Overcoming Proteolytic Instability in a β-Turn Antimicrobial Peptide via Cyclization and Stereochemical Inversion to Combat MDR Bacteria

Today, we are sharing an important study published in the Journal of Medicinal Chemistry, titled "Overcoming Proteolytic Instability in a β-Turn Antimicrobial Peptide via Cyclization and Stereochemical Inversion to Combat MDR Bacteria." Led by the team of Qingbin Meng, this research aims to address the core bottleneck in the clinical application of antimicrobial peptides (AMPs)—their proteolytic instability. Targeting the linear β-turn antimicrobial peptide P-07 previously developed by the group, the researchers constructed a library of derivatives through systematic strategies of disulfide/lactam bond cyclization and D-amino acid substitution. Among them, the lead candidate, PT-17, not only maintained the potent, broad-spectrum antibacterial activity comparable to its parent peptide P-07 but also demonstrated significantly enhanced proteolytic stability (with half-lives increased by 54.3-fold and 25.5-fold against chymotrypsin and trypsin, respectively), plasma stability, and in vivosafety. PT-17 functions by rapidly disrupting bacterial membranes, is less prone to inducing resistance, and exhibits synergistic effects with conventional antibiotics. It demonstrated excellent therapeutic efficacy in a mouse systemic infection model. This work provides a systematic strategy and empirical evidence for the rational design of stable β-turn antimicrobial peptides with clinical translation potential.

01 Research Background

The global spread of multidrug-resistant (MDR) bacteria poses a serious threat to public health, urgently necessitating the development of new-generation antibacterial agents. Antimicrobial peptides (AMPs) are considered ideal candidates due to their unique membrane-disrupting mechanism and low tendency to induce resistance. However, the vast majority of AMPs are rapidly degraded by proteases in physiological environments, leading to extremely short in vivohalf-lives and greatly diminished efficacy, which is the primary bottleneck hindering their clinical translation. To enhance stability, D-amino acid substitution (exploiting the differential recognition of D-amino acids by proteases) and peptide cyclization (spatially shielding proteolytic sites and stabilizing the active conformation) are two commonly employed strategies. While cyclization has been successfully applied to stabilize random coil and α-helical AMPs, its structure-activity relationship impact on AMPs with the specific β-turn secondary structure lacks systematic and in-depth study. Our group previously reported a symmetric β-turn antimicrobial peptide, P-07, which exhibited potent activity against MDR bacteria. This study aims to significantly enhance the proteolytic stability of P-07 through the rational design and synergistic integration of cyclization and D-amino acid substitution. It also seeks to comprehensively elucidate how these modifications affect the structure, activity, stability, and in vivobehavior of β-turn AMPs, thereby overcoming the key obstacle to their clinical translation.

02 Innovative Highlights

2.1 Systematic Investigation of Cyclization's Impact on β-Turn AMPs:

The study, for the first time, systematically applied two cyclization strategies—disulfide bond and lactam bond formation—to the β-turn antimicrobial peptide P-07. It investigated the effects of different cyclization sites (near the turn, center, termini) on activity. The research found that centrally cyclized derivatives (e.g., PT-08) generally possessed optimal antibacterial activity, and disulfide bond cyclization was superior to lactam bond cyclization in preserving activity.

2.2 Revealing the Synergistic Stabilization Mechanism of Cyclization and D-Amino Acid Substitution:

The study not only applied the two strategies individually but also combined them. The results showed that cyclization alone (PT-08) or D-amino acid substitution each had specific focuses for stability improvement (e.g., PT-08 showed significant improvement against trypsin). In contrast, the combination of both in the lead peptide PT-17 achieved synergistic enhancement, with stability against chymotrypsin and trypsin increased by tens of fold, and exhibited optimal plasma stability.

2.3 Achieving Stability Enhancement Without Compromising Activity:

Through rational design, the peptide's positive charge and hydrophobic profile were deliberately maintained during cyclization and D-amino acid substitution. This allowed the optimal candidate PT-17 to achieve excellent stability while its antibacterial activity (GM ~5.66 µM) and therapeutic index (TI=90.46) remained comparable to or even better than the parent peptide P-07, successfully breaking the dilemma where "stability improvement often accompanies activity loss."

03 Results and Discussion

Through a series of experiments including design and synthesis, activity screening, stability testing, mechanism investigation, and in vivoevaluation, the study progressively screened and fully validated the lead peptide PT-17.

