Gut microbiota is a complex microbial ecosystem that resides in the human intestine. Under normal physiological conditions, gut microbial participate in the body's metabolism and establish a mutually beneficial symbiotic relationship with both the internal physiological environment and the external surroundings, thus supporting vital functions such as metabolism, immunity, and digestion [[1], [2], [3], [4]]. However, factors such as high-intensity psychological stress, antibiotic usage, and parasitic infections can disrupt the native microbial populations in the large intestine, leading to dysbiosis. This dysbiosis, mediated by small-molecule metabolites, can contribute to diseases such as obesity, type II diabetes mellitus, and colorectal cancer [5,6]. Recent research has highlighted that gut microbes encode various metabolic enzymes crucial for the metabolism of endogenous and exogenous substances [7]. These enzymes are closely linked to disease pathogenesis, drug absorption, and metabolism, positioning them as potential therapeutic targets and garnering significant research interest in biomedicine.

Gut microbial β-glucuronidase (GUS) plays a crucial role in metabolizing various endogenous and exogenous molecules and is linked to disease pathogenesis and drug metabolism. Elevated GUS activity in the gut can often lead to a range of intestinal diseases. Although antibiotics can help eliminate GUS-producing bacteria to reduce the toxic side effects of certain medications, their overuse can severely disrupt the gastrointestinal system, potentially causing fatal damage to the liver, kidneys, and other organs. In contrast, gut microbial GUS inhibitors demonstrate high selectivity in blocking the hydrolytic activity of GUS, showing effective alleviation of the toxicity associated with irinotecan (CPT-11) and nonsteroidal anti-inflammatory drugs (NSAIDs). Additionally, these inhibitors have minimal impact on gut microbiota homeostasis, highlighting their significant scientific relevance and potential for practical application [[8], [9], [10]].

The application of gut microbial GUS inhibitors can be traced back to 1982, when Takada et al. pioneered the use of C-GAL for the prevention of neurotoxic compound azoxymethane-induced colon cancer in rats [11]. In 1996, Kamataki et al. found that the degree of damage to intestinal tissues caused by CPT-11 showed a positive correlation with the activity of gut microbial GUS, and the use of antibiotics could inhibit GUS activity and ameliorate CPT-11-induced intestinal damage [12]. Subsequently, accumulated studies have shown that there is a correlation between the decomposition of glucuronide conjugates by gut microbial GUS and the adverse effects of drug therapy. In 2010, Wallace et al. reported the crystal structure of Escherichia coli β-glucuronidase (EcGUS) for the first time, and identified potent EcGUS inhibitors by high-throughput screening based on the unique “bacterial loop” structure of EcGUS [13]. These inhibitors selectively enhance the pharmacokinetics and bioavailability of chemotherapeutic drugs, reducing CPT-11 toxicity without affecting mammalian cells, thus advancing the precise development of GUS inhibitors. Additionally, the clinical antidepressant drug amoxapine has shown GUS inhibitory activity, offering significant clinical potential due to its established pharmacokinetic and toxicity profiles [14]. Recently, GUS-specific inhibitors of enterobacteria can be obtained from natural products or plants as well as clinical drugs by high-throughput screening and computer-aided design [15,16]. Despite these progresses, the limited potency of known GUS inhibitors suggests that it is still necessary to develop new GUS inhibitors for maintaining intestinal health.

Molan et al. found that the inhibition of GUS activity in rats gavaged with selenium-containing green tea (selenium 1.44 mg/kg of dry leaves) was significantly higher than that in rats treated with Chinese green tea (selenium 0.13 mg/kg of dry leaves) [17], indicating the great potential of selenium compounds as GUS inhibitors. Ebselen is a multifunctional drug in active clinical research for treating depressive disorder and hearing loss (Fig. 1B). Particularly, it has been identified as a potential inhibitor of SARS-CoV-2 main protease (Mpro) by covalent modification of cysteine 145 (Cys145) [18]. Therefore, its potent biological activity, stable chemical structure, potential covalent interaction, favorable safety and pharmacokinetic profiles [19] has promoted its core structure 1,2-Benzoselenazol-3-one (BSEA), to be conceived as a “privileged structure” in new drug research. Structurally, BSEA is the bioisostere of the core structure of some known GUS inhibitors (Fig. 1A), e.g., benzothiazole (compound I) and benzimidazole (II) [20].

Due to the advantages of stronger potency, extended duration of action, and reduced risk of developing drug resistance, covalent inhibitor has become an effective and safe paradigm in modern new drug research and development [21]. Previously, through site-directed mutagenesis and mass spectrometry experiments, BSEA derivatives were identified as the first-generation covalent allosteric modulators of fructose-1,6-bisphosphate aldolase [22]. To date, most known β-glucuronidase (GUS) inhibitors function through non-covalent interactions [9], while studies on covalent GUS inhibitors remain scarce and are still in an exploratory stage. This suggests the promise of developing novel GUS covalent inhibitors from BSEA to mitigate the adverse effects of related chemotherapeutic agents.

Take these considerations in mind, and in continuation of our research interest in GUS inhibitor [23,24] and organoselenium drug development [[25], [26], [27], [28], [29], [30], [31], [32]], in this study, we have therefore synthesized 50 BSEA derivatives and evaluated their in vitro inhibitory activities against intestinal microbial β-glucuronidase, and we further analyzed their structure-activity relationships. The specific covalent binding sites of BSEA derivatives with EcGUS were identified by tandem mass spectrometry (LC-MS/MS). Finally, the inhibitory behavior of these compounds was investigated through jump dilution assays and kinetic assays, and their molecular determinants against EcGUS were explored using molecular docking studies.