Despite the advances in modern medicine, antimicrobial and chemotherapy resistance remains a global health challenge. The World Health Organization (WHO) deemed antimicrobial resistance as one of the top global public health risks in 2019 [1]. With all currently approved antibiotic classes being limited by resistance in at least some of the bacteria they work against, it is no surprise that antimicrobial resistance will bring about over 10 million global annual deaths by 2050 [1,2]. Yet, multidrug resistant bacteria are in closer proximity to us than we think. Furthermore, in both more and less developed countries, multi-drug resistant bacteria are often detected in humans and animals [3]. In fact, antibiotic resistance genes have been detected in wastewater and its treatment plants, fresh produce, and even ambient air [[3], [4], [5], [6]]. Alarmingly, antibiotic resistant bacterial strains including Methicillin-resistant Staphylococcus aureus have been detected in minced meat samples [7]. This being said, we are far from keeping up with antimicrobial resistance. Since the 1960s, only two antibiotic classes have been introduced to the market [8]. Accordingly, the quest for novel antibacterial compounds is one of paramount importance. Furthermore, chemotherapy resistance is another global health challenge that significantly hinders the success of cancer treatment [9].This hurdle makes tumour cells intrinsically resistant to chemotherapy and this limits the effectiveness of treatment. To surmount this resistance, there is a need to develop new classes of antitumor drugs with minimal toxicities and novel mechanisms of action.

Targeting deoxyribonucleic acid (DNA) is an efficient therapeutic strategy to hinder microbial or cancerous cells division whether through inserting intercalators between DNA bases, alkylating and cleaving DNA, or binding to its minor groove as well as major groove [[10], [11], [12], [13], [14], [15], [16]]. With their reported antitumor, antibacterial, antiviral, and antiparasitic properties [17], DNA minor groove binders (MGBs) pose an advantageous niche to explore. In general, these agents may induce permanent damage or temporary inactivation of DNA depending on whether they bind in an irreversible or reversible manner with DNA [18]. Following the discovery of the DNA minor groove binder, distamycin, numerous research groups have embarked on developing different classes of DNA minor groove binders (Fig. 1) for various therapeutic purposes such as Wilson [19,20], Thurston [21,22], Lown [23,24], Suckling [[25], [26], [27], [28], [29], [30], [31]], and Dervan [[32], [33], [34], [35]] research groups. Some of the MGBs were formulated as organometallic complexes, which feature metal centres coordinated to organic ligands that effectively recognize and stabilize specific DNA sequences [[36], [37], [38]]. Notably, certain MGBs were reported to be effective against Mycobacterium tuberculosis, Acanthamoeba castellanii, and Leishmania donovani [17,39,40] and two MGB compounds, ridinilazole [[41], [42], [43]] and MGB-BP-3 [44] have recently been approved for Phase III clinical trials for the treatment of Clostridioides difficile infections. Tallimustine and Brostallicin showed a promising antitumor activity in phase I and II clinical trials, however, the observed myelotoxicity was the key factor for the cessation of clinical trials [45].

The unique functions of DNA in various bioprocesses make it an important target in modern drug design. In the past, the early approaches of drug discovery targeted the whole pathogen without an understanding of cell biology. There was only a few discoveries of natural products, such as penicillin by Fleming in 1929, that shed some light on the mechanism of antibiotics [46]. With the advances in biochemistry and the instrumental methods in the mid-twentieth century, targeting particular enzymes and receptors became more feasible [47]. The discovery of the secondary structure of DNA by Watson and Crick in 1953 [48] paved the way for the development of therapeutics designed to target DNA, addressing illnesses related to its replication including cancerous, viral and bacterial diseases [18,[49], [50], [51], [52], [53]].

