Recognized as the ‘guardian of the genome’, the p53 protein has been an important drug target since its discovery in 1979 [1]. The p53 protein is a tetrameric transcription factor that regulates the expression of target genes responsible for cell cycle arrest, DNA repair, and apoptosis of damaged cells, and by these means induces an antiproliferative cellular response [[2], [3], [4], [5], [6], [7]]. Essential to the stability of the p53 protein is a structural Zn2+, which is tetrahedrally coordinated in a Zn-finger binding motif in the DNA-binding (DBD) domain. Due to the significance of p53 in the maintenance of normal cell function, mutations that compromise p53 activity can have serious consequences, with over 50% of cancers resulting from point mutations to p53 [8,9]. The majority of these mutations are localized to the protein's DBD and can be broadly classified as impairing DNA interactions and/or overall stability [[10], [11], [12]]. Wild-type (WT) p53 DBD exhibits limited kinetic and thermodynamic stability [13], and mutations lead to further destabilization and loss of function due to impaired Zn2+ binding, protein unfolding, aggregation and amyloid formation [14]. Given that p53 is the most frequently mutated protein in cancer and almost all cancers exhibit malfunction along the p53 pathway [15,16], there has been considerable interest in the development of therapeutic interventions targeting the p53 pathway [[17], [18], [19], [20], [21], [22], [23], [24]]. In addition, mutant p53 represent a particularly attractive target due to its selective presence in cancer cells, hence hypothetically limiting side effects on healthy tissues.

Previous studies have shown that p53 is delicately partitioned between folded and unfolded conformations in the cell, and available Zn2+ is determined to be a major factor in folding and activity [12]. Targeting metal ion chelation and redistribution has shown utility as an anticancer strategy [[25], [26], [27], [28], [29], [30], [31]], and re-population of the p53 structural Zn2+ site via metallochaperones has been shown to restore function to common p53 mutants (Fig. 1) [[32], [33], [34], [35], [36], [37], [38], [39], [40], [41]].

Further studies showed that the mechanism of action of the ZMCs is in part due to the generation of intracellular ROS. ROS generation contributes to the reactivation of p53 via the induction of post-translational modifications but can also result in off-target effects and general cytotoxicity [36]. ROS generation was attributed to internalization of redox-active metals (e.g. Cu+/2+ and Fe2+/3+) and highlights the difficulty of selective metal ion binding and redistribution in cancer therapy. For example, recent work has shown that Cu uptake via ionophores can promote targeted lipoylation (and aggregation) of TCA cycle proteins leading to cell death [42]. A structure-activity relationship (SAR) study on the thiosemicarbazone framework identified a benzimidazole (Fig. 1, C85) that exhibits a similar reactivation profile to ZMC1 (Fig. 1) with lower ROS generation, which the authors ascribed to reduced Cu2+ affinity and lower Cu2+/Zn2+ selectivity ratio. To further test the ROS hypothesis, a series of cell-permeable derivatives of nitrilotriacetic acid (NTA) were designed (Fig. 1). These derivatives exhibited diminished Cu2+ affinity, less ROS generation, and exhibited synergistic activity with chemotherapies and radiation therapies without the need for the administration of additional ROS scavengers [43]. Thus, previous studies have identified a Cu2+:Zn2+ binding ratio of 10–103 to be optimal to minimize ROS generation and off-target toxicity [43].

Our interest centers on the p53-Y220C mutant, a common destabilizing mutant that results in a cavity at the protein surface and decreases the protein melting point by ∼2 °C [44]. We have reported on a series of multifunctional ligands which combine a p53-Y220C binding diiodophenol core [44] and di-(2-picolyl)amine (DPA) as the Zn2+ binding group to promote metallochaperone activity [32]. However, our multifunctional compounds exhibited non-specific mutant and WTp53 cytotoxicity in cancer cell lines, which we hypothesize is a result of ROS generation. Herein, we report modifications to the original compound series that feature different Zn2+ binding groups to tune metal affinities in an effort to reduce off-target ROS generation (Fig. 2). An approximately 10 [3]-fold reduction in Cu2+ binding affinity has been reported for iminodiacetic acid (IDA) in comparison to DPA, while the Zn2+ binding affinity remained constant [45]. A similar trend in relative binding affinities is reported for tripyridylamine (TPA) and NTA [45]. We have thus prepared and studied the substitution of pyridines for carboxylates in the ligand series shown in Fig. 2. While we have previously reported on L4-P and L4-AP [32], we now test the full series for both Zn2+ and Cu2+ binding affinity, ZMC activity, cytotoxicity in two cancer cell lines with differing p53 mutation status, and finally ROS generation to aid in the development and optimization of our multifunctional ligand design.