Cancer is increasingly recognized as a significant global health threat and ranks among the foremost causes of mortality [1]. The development and progression of cancer involve various factors [2], such as gene mutations, tissue microenvironments, immune system responses, environmental effects, aging, metabolism, biological rhythms, the nervous system, and microbiomes. Tumor cell evolution occurs through multiple stages, marking the gradual transition from normal to invasive cancer cells. These tumor cells exhibit distinct alterations in several areas, including sustained proliferative signals, evasion of growth suppression, resistance to programmed cell death, angiogenesis, invasion, metastasis, and other key characteristics that promote tumor progression [[3], [4], [5]]. While the hallmark traits of a tumor dictate its malignant phenotype and ongoing proliferation of cancer cells, they also create vital targets for therapeutic interventions [[6], [7], [8], [9], [10], [11]]. By focusing on these traits, tumor biological processes can be effectively disrupted, enhancing the accuracy and effectiveness of treatment (Fig. 1).

Topo is an essential group of enzymes that modulate superhelical structure by cleaving and reconnecting DNA strands, ensuring genomic stability during DNA replication, transcription, and repair [12]. Human topoisomerases are categorized into type I and type II based on their action mechanisms (Fig. 2). Topo I alleviates superhelical tension by temporarily cutting single-stranded DNA [13], Topo II (including isoforms IIα/IIβ) relies on ATP hydrolysis energy to cleave double-stranded DNA to unwind or relieve superhelical tension on chromosomes [14].

Specifically, (1) Topo I comprises 765 amino acids organized into five functional regions. Among these, the Capping Domain (CAP) and the Catalytic Domain (CAT) form the catalytic core structure, which remains open without DNA binding, resulting in a V-shaped lumen that obstructs DNA from reaching the active site [15,16]. Upon the entry of the DNA double strand into the lumen, the CAP domain closes around the CAT, forming a ring through the flexible movement of the hinge region. In this closed configuration, the CAT domain catalyzes a tyrosine attack on the DNA phosphodiester bond (the highly conserved Tyr723 within the CAT region is a critical residue for this cleavage reaction [17]), producing a 3′-phosphotyrosine intermediate that cleaves the single strand. In contrast, the rotation of the DNA strand alleviates superhelical stress, ultimately completing the reconnection through a transesterification reaction. The Linker region, enriched in hydrophobic residues, takes on an α-helical conformation that grants CAT spatial flexibility, aiding in regulating the catalytic pocket's open state. Conversely, the N-terminal and C-terminal regions partake in non-catalytic roles such as mediating protein-protein interactions [18], determining subcellular localization, and binding to regulatory factors (Fig. 3A).

(2)Topo IIα is a homodimeric protein; in contrast to Topo IIβ, it is overexpressed in proliferating cells and can serve as a biomarker for cell proliferation [19]. Topo IIα consists of four distinct parts: the N-gate (which contains the ATP-binding site of the GHKL ATPase family), the DNA-gate, the C-gate, and the C-terminal structural domain. The first three are involved in the Topo II catalytic cycle. At the same time, the C-terminal structural domain is primarily responsible for nuclear localization and DNA geometric recognition during the catalytic cycle [20]. Topo II undergoes an ATP-dependent gating mechanism during catalysis. First, the substrate DNA binds at the DNA gate, closing the N-gate and inducing a conformational change via ATP binding. It is followed by G-segment DNA double-strand cleavage, allowing the T-segment DNA to traverse through the cleavage and enter the DNA gate, completing the untangling process. Ultimately, ATP hydrolysis triggers the opening of the C-gate, releasing the T-segment DNA while the G-segment is reattached and closed [21], thus completing the entire unwinding cycle (Fig. 3B).

Notably, the ATPase domain (N-gate) and the DNA-binding/cleavage domain (DNA-gate) are connected by a 27-residue α-helical linker that mediates long-range allosteric communication. Conformational oscillation of the DNA-gate between “closed” and “pre-open” states induces a corkscrew-like rotation of the N-gate, thereby coordinating the catalytic cycle. Moreover, the previously uncharacterized C-terminal domain (CTD) linker region (residues 1192–1215) has been resolved, which lies adjacent to the G-segment DNA and stabilizes its bent conformation, significantly enhancing catalytic activity and suggesting a direct role of the CTD in fine-tuning enzyme function [22].

Research indicates that Topo is a vital nuclease involved in the structural remodeling and separation of chromosomes during mitosis. It is essential for chromosome condensation, spindle attachment, and the separation of sister chromatids, making it a key factor for cell cycle regulation, particularly in the G2/M phase [23]. When Topo fails to function correctly, it leads to chromosome abnormalities and cell cycle arrest, and it is closely linked to genome instability and the development of malignant tumors. This relationship underscores its significance as a primary molecular target for anticancer treatments [24].

