ldt_Mab2 can be explained through the nucleophilic attack of the cysteine residue at the carbonyl carbon of the β-lactam ring in sulopenem, with the help of activation from His333 (1). This nucleophilic attack results in the opening of the β-lactam ring and the formation of a thioester bond between the enzyme and sulopenem (2). Steps (3) and (4) are intermediate steps in the reaction mechanism suggested by the MDS results, where three water molecules were recruited into the active site. The end adduct of 87 Da (5) was observed after 5 minute incubation of Ldt_Mab2 with sulopenem. When sulopenem is incubated with Ldt_Mab3, after 2.5 h, the adduct with the intact sulopenem (2) was observed in the MS. The molecular modeling of sulopenem and Ldt_Mab3 does not result in water molecules recruited into the active site. This may suggest that the reaction mechanism of sulopenem with Ldt_Mab3 ends after step 2, with the release of the intact sulopenem.

FIG 7

Ldt_Mab2 and sulopenem as Michaelis-Menten (A) and acyl enzyme (B) complexes. During MDS simulation of the acyl-enzyme (C, D) and sulopenem fragment (E) complexes, three water molecules are recruited into the active site of Ldt_Mab2. Initially, (A) H333 is at H-bond distance from Cys351 and ready to activate it for acyl-enzyme formation. After the acyl formation (B), the His333 is moving away from Cys351, and sulopenem carbonyl is positioned outside of the oxyanion hole. The potential H-bonds interactions are represented with green, hydrophobic interaction with pink, and sulfur with yellow.

FIG 8

Ldt_Mab3 and sulopenem molecular docking as Michaelis-Menten (A, B) and acyl enzyme (C, D) complexes. The sulopenem carbonyl is positioned toward the Ldt_Mab3 oxyanion hole formed by Cys351:NH and Gly350:NH, ready for acylation. However, His333:NE2 is more than 5 Å away from the catalytic Cys351, and the sulopenem carbonyl makes H-bonds with Gly329 (A, B). This suggests that the acyl enzyme complex may take longer to form. When the acyl enzyme is formed (C), the complex is held and stabilized in the active site of Ldt_Mab3 through a network of H-bonds (green) and hydrophobic interactions (pink) (C, D).