Target-based drug discovery (TDD) has been the primary approach leveraged by the pharmaceutical industry in recent decades [1,2]. The “single molecule, single target” strategy has led to the successful development of many drugs [3,4]. However, drugs acting on an individual molecular target are usually insufficient for effective treatment of cancer [5]. In addition, long-term excessive activation or inhibition of a specific target can lead to drug resistance and even toxic adverse effects, due to the activation of compensatory mechanisms or interference with normal biological functions [2,6]. Fortunately, multi-target drugs have been proposed as a potential solution. Conjugating two anticancer drugs into a single molecule (the multi-target drug strategy) may offer advantages such as improved patient compliance, reduced drug-drug interactions and simplified pharmacokinetics [7]. So far, several dual-target conjugates have been developed, such as those targeting epidermal growth factor receptor (EGFR)/Src kinase [8], estrogen receptor α (ERα)/EGFR [9], histone deacetylase (HDAC)/EGFR [10], bromodomain-containing protein 4 (BRD4)/HDAC [11] and enhancer of zeste homologue 2 (EZH2)/(BRD4) [12]. Among them, the conjugate of erlotinib and vorinostat, known as CUDC-101, has reached Phase I clinical trials [13,14], indicating the potential for further development in this field.

Breast cancer (BC) is the most prevalent malignant neoplasm among women, including 31 % of female tumor cases, and it is the primary cause of cancer-related mortality in women aged 20–49 [15]. TNBC, accounting for 9%–19 % of primary BC [16], is characterized by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) expression [17,18]. Due to limited treatment options and high invasiveness, as well as a high risk of relapse, TNBC is often associated with poor prognosis and low survival rates [19,20], therefore, new effective drugs for TNBC treatment are urgently required. A genome sequencing study has revealed extensive inter- and intra-tumor heterogeneity in TNBC and no potential therapeutic targets for recurrent mutations has been identified [21], suggesting that combination therapy may be a promising strategy for TNBC [22].

BRD4 contributes to cancer pathogenesis by reading acetylated lysine residues on histones to promote oncogene transcription [23,24]. A large-scale genomic analysis of TNBC has revealed that dysregulated BRD4 expression is crucial for the survival of TNBC cells [25], and BRD4 inhibitors have been identified as potential therapeutic drugs for TNBC [26]. However, acquired resistance to BRD4 inhibitors has been observed [27]. Src, a non-receptor tyrosine kinase, is closely associated with tumor progression and metastasis [28]. Several studies have reported the effectiveness of Src inhibitors for treating BC, especially the aggressive TNBC subtype [[29], [30], [31]]. Downregulation of key oncogenes such as c-MYC is an important mechanism for the anti-tumor activity of BRD4 inhibitors [32], while Src has been reported to activate the transcription of c-MYC and stabilize c-MYC mRNA to promote cell proliferation and cell cycle progression [[33], [34], [35]]. Moreover, the combination therapy of Src inhibitor dasatinib and BRD4 inhibitor JQ1 was reported to exhibite synergistic effect on JQ1-resistant TNBC cell line SUM159R [36]. Our preliminary experimental results also validated the additive or synergistic anti-tumor effects of the combination therapy in several solid cell lines (Table S1). Therefore, we hypothesized that simultaneous inhibition of BRD4 and Src could not only enhance anti-tumor efficacy but also suppress metastasis of TNBC and potentially reduce the occurrence of acquired resistance. To this end, we designed and synthesized a class of dual BRD4/Src inhibitors and evaluated their efficacy against TNBC both in vitro and in vivo. Structure-activity relationship studies led to the identification of the optimal dual-target inhibitor HL403, which not only effectively suppressed migration and invasion of MDA-MB-231 cells but also inhibited the proliferation with an IC50 of 34 nM in vitro and achieved a TGI of 70.7 % in a mouse MDA-MB-231 xenograft model, outperforming the single-target inhibitors or the combination. Our study provides an example of BRD4/Src dual inhibitors for effective TNBC treatment.