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.