## 1 Introduction  
The use of herbicides has played a crucial role in effectively managing weed infestation, thereby facilitating agricultural progress [1]. Nevertheless, the escalating emergence of herbicide resistance in weeds poses significant challenges to their efficacy [2; 3]. The repeated utilization of herbicides with similar modes of action often leads to the development of resistance in weeds. This phenomenon has resulted in the proliferation of herbicide-resistant weed species, which can cause substantial reductions in agricultural yields, ranging from 40% to 90% [4].  
Considering the detrimental impacts of current synthetic herbicides and the formidable obstacles presented by resistant weeds, the replacement of harmful chemicals with promising natural products emerges as a viable long-term approach for weed management [5]. In this context, the utilization of allelochemicals obtained from plants presents a pathway towards sustainable agriculture. Allelochemicals refers to secondary metabolites produced by different plant parts, which have the ability to influence the growth and development of adjacent plants [6].  
Alleochemicals consist of phenolic acids, flavonoids, alkaloids, steroids, and fatty acids, among other compounds [7]. These substances exert toxicity on weeds by disrupting their photosynthetic system, respiratory system, cell division, and lignin content [8]. A sulfur-containing compound called (-)-Spirobrassinin has been isolated from broccoli, and it is unique to the Brassicaceae family (Figure 1). (-)-Spirobrassinin has demonstrated inhibitory effects on the growth of _Panicum miliaceum_ L. and _Amranthus retroflexus_ L.  
(Figures S2 and S3). This compound exhibits potential for development as a novel plant-based herbicide. However, the mode of action and specific target site of (-)-Spirobrassinin have not been investigated yet.  
Currently, herbicides available in the market act through various modes of action. One of the common targets of herbicides is the enzyme acetolactate synthase (ALS), which is responsible for catalyzing the formation of acetohydroxybutyrate and acetolactate [9]. Another target is dihydroxyacid dehydratase (DHAD), which catalyzes the \(\alpha\),\(\beta\)-dehydration reaction in the biosynthetic pathway of branched-chain amino acids, producing \(\alpha\)-keto acids that serve as precursors for leucine, isoleucine, and valine [10; 11]. Inhibitors of hydroxyphenylpyruvate dioxygenase (HPPD) target the photosynthesis process by interfering with the conversion of hydroxyphenylpyruvate to homogenistate. This disruption affects the synthesis of tocopherols and plastoquinones, ultimately impacting the biosynthesis of carotenoids in the target organisms and leading to plant death [12]. Protochlorophyllide oxidase (PPO), found in both mitochondria and chloroplasts, is another important enzyme involved in tetrapyrrole biosynthesis, specifically in the synthesis of proteome and chlorophyll [13; 14]. Herbicide targets also include components of photosystem II (PSBD1), where they compete with plastoquinone for binding sites, disrupt the electron transport chain, and interfere with light reactions [15]. Furthermore, there are targets related to plant growth regulation, including binding sites for auxins (TIR1) [16], jasmonic acid (COLI-ASK1) [17], abscisic acid (PLY2) [18], Strigolactone (D14-D3-ASK1) [19] and gibberellins (GIDI) [20].  
In this study, we employed molecular docking, virtual screening, biological validation experiments, and molecular dynamics simulations to investigate the mode of action and binding sites of (-)-Spirobrassinin. The primary objective was to establish a theoretical basis for the potential development of (-)-Spirobrassinin as a natural product herbicide.