Biophysical and biochemical characterizations of drug-nucleic acids complexes play a pivotal role in the exploration of 3D structure, sequence selectivity and the molecular basis of nucleic acids interaction with small molecules. Among these techniques, NMR spectroscopy, isothermal titration calorimetry (ITC), circular dichroism and DNase footprinting [54] have been employed to study the interaction of the DNA minor groove binders, thiazotropsin A and AIK-18-51, [12,15,25,55] which were developed as analogues of the natural product, distamycin. The findings of these investigations showed that both compounds bind selectively with the 5′-ACTAGT-3′ binding site as anti-parallel side by side dimers. Therefore, in the current study, the DNA duplex sequence d(CGACTAGTCG)2 was selected as a target for the investigated analogues of AIK-18-51.

Modern approaches of drug discovery have focused on the concept of structure-based drug design [56], which aims to design drugs based on our prior knowledge of the interactions between a lead compound and the target binding site. This requires solving the 3D structure of the complex between a lead compound and the target binding site using analytical techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), or NMR spectroscopy. Moreover, this process involves employing a molecular modelling tool in which the experimental 3D structure of the complex is used as a basis to design novel compounds that may bind to the target site [[56], [57], [58]]. The approach of structure-based drug design expedites the drug discovery process and decreases experimental costs by reducing the number of investigated ligands, as these ligands are designed based on the experimental 3D structure of the target binding site. Thus, this rational approach of drug design enhances the success rate of lead identification/optimization [59].

In a previous study, we solved the NMR structure (Fig. 2) of the complex between the distamycin analogue “AIK18-51” and a 10-mer DNA sequence “d(CGACTAGTCG)2” (PDB code:2mne), [25], and the interaction was fully characterized using a variety of biophysical techniques [25,27]. In another study, we investigated the structural features that dictate the preferred orientation of MGBs in the minor grooves with respect to the 3′- and 5′-ends [15]. The findings of this study revealed that the position of the basic tail moiety and the orientation of the amide groups of the ligand with respect to the 5′–3′-ends; determine the preferred orientation of the ligand dimer in the minor grooves. The amide links between the aromatic rings of the ligand are not only important to form hydrogen bonds with the DNA bases, but also are essential to maintain curvature of the ligand to match the helical structure of DNA in the minor groove. Since hydrogen bonds are directional and their strength is greatly influenced by the distance between the hydrogen bond acceptor and donor, the amide links of the ligand must be correctly oriented in the minor groove with respect to the 5′- and 3′-ends to be capable of forming hydrogen bonds with DNA bases.

In the current study, we used the 3D NMR structure (2mne) to design and synthesize 11 MGB analogues (Table 1) to the lead compound “AIK18-51”. The tail and head positions in the AIK18-51 structure were modified to include either dimethylamine or morpholine moieties at the tail position. These basic cationic groups are important for DNA binding via electrostatic interactions. Furthermore, the pka's of these moieties affect the ionization state of MGB compounds at physiological pH, and this has an implication on their cell permeability and biological activity. Different aromatic rings were used at the head position including phenyl, naphthyl, thiazole and imidazole, in addition to a variety of linkage groups such as amide, alkene, alkyne and thioether (Fig. 2C). These modifications aimed to improve the ligand's lipophilicity, which is important for biological activity. Moreover, the structural modifications were mainly done on the terminal head, tail, and amide linkage positions to maintain the crescent shape of the ligand, which is crucial for DNA minor groove binding. For example, the incorporation of a rigid triple bond (possess a linear geometry) in the centre of the MGB structure disrupts its crescent shape, which is essential for complementing the helical nature of the binding site. Consequently, such structures were excluded during the initial stages of ligand design and hence were not considered for the synthesis.

In the present study, we evaluated the antitumor activity of the designed MGB compounds through the NCI screening protocol. The efficacy of the prepared MGBs was also tested against various multidrug resistant bacterial strains through bactericidal, cytopathogenicity, and cytotoxicity assays. The minimum inhibitory concentrations of these agents were also determined. Furthermore, molecular docking, molecular dynamic simulations, DNA melting, and ITC studies were employed to investigate the binding mode/interactions of the most active MGBs with the DNA duplex d(CGACTAGTCG)2. The structural features of MGBs and their relationship to the antibacterial and antitumor activity were also discussed. The findings of this study could be useful for future optimization of MGBs as potential antitumor and antibacterial agents.