Research paper
An optimized 2,2′-dipicolylamine-rutaecarpine quaternary ammonium derivative: targeting bacterial membrane disruption for enhanced anti-methicillin-resistant Staphylococcus aureus (MRSA) activity

https://doi.org/10.1016/j.ejmech.2025.117975Get rights and content

Highlights

  • •
    A series of antibacterial amphiphilic rutaecarpine derivatives were synthesized.
  • •
    IV4 shows best anti-MRSA activity with excellent biosafety and membrane selectivity.
  • •
    IV4 targets bacterial cell membrane resulting in a low resistance frequency.
  • •
    The unique component PG on the bacterial membrane may be the specific target of IV4.
  • •
    IV4 is highly effective in MRSA infection with excellent safety profiles in vivo.

Abstract

The escalating threat of antibiotic resistance necessitates innovative strategies to combat multidrug-resistant pathogens. Herein, we reported the rational design of amphiphilic rutaecarpine derivatives through structural modular optimization, aiming to enhance antibacterial efficacy. A quaternary ammonium derivative IV4, bearing a 2,2′-dipicolylamine group, was found to be the most potent candidate, exhibiting remarkable activity against methicillin-resistant Staphylococcus aureus (MRSA) with MIC values of 2–4 μg/mL, demonstrated rapid bactericidal kinetics, effective biofilm eradication, and exceptional plasma stability. Its superior selectivity was evidenced by low hemolytic activity (HC50 > 640 μg/mL) and minimal cytotoxicity toward mammalian cells. In a murine skin infection model, IV4 outperformed vancomycin in reducing bacterial load and attenuating inflammation without systemic toxicity, highlighting its strong therapeutic potential and favorable safety profile. Mechanistic studies revealed that IV4 specifically binds to phosphatidylglycerol (PG) on bacterial membranes, leading to membrane disruption, excessive production of reactive oxygen species (ROS), and metabolic collapse, ultimately resulting in bacterial cell death. Collectively, these findings establish IV4 as a promising membrane-targeting antibacterial agent that combines potent anti-MRSA activity with favorable biosafety, offering a novel framework for addressing antimicrobial resistance.

Introduction

Antimicrobial resistance (AMR) is widely recognized as one of the most significant threats to global public health, resulting in over 1 million deaths annually [1]. In clinically common multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing hospital-acquired and community-acquired infections, which can lead to various lethal infections [2,3]. More severely, currently, clinical drug candidates targeting MRSA mainly focus on classical biological targets such as cell-wall synthesis inhibitors, including vancomycin and daptomycin, protein synthesis inhibitors represented by linezolid and tedizolid, folate metabolism inhibitors such as sulfamethoxazole/trimethoprim, and RNA polymerase inhibitors like rifampin. However, these agents generally suffer from rapid emergence of resistance, narrow therapeutic windows, and adverse effects such as nephrotoxicity and bone marrow suppression [4]. Furthermore, novel targets like fatty acid synthesis inhibitors and teichoic acid synthesis inhibitors have advanced into clinical studies, but their clinical application has been limited due to insufficient efficacy and rapid resistance development. Therefore, there is an urgent need to develop anti-MRSA drugs with innovative mechanisms and reduced potential for resistance [5].
The bacterial cell membrane has emerged as an ideal novel antibacterial target due to two main advantages: first, the bacterial cell membrane is not likely to develop drug resistance of its stable structure and a low mutation rate [6]; second, the bacterial cell membrane contains negatively charged phospholipids such as phosphatidylglycerol (PG) and cardiolipin (CL), which makes it more electronegative than the normal host cell membrane, providing potential targeting selectivity [7]. Some amphiphilic cationic molecules have achieved specific disruption of the bacterial cell membrane based on this characteristic, in which their cationic parts are first attracted by the negative charge components on the membrane as mentioned above, and then the hydrophobic parts take the response of inserting, thereby disturbing its membrane homeostasis, causing the leakage of contents, and ultimately killing the bacteria [8]. Such completely artificially designed amphiphilic cationic molecules, such as Brilacidin and LTX-109, have already entered clinical trials but have not yet been used in clinical practice to date for their systemic off-target toxicity in vivo [9,10]. How to improve the selectivity of this kind of antibacterial agent is the key for their clinical translation.
Usually, compared to the more variable cationic parts mainly based on amine structures, the hydrophobic core in this kind of structure often occupies a larger chemical space and is more complex, which makes it more difficult to conduct de novo design based on the differences between bacterial cell membranes and host cell membranes. Natural products are an important source for the discovery of new antibacterial agents, containing extremely diverse hydrophobic structures and naturally having advantages in terms of safety [11,12]. Recently, amphiphilic cationic molecules constructed based on natural products, such as osthole [7], xanthohumol [13], and nonivamide [14], have displayed better effects in terms of antibacterial activity and selectivity. However, the reasons for choosing these natural hydrophobic components are usually not strongly related to the direct interaction mechanism with bacterial cell membranes or are just based on the reported antibacterial activity with unknown mechanisms. Therefore, it is difficult to obtain guiding rules for the selection of natural core structures from these works.
Based on the mode of action of amphiphilic cationic molecules on the bacterial cell membranes, we hypothesized that the natural hydrophobic structures with better affinity for the membrane structure might be crucial. Therefore, we identified rutaecarpine, a kind of natural active component with significant cardiovascular benefits, mainly by protecting the heart and maintaining its metabolic homeostasis [15]. More importantly, a recent study has found that it is sensitive to the negatively charged phosphate part of phospholipids and has good affinity to negatively charged phospholipids [16]. This prompted us to regard it as a paradigm for exploring the natural hydrophobic core with membrane affinity to serve as an antimicrobial lead. Specifically, in this work, we introduced diverse linkers and hydrophilic cationic amine fragments onto the B ring of rutaecarpine (Fig. 1), modularly constructing 32 rutaecarpine derivatives, and explored the in vitro and in vivo antibacterial activity, biological safety, bactericidal performance, and resistance to drug resistance of the best candidate IV4 (Fig. 1), as well as its bacterial membrane-targeting action mode. We hope that this study can provide valuable insights into the screening of this new class of novel membrane-targeting natural antibacterial agents, thereby further providing a basis for the development of antibacterial drugs with clinical application potential.

