Toxin-Activating Stapled Peptides Discovered by Structural Analysis Were Identified as New Therapeutic Candidates That Trigger Antibacterial Activity against Mycobacterium tuberculosis in the Mycobacterium smegmatis Model

Abstract

The structure-function relationships of toxin-antitoxin (TA) systems from Mycobacterium tuberculosis have prompted the development of novel and effective antimicrobial agents that selectively target this organism. The artificial activation of toxins by peptide inhibitors can lead to the growth arrest and eventual death of bacterial cells. Optimizing candidate peptides by hydrocarbon α-helix stapling based on structural information from the VapBC TA system and in vitro systematic validation led to V26-SP-8, a VapC26 activator of M. tuberculosis. This compound exhibited highly enhanced activity and cell permeability owing to the stabilizing helical propensity of the peptide. These characteristics will increase its efficacy against multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Similar approaches utilizing structural and biochemical information for new antibiotic targets opens a new era for developing TB therapies.

Stapled Phd Peptides Inhibit Doc Toxin Induced Growth Arrest in Salmonella

“…One such class of targets is bacterial toxins, which are involved in stress responses. Peptide ligands have been developed which work to activate toxins with the aim of initiating cell death. ,, However, we propose an alternative approach. Given that toxin activation may result in the increased formation of antibiotic tolerance persister cells, we have instead focused on the development of toxin inhibitors.…”

Focused Overview of Mycobacterium tuberculosis VapBC Toxin–Antitoxin Systems Regarding Their Structural and Functional Aspects: Including Insights on Biomimetic Peptides

“…If the peptides bind with high affinity, the binding between VapB26 and VapC26 would be disrupted, and free VapC26 would become more predominant, leading to increased RNase activity. In fact, it has been confirmed that the ribonuclease activity of VapBC26 increases as a result of peptide addition [ 49 , 62 ]. Effective peptides were those that mimicked the binding region of VapC26, specifically the α3 and α4 helices ( Figure 6 B).…”

“…This led to the discovery of an inhibitory peptidomimetic, ‘V26-SP-8’, which specifically targeted the VapC26 α4 helix. ‘V26-SP-8’ was engineered from the initial peptide through hydrocarbon α-helix stapling, resulting in enhanced VapC26 activity even at significantly lower concentrations [ 62 ]. Circular dichroism spectroscopy confirmed the increased α-helical propensity of ‘V26-SP-8’, and isothermal titration calorimetry determined a dissociation constant ( K d ) of approximately 604 ± 18.2 nM for the VapB26-’V26-SP-8’ interaction.…”

“…In experiments with M. smegmatis , fluorescence-labeled ‘V26-SP-8’ demonstrated uptake by bacterial cells and inhibited their growth effectively. Moreover, ‘V26-SP-8’ exhibited stability in human and mouse blood/serum/plasma, with a half-life exceeding three hours [ 62 ].…”

Peptide Stapling Applied to Antimicrobial Peptides

“…Finding critical interaction domains allows for the rational design of PPI inhibitors [ 24 , 59 , 60 ]. Matching the antimicrobial resistance context and protein–protein interaction strategies, Kang et al [ 54 ] took advantage of the toxin–antitoxin (TA) system VapBC26 from Mycobacterium tuberculosis to develop an AMP agent that can act as an inhibitor of intermolecular protein–protein interactions. Firstly, non-crucial residues were selected for stapling modification, and the stapled peptide V26-SP-8 was synthesized and derived from VapC26 α4 54–65 , previously described as inhibiting the VapBC26 TA system.…”