3.1 Peptide Library Design, Synthesis, and Structural Characterization

To enhance the stability of P-07, 17 derivatives (PT-01 to PT-17) were designed. Strategies included: (1) Symmetric substitution of Ile with Cys or Lys/Glu pairs for disulfide or lactam bond cyclization (PT-01-PT-06); (2) Removal of Pro from the above cyclic peptides to verify the contribution of the disulfide bond to maintaining the β-turn (PT-07-PT-09); (3) Systematic D-amino acid substitution based on the optimal cyclic peptide PT-08 (PT-10-PT-17). All peptides were verified for purity and molecular weight by HPLC and MALDI-TOF-MS. Circular dichroism (CD) spectroscopy confirmed that the cyclic peptides maintained the β-turn conformation, while the introduction of D-amino acids, especially in the β-turn region, caused inversion of the CD spectra.

3.2 Antibacterial Activity, Hemolytic Activity, and Cytotoxicity

Antibacterial activity testing (MIC) showed that cyclization typically slightly reduced activity, but the centrally disulfide-cyclized PT-08 had the best activity. PT-17, obtained by performing a complete D-amino acid substitution on the basis of PT-08, regained activity comparable to P-07 and exhibited potent inhibition against all tested MDR Gram-positive and Gram-negative bacteria. All cyclic peptides showed hemolysis rates below 10% at 256 µM, indicating high selectivity. Cytotoxicity experiments revealed that PT-17 was less cytotoxic than both PT-08 and P-07.

3.3 Significant Stability Enhancement

PT-17 demonstrated excellent salt tolerance (MIC unchanged in the presence of various physiological salt ions). In 50% plasma, PT-17's activity remained intact up to 12 hours of incubation (unchanged MIC), with the MIC only doubling after 24 hours, showing far superior stability compared to P-07 (activity began to decline after 6 hours). The most critical enzyme stability data showed: The half-lives (t1/2) of PT-17 against chymotrypsin and trypsin were as high as 21.7 hours and 10.2 hours, respectively, which are 54.3-fold and 25.5-fold greater than those of P-07 (0.4 hours), and also significantly longer than the cyclized-only PT-08. This quantitatively proves the synergistic power of the "cyclization + D-amino acid" strategy.

3.4 Bactericidal Kinetics, Resistance Induction, and Synergistic Effects

Time-kill curves indicated that PT-17 could completely eradicate bacteria within 6 hours. After 20 serial passages, the MIC of PT-17 against the tested strains showed no significant change (≤2-fold), whereas amoxicillin showed up to a 32-fold increase in MIC, demonstrating that PT-17 is highly unlikely to induce resistance. Checkerboard assays revealed that PT-17, in combination with various clinically used antibiotics (e.g., polymyxin B, amoxicillin, cefoperazone), exhibited synergistic or additive effects against MDR strains.

3.5 In Vivo Safety and Efficacy Validation

In acute toxicity experiments, the LD50 of PT-17 was as high as 38.0 mg/kg, which is 13.1-fold and 3.2-fold higher than that of P-07 (2.9 mg/kg) and polymyxin B (12.0 mg/kg), respectively, indicating a remarkably superior in vivosafety profile. In a mouse systemic infection model induced by MDR E. coli, intravenous administration of PT-17 (5 and 10 mg/kg/day for 3 days) significantly reduced bacterial loads in the liver, spleen, lungs, and kidneys (by approximately 2-4 log units) and effectively alleviated infection-induced histopathological damage. The clearance effect of the high dose of PT-17 (10 mg/kg) was even superior to that of the positive control polymyxin B (5 mg/kg), and no toxic reactions were observed during the treatment.

04 Conclusion and Future Perspectives

This study successfully combined rational structural biology design with chemical biology validation. By integrating the two major strategies of "central disulfide bond cyclization" and "complete D-amino acid substitution," the linear β-turn antimicrobial peptide P-07 was transformed into the lead candidate PT-17. PT-17 perfectly balances potent, broad-spectrum antibacterial activity, exceptional proteolytic/plasma stability, a very low tendency to induce resistance, excellent in vivosafety, and therapeutic efficacy, meeting nearly all core attributes of an ideal antimicrobial peptide drug.

The paradigmatic significance of this work lies in its clear "stepwise optimization" logic: first constructing a stable, degradation-resistant peptide backbone framework via cyclization (accepting a temporary activity cost), followed by "activity rescue" and "further stability enhancement" through D-amino acid substitution, ultimately achieving comprehensive performance superiority. This provides a replicable R&D pathway for optimizing other unstable but promising bioactive peptides. Future research based on PT-17 can delve deeper into the following directions: ① Conduct systematic pharmacokinetic/pharmacodynamic studies to clarify its in vivodistribution, metabolism, and excretion patterns; ② Explore its efficacy in more complex, clinically relevant infection models (e.g., pneumonia, burn infections, biofilm-associated infections); ③ Develop scalable production processes and formulation studies to advance it towards clinical research. In summary, this study not only contributes a highly clinically promising antimicrobial peptide candidate drug but also provides a validated, efficient methodology for developing stable peptide-based therapeutics.