Dual Inhibitors Targeting DNA and Histone Deacetylases

Over the last decade, chemotherapeutics and targeted drugs have been the most common and effective approaches for cancer treatment. Due to activation of compensatory mechanisms and multiple signaling pathways, cancer cells become resistant to single-target drugs.1 Thus, drug combination treatment gradually attracts the interest of researchers by its advantages such as synergistic effects and less resistance.2 Furthermore, lower doses of the individual drugs are used, which means fewer side effects than single-drug treatments.3 Currently, drug combination therapieshavemoved toward the dualor multi-targeting drugs,4 which has raised scientific interest through some advantages such as the more predictable pharmacokinetics and the less risks of drug interactions.5 DNA is themost important storage unit of genetic information and plays key roles in replication, transcription, and translation.6 Histone deacetylases (HDACs) get rid of acetyl groups from histones and regulate the structure of chromatin. It is reported that thestructureofchromatinbecomes relaxedafter inhibition of HDAC, leading to DNA damage.7 Several dual DNA/HDAC inhibitors have been reported, which possess obvious anticancer activity.8 This article describes the dual DNA/HDAC inhibitors and their biology activity studies.


Introduction
Over the last decade, chemotherapeutics and targeted drugs have been the most common and effective approaches for cancer treatment. Due to activation of compensatory mechanisms and multiple signaling pathways, cancer cells become resistant to single-target drugs. 1 Thus, drug combination treatment gradually attracts the interest of researchers by its advantages such as synergistic effects and less resistance. 2 Furthermore, lower doses of the individual drugs are used, which means fewer side effects than single-drug treatments. 3 Currently, drug combination therapies have moved toward the dual-or multi-targeting drugs, 4 which has raised scientific interest through some advantages such as the more predictable pharmacokinetics and the less risks of drug interactions. 5 DNA is the most important storage unit of genetic information and plays key roles in replication, transcription, and translation. 6 Histone deacetylases (HDACs) get rid of acetyl groups from histones and regulate the structure of chromatin. It is reported that the structure of chromatin becomes relaxed after inhibition of HDAC, leading to DNA damage. 7 Several dual DNA/HDAC inhibitors have been reported, which possess obvious anticancer activity. 8 This article describes the dual DNA/HDAC inhibitors and their biology activity studies.

DNA and DNA Binders
Covalent and noncovalent interactions are main processes happening between small molecules and DNA. The interac-tion between alkylating agents and DNA is a covalent interaction (►Fig. 1). The formation of covalent bonds between alkylating agents and DNA leads to the inhibition of replication or transcription. Earlier, doctors used nitrogen mustards, the first-generation alkylating agents, for therapy of leukemias and lymphomas. The nitrogen mustard molecules attack the N7 of guanine to form an aziridinium ion, thereby causing DNA interstrand cross-linking. Other well-known alkylators include platinum derivatives, 9 oxazaphosphorines, 10 nitrosoureas, 11 triazenes, 12 hydrazines, 13 and so on.
Intercalation and groove binding belong to noncovalent interactions. DNA intercalators could intercalate and stack between the adjacent DNA base pairs, 14 which resulted in elongation of the DNA, 15 finally interrupting the replication, transcription, and DNA repair processes. 16 Topoisomerases (Topo) could combine with DNA to form a reversible covalent Topo-DNA complex and modulate DNA supercoiling. 17 Topo inhibitors could intercalate into DNA base pairs and maintain the structure of the DNA-enzyme cleavable complex. 18 Thus, most of Topo inhibitors are also DNA intercalators (►Fig. 2).
DNA groove binders could bind to the edges of DNA base pairs via reversible noncovalent interactions, 19 yet the conformation of DNA duplex was changeless. 20 To adapt with the shape of the minor groove, minor groove binders (MGBs) are often designed as isohelical and crescent-shaped molecules. The common MGBs are listed in ►Fig. 3.

HDACs and HDAC Inhibitors
As a crucial enzyme in epigenetics regulation, HDACs eliminate the acetyl groups of lysine residues, resulting in a condensed chromatin structure and transcriptional suppression. Eighteen members of human HDACs have been found, and they are subdivided into four classes (►Table 1).
As the structures of various HDACs were elucidated, lots of HDAC inhibitors have been developed. The common pharmacophore model of HDAC inhibitors comprises a cap group, a linker and a zinc-binding group (ZBG). The linker is usually a saturated or unsaturated fatty chain, or an aromatic and heterocyclic ring, which connects the cap group to ZBG and interacts with a hydrophobic channel of active pocket. ZBG, including hydroxamic acid, carboxylic acid, boric acid, and so on, reversibly or irreversibly chelates to Zn 2þ at the bottom of the active pocket.
HDAC inhibitors based on hydroxamic acid are the most representative HDAC inhibitors and three drugs have been approved by Food and Drug Administration. Vorinostat, belinostat, and panobinostat were respectively approved for the treatment of relapsed/refractory cutaneous T cell lymphoma, peripheral T cell lymphoma (PTCL), and multiple myeloma. They are all pan-HDAC inhibitors (►Fig. 4).
Valproic acid (VPA) and benzenebutyric acid are weak inhibitors of class I and class II HDACs (►Fig. 5). Among them, VPA is used for the treatment for epilepsy, bipolar disorder, and migraine.
Entinostat (MS-275) and tacedinaline (CI-994) are class I selective HDAC inhibitors. Entinostat is in the third phase of  clinical treatment for breast cancer. Chidamide is a Chinese original class I selective HDAC inhibitor against relapsed/ refractory PTCL. Their structures are listed in ►Fig. 6.
As a prodrug, romidepsin (►Fig. 7) reduces the disulfide bond to a thiol, reaching the active center of class I HDACs, in turn sequestering Zn 2þ to exert HDAC inhibitory activity.