Under typical physiological conditions, topoisomerase-induced DNA strand breaks are reversible regulatory events that can be swiftly repaired to maintain an orderly cell cycle. In contrast, tumor cells often exhibit significantly elevated levels of Topo I and Topo IIα isoforms due to their heightened proliferative activity, accumulating DNA breaks, and resulting in genomic instability. Additionally, the abnormal activation of Topo I and II can disrupt the cell's ability to recognize and repair DNA damage, adversely influencing the homeostatic control of critical signaling pathways like p53 and ATM/ATR, facilitating the cells' malignant transformation [25]. Topo I plays a crucial role in DNA replication and repair, and its elevated expression has been observed in various malignancies, including breast cancer. Several studies suggest that high Topo I levels correlate with increased invasiveness and metastatic potential in tumor cells, potentially through enhanced genomic instability and disrupted DNA damage responses. However, while this association positions Topo I as a promising prognostic biomarker and therapeutic target, its causal role in driving metastasis remains to be fully elucidated [26]. Topo IIα is frequently overexpressed in various solid tumors, such as breast, lung, gastric, and bladder cancers, and is often regarded as a biomarker for poor prognosis [27,28].

According to their therapeutic mechanisms, Topo inhibitors can be broadly classified into two categories (Fig. 4A). (1) Poison inhibitors: These agents stabilize the Topo–DNA cleavage complex [29], thereby preventing the breakage–reunion process, inducing DNA breaks, and ultimately leading to cell death. Well-known examples include the Topo I inhibitors topotecan and irinotecan, as well as the Topo II inhibitors etoposide and doxorubicin [30,31]. (2) Catalytic inhibitors: In particular, catalytic inhibitors of Topo II interfere with key enzymatic steps of the catalytic cycle, thereby disrupting topological regulation. Representative compounds include Merbarone (inhibits the cleavage activity of Topo II), ICRF193, Dexrazoxane, and ICRF-154 (block ATP hydrolysis), Aclarubicin and Suramin (inhibit DNA binding), as well as Salvicine, Novobiocin, and QAPI (compete with ATP binding) [32]. Currently, the majority of Topo inhibitors in clinical use belong to the poison type, which form stable “enzyme–drug–DNA” cleavage complexes. It leads to irreversible DNA damage, blockade of the G1–M transition [33,34], and induction of apoptosis (Fig. 4B). By contrast, reports on catalytic inhibitors targeting Topo, particularly Topo I, remain relatively scarce; nevertheless, this provides new potential targets for the development of small-molecule inhibitors.

Although topoisomerase inhibitors have achieved significant success in cancer therapy, their development continues to face challenges, including limited selectivity, severe toxicity, drug resistance, and unfavorable pharmacokinetic properties. The high homology between Topo IIα and Topo IIβ makes it difficult for inhibitors to achieve isoform- and cell-specific selectivity, thereby leading to pronounced off-target effects. Of particular concern, most clinically used topoisomerase inhibitors exert their activity by stabilizing the DNA cleavage complex, but this mechanism also causes uncontrollable DNA damage and subsequent toxic side effects. For example, doxorubicin acts on both Topo IIα and Topo IIβ, resulting in dose-dependent cardiotoxicity [35]. At the same time, etoposide disrupts Topo IIα-mediated chromosome segregation and markedly increases the risk of secondary leukemia [36]. In addition, topoisomerase inhibitors are hindered by drug resistance and suboptimal pharmacological properties, including overexpression of efflux pumps and drug inactivation in the tumor microenvironment. For instance, camptothecin derivatives readily undergo hydrolytic ring opening under acidic conditions (pH < 6.5), thereby reducing efficacy in the hypoxic core of solid tumors [37].

Isoform-selective inhibition: targeting Topo IIα while sparing Topo IIβ to reduce cardiotoxicity; for example, a recent study identified an ‘obex’ pocket within the Topo II ATPase domain and developed the allosteric inhibitor Topobexin, which selectively acts on Topo IIβ by serving as a physical obstacle that blocks the conformational changes required for ATP hydrolysis, thereby exerting allosteric catalytic inhibition [38].

Catalytic inhibitors: avoiding accumulation of the DNA cleavage complex, thereby mitigating DNA damage while maintaining antitumor activity.

Dual- or multi-targeting strategies: combining topoisomerase inhibition with other oncogenic pathways (e.g., microtubule polymerization, HDAC, PARP-mediated repair, MYC-driven transcription) to reduce reliance on direct DNA scission and alleviate DNA toxicity burden.

Structural and pharmacokinetic optimization: improving solubility and molecular stability while minimizing toxic metabolites, thus retaining antitumor efficacy while controlling the extent of DNA damage.

Building upon these strategies, this review systematically summarizes recent progress in the structural optimization of Topo I/II inhibitors. Further, it explores emerging trends such as catalytic inhibitors and dual-target drug design. We aim to provide a comprehensive perspective to support the rational design and application of next-generation small-molecule topoisomerase inhibitors.