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Section snippets

Chemistry

As shown in Scheme 1, using rutaecarpine (I) as a starting material, the N–H position of I was reacted with three different brominated alkanes (n = 3, 4, 5) in the presence of potassium carbonate (K2CO3) to produce bromo-rutaecarpine intermediates II1−3, respectively. Subsequently, the intermediates II1−3 were reacted with 5-fold equivalents of different aliphatic amines and heterocyclic amines, respectively, yielding 27 rutaecarpine-tertiary amine derivatives III1−27. In order to further

Conclusion

Bacterial cell membrane-targeting molecules based on natural products have demonstrated strong translational potential, with excellent antibacterial activity, anti-resistance properties, and biological safety. However, there are still many research gaps before they can be truly applied in practice. Among them, exploring the molecular construction mechanism and rules of these molecules is crucial for further optimizing and expanding these molecules to facilitate clinical translation. In this

General chemistry

The chemical reagents, including rutaecarpine, tertiary amine derivatives, 1,3-dibromopropane, 1,4-dibromobutane, and 1,5-dibromopentane were procured from Energy-Chemical Co., while analytical grade solvents and auxiliary chemicals were acquired from Shanghai McLean Biochemical Technology Co., Ltd (China). Structural characterization was performed using Nuclear magnetic resonance (NMR) spectra recorded on a Bruker Avance 400/100 MHz spectrometer (Germany) operating at 400 MHz for 1H and

CRediT authorship contribution statement

Ting Xu: Writing – review & editing, Writing – original draft, Validation, Methodology, Data curation, Conceptualization. Tingting Wang: Validation, Software, Methodology, Data curation. Yue Tian: Validation. Xinhui Li: Validation. Yan Zhong: Validation, Methodology. Jifeng Liu: Writing – review & editing. Ruige Yang: Writing – review & editing, Supervision, Conceptualization. Yong Guo: Conceptualization, Data curation, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are thankful to the financial support from the financial support from the National Natural Science Foundation of China (82373754), the Natural Science Foundation of Hunan Province-Department Joint Fund (2024JJ8141), the Outstanding Youth Science Foundation of Henan Province (242300421078), and the Startup Research Fund from University of South China (5524GC003 and 5524GC001).

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    T. Xu and T. Wang contributed equally to this work.
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