Exposure to a high dose of ionizing radiation (IR) can cause dysfunction in functional systems [1], and damage to the digestive system, nervous system and blood-forming organs is most obvious [2]. Ionizing radiation directly acts on or produces reactive oxygen species, which change the structure and function of biological macromolecules such as DNA and proteins in cells [3], thus leading to apoptosis and necrosis of cells, especially for organs with high proliferation [4]. IR has become an invisible “killer” to endanger human health [5]. When the cumulative exposure dose of the human body exceeds 1 Gy, various uncomfortable symptoms will appear, such as nausea, vomiting and dizziness [6].

Ex-RAD, also known as ON 01210. Na (4-carboxystyryl-4-chlorobenzyl sulfone, sodium salt) is a chlorobenzyl sulfone derivative developed by Onconova Therapeutics as a radioprotector and mitigator [7]. Unlike most radioprotectors, Ex-RAD is not a free-radical scavenger or responsible for cell cycle arrest [8]. Available data suggest that Ex-RAD has a novel mechanism for radiation protection involving DNA repair pathways [9]. It is a water soluble, nontoxic, synthetic molecule with potent radioprotective properties both in vitro and in vivo [10]. Moreover, preclinical pharmacokinetic studies in rats, dogs, rabbits and monkeys have demonstrated that Ex-RAD is well absorbed following extravascular administration, resulting in significant plasma exposure [11]. Mechanistically, the radioprotective effects of Ex-RAD may involve the prevention of p53-dependent apoptosis [12]. Onconova Therapeutics has completed four phase I trials with Ex-RAD, three trials with subcutaneous Ex-RAD in more than 50 healthy adults and one trial with oral Ex-RAD in nine healthy adults [13]; none of these trials reported evidence of systemic side effects [14].

Toll-like receptors (TLRs) are important pattern recognition receptors in innate immunity and have been widely studied in recent years [15]. TLRs control adaptive immune responses through the upregulation of costimulatory molecules present on antigen-presenting cells (APCs) and then release cytokines that guide the activation, expansion, and differentiation of T cells. For example, TLR2 agonists can bind to TLR2/TLR1 or TLR2/TLR6 heterodimers and recruit the adaptor proteins MyD88 and TRIF to activate specific protein kinases, such as MAPK, IκB kinase, and PI3K, which in turn activate certain transcription factors, such as NF-κB and IRFs [16]. Accumulating evidence shows that TLRs have lower side effects and higher protective efficiency than traditional radiation protection drugs [17], making TLR agonists potential candidate drugs for ionizing radiation protection [18]. In 2012, Vijay K. Singh et al. reported that the activation of TLR2/6 exhibited strong radioprotective effects through activation of nuclear factor-κB (NF-κB) [19]. However, TLR2 mainly recognizes non-small-molecule lipopeptides or bacterial cell wall-related components from Gram-positive bacteria [20]. An insurmountable obstacle that prevents the clinical development, is their poor pharmacokinetics as they typically exhibit low bioavailability and are highly susceptible to proteolytic cleavage in serum. In contrast, small molecular of TLR2 agonist have several assets including stability to proteases, increased selectivity, easy synthesis, cost-effectiveness and reduced toxicity that make them excellent prospects as potential agonist. Those promising potential applications motivate us to explore and discover novel small molecules to replace these TLR2 macromolecular activators.

Conventional single-target drugs tend to develop resistance and are less effective against the disease [21]. Therefore, new approaches, such as combination therapy, multitargeted drugs and proteolytically targeted chimaeras, have emerged to address this challenge [22]. Undoubtedly, drug combination therapies may have powerful therapeutic implications [23], but they also bring some problems, such as drug‒drug interactions and poor medication adherence [24]. However, multitargeted drugs can avoid some of the disadvantages of drug combinations [25]. This discovery provides new insights into radioprotector development. Based on these findings, we attempted to find small molecules with high efficiency and low toxicity to regulate both the TLR2 and p53 pathways.