Colorectal cancer (CRC) ranks as the third most deadly cancer worldwide. While drugs like 5-fluorouracil (5-FU), cisplatin, and irinotecan (CPT-11) form the backbone of treatment, their effectiveness is increasingly limited by a process called chemoresistance - where cancer cells adapt and survive drug treatment.
Irinotecan (CPT-11) is one of the most important chemotherapy drugs for CRC. It works by inhibiting an enzyme called topoisomerase I (TOP1), which cancer cells need to manage the physical stress in DNA during rapid replication. However, CPT-11 itself is inactive - it must be converted by the body into its active form SN-38, and this conversion is inefficient, achieving only 2-8% efficiency in patients.
Researchers have therefore sought to develop improved compounds that can bypass the limitations of CPT-11 while potentially engaging additional cancer-killing mechanisms. This study introduces ZBH-01, a newly designed drug that directly inhibits TOP1 without needing activation, and that also targets a second vulnerability in cancer cells: structures in DNA called G-quadruplexes.
DNA G-quadruplexes (G4s) are unusual four-stranded structures that form in DNA-rich regions of guanine nucleotides. Unlike the standard double helix, G4s are particularly abundant in the promoter regions of cancer-driving genes such as hTERT (which controls telomerase), MYC, and KRAS - genes that are overactive in most colorectal cancers.
When G4 structures are stabilized by drugs, the transcription factors that normally bind the DNA to switch on these cancer-promoting genes cannot access their binding sites. This effectively silences oncogene expression, making G4 stabilization an attractive anti-cancer strategy. However, prior G4-targeting drugs had poor stability in the body and unclear effects in real tumors.
The hTERT gene is the catalytic core of telomerase, an enzyme that allows cancer cells to replicate indefinitely by maintaining their chromosome ends (telomeres). It is overexpressed in over 85% of all cancers. Its promoter contains conserved G4 motifs, making it a prime target for G4-stabilizing drugs like ZBH-01.
ZBH-01 was engineered through structure-based optimization of the camptothecin scaffold - the same chemical backbone as irinotecan. The key modifications added an N-methylpiperazine group to dramatically improve solubility (more than 2,600-fold better than SN-38) and an N-formylglycine linker to fine-tune TOP1 binding.
Unexpectedly during development, these structural changes were found to also enable G4 stabilization - the compound could bind and stabilize DNA G-quadruplex structures. This serendipitous dual activity created a single molecule capable of targeting two independent cancer vulnerabilities simultaneously: TOP1 inhibition that causes DNA breaks, and G4 stabilization that silences cancer-promoting genes.
Structural studies using NMR spectroscopy revealed that ZBH-01's crescent-shaped molecular scaffold stacks onto the outer G-tetrad layer of the hTERT G4 structure. This binding physically displaces the transcription factors SP1 and MYC from the promoter region, preventing them from turning on hTERT expression in cancer cells.
In anti-cancer potency tests across eight cancer cell lines (including colorectal cancer lines LS174T, HCT116, HCT8, and SW480), ZBH-01 showed activity equal to SN-38 and significantly superior to CPT-11. Unlike CPT-11, ZBH-01 does not need metabolic conversion - it kills cancer cells directly.
Using a specialized assay called the immunocomplex of enzyme (ICE) assay, researchers confirmed that ZBH-01 robustly stabilizes TOP1-DNA complexes (the mechanism by which TOP1 inhibitors kill cancer cells). Its performance was superior to CPT-11 and comparable to SN-38, validating its direct TOP1-inhibitory activity.
Molecular docking analysis showed ZBH-01 binds within the TOP1-DNA cleavage site through multiple stabilizing interactions including hydrogen bonds with residues Asp533 and Thr718, and additional contacts unique to ZBH-01's modified structure. These extra binding contacts explain its enhanced potency compared to the unmodified camptothecin core.
Cellular senescence is a permanent growth arrest state - a cell enters it and stops dividing but does not die. Inducing senescence in cancer cells is a powerful strategy because it permanently removes them from the pool of dividing, spreading cells. RNA sequencing showed that ZBH-01 altered the expression of nearly 5,600 genes in CRC cells, with the most enriched pathways being cellular senescence, DNA damage response, and telomere maintenance.
Among the most significantly downregulated genes were hTERT and MYC - both known regulators of telomere maintenance and cell cycle progression with G4-rich promoters. ZBH-01 also reduced Cyclin B1, Cyclin A2, CDC25A, FOXM1, and other cell cycle drivers, effectively putting the brakes on cancer cell proliferation from multiple angles.
Network analysis identified eight central hub genes regulated by ZBH-01, including CDK2, CDK4, CHEK1, TERT, and MYC. Importantly, analysis of clinical CRC data showed that high hTERT expression significantly correlates with reduced overall survival, making ZBH-01's ability to suppress hTERT particularly clinically relevant.
The most compelling finding was ZBH-01's performance in drug-resistant CRC models. In cisplatin-resistant CRC cells, ZBH-01 was 14-fold more potent than CPT-11 and 7-fold more potent than SN-38. In 5-FU-resistant cells, it outperformed CPT-11 by 61-fold and SN-38 by 2.4-fold.
This exceptional activity in resistant cells is explained by ZBH-01's dual mechanism: when cancer cells develop resistance to one drug by altering a single pathway, ZBH-01 simultaneously attacks a second pathway. Cells that become resistant to TOP1-based DNA damage still remain vulnerable to G4-mediated transcriptional suppression of hTERT and MYC.
Telomere shortening and increased DNA damage markers (gamma-H2AX foci) were confirmed in ZBH-01-treated cells, consistent with both TOP1 inhibition causing replication-associated DNA breaks and G4 stabilization preventing telomere maintenance. This combination of genomic destabilization and transcriptional suppression creates multiple layers of anti-cancer activity.
This study establishes dual TOP1/G4 targeting as a promising strategy for colorectal cancer treatment, particularly for patients who have failed standard chemotherapy. Rather than switching from one single-target drug to another, ZBH-01 engages two independent cancer vulnerabilities in the same molecule.
The convergence of DNA damage induction (via TOP1 trapping) with epigenetic/transcriptional modulation (via G4 stabilization) represents a more comprehensive attack on cancer cell survival. Cancers typically develop resistance by becoming adept at repairing one type of damage, but dealing with simultaneous damage from different mechanisms is much harder.
Future studies will need to evaluate ZBH-01 in animal models and eventually clinical trials to confirm safety and efficacy. The drug's dramatically improved solubility compared to SN-38 is an important advantage for clinical formulation. These findings establish a proof-of-concept that rational integration of multiple mechanisms into a single drug molecule can overcome the persistent problem of chemoresistance in colorectal cancer.