Dual Inhibitors Targeting DNA and HDACs Dual Inhibitors Based on Nitrogen Mustards
In 2015, Liu et al developed and characterized a novel dualtargeting HDAC/DNA drug based on bendamustine. 21 The representative drug CY190602 showed significantly improved anticancer activity in vitro and in vivo (Scheme 1). Meanwhile, CY190602 was used as a tool to explore the role of HDAC in DNA damage and repair. As a result, it was found that the expression of TYMS, Tip60, CBP, EP300, and MSL1, which participate in DNA synthesis and repair, was related to HDAC activity. These findings provide rationales for dualtargeting inhibitors, to overcome the resistance of cancer cells.
Based on the strategy of combination of nitrogen and hydroxamic acid, Xie et al reported a series of chlorambucil derivatives with a hydroxamic acid tail and assayed the inhibitory activities of these derivatives on DNA and total HDACs. 22 The results showed that vorambucil (Scheme 2) possessed good HDAC1, HDAC2, and HDAC6 inhibitory activities with micromolar level IC 50 values. In addition, vorambucil showed potent antiproliferative activity against A375 cancer cells and could significantly inhibit their colony formation. Meanwhile, vorambucil remarkably affected cancer cell apoptosis and cycle.
In 2018, a dual-targeting inhibitor named chlordinaline was reported (Scheme 3). 23 Chlordinaline exhibited moderate total HDAC inhibitory activity and selectivity and HDAC3

Dual Inhibitors Based on Metal Complexes
In 2009, Griffith et al reported the first Pt complex with dual DNA-binding and HDAC inhibitory activity. 24 Agarose gel electrophoretic assay verified that the Pt complex 3 (►Fig. 8) bound to nucleotides leading to the DNA strands unwrapping. Pt complex 3 showed moderate inhibitory activity against HDAC1 with IC 50 values as low as 1 μmol/L. Furthermore, for A2780P cell lines, Pt complex 3 had a similar cytotoxicity (IC 50 9 μmol/L) as compared with cisplatin (IC 50 3 μmol/L), but for   McGivern et al designed and developed a series of Cu (II) prodrugs containing SAHA and phenanthrene ligands as DNA intercalators (►Fig. 9). 25 It was proved that the complex preferred to intercalate at both A-T-and G-C-rich sequences, resulting in DNA damage by yielding reactive oxygen species. In addition, the prodrug displayed promising antiproliferative effects against two p53-mutated cell lines possessing SK-OV-3 and DU145, with IC 50 values of as low as 1 μmol/L. It was verified by confocal imaging and gene expression analysis that the cytotoxicity of this metallodrug came from an apoptotic pathway.

Dual Inhibitors Based on Intercalators
In 2018, Chen et al reported a new series of acridine hydroxamic acid derivatives targeting both Topo and HDAC. 26 Among these compounds, compound 8c (►Fig. 10) showed the best enzyme inhibitory activity. In addition to having micromolar level enzyme inhibitory activity, it also showed nanomolar IC 50 values against U937 cells. What's more, 8c interacted with DNA and induced U937 apoptosis through both endogenous and exogenous pathways.
Ling et al reported a novel series of hybrid derivatives as dual inhibitors. 27 The most potent compound YL-11c (►Fig. 11) showed good HDAC inhibitory antiproliferative effects in vitro. Meanwhile, YL-11c cleaved both PARP and caspase 3, triggering cancer cell apoptosis. Furthermore, YL-11c enhanced expression of histone H2AX phosphorylation and p-p53 (Ser15), which were usually used as DNA damage markers.

Conclusion
Due to the importance of DNA and HDAC in cancer therapy, some dual inhibitors targeting DNA and HDACs have been developed and evaluated. In this review, we overviewed the newest studies and summarized their biology activity. Several molecules targeting DNA and HDACs showed excellent activity against cancer cell lines. With their excellent activity, they may become hopeful candidates and valuable tools to illuminate their mechanism for cancer therapy.