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A Marine Anticancer Cinnamyloxyl Derivative with Unique Binding Sites at Carbonic Anhydrase IX (CAIX) Inhibits

Shailaja Vommi Lakshmipathy¹, Christina Vijayaraghavan Sathyanathan¹, Mohanapriya Dandapani Chinambedu²

¹Department of Biotechnology, Faculty of Biomedical Sciences and Technology (FBMS&T), Sri Ramachandra Institute of Higher Education and Research (SRIHER), Deemed to be University (DU), Porur, Chennai 600116, Tamil Nadu, India.

Pharmaceuticals (Basel, Switzerland)

|January 28, 2026

Abstract:

Background: Many inhibitors have been discovered to target hypoxia-induced carbonic anhydrase IX (CAIX) due to its critical role in lung cancers. This study discovers a novel compound, 3-(E-3,4-dihydroxycinnamaoyloxyl)-2-hydroxypropyl-9Z,12Z-octadeca-9,12-dienoate, which is produced by the seagrass Cymodocea serrulata and has binding sites at CAIX that are distinct from those of current inhibitors. Methods: Compound and reference drug treatment for cell lines; Cell viability: MTT; Staining: Ao/PI/DAPI; MMP shifts and cell cycle: FACS; Gene and protein expression of CAIX, BAX, BAD: qPCR and Western blotting. Results: The compound binds to the CAIX protein, raises extracellular pH, and kills A549 cells [IC₅₀: 11.61 µM], producing results that are lower than those of the reference drug doxorubicin [13.7 µM]. The substance depolarised the electrical potential of the mitochondrial membrane, caused S-phase arrest, and fragmented DNA. Additionally, it downregulated CAIX by 0.9 times while increasing apoptotic mRNA, BAX and BAD by 5.2 and 3.08 times, respectively, as demonstrated by qPCR. Between 0 and 24 h, the untreated hypoxic cells had a ΔpHe of 0.15, but the compound-treated cells had a ΔpHe of 0.6 indicative of intracellular acidosis. MD simulations verify the stability of the CAIX-C1 complex for more than 100 ns, and in silico studies show a strong binding affinity of the molecule to CAIX [-7.55 kcal/mol]. Conclusions: This implies that the amount of extracellular alkalosis was increased by the combination of treatment and hypoxia induction. As a result, when the cells were deprived of O₂, the compound provided less defense against ROS. The compound binds to the glutamine and alanine amino acids at positions 242 and 392, respectively, at the central Zn atom of CAIX, which sets it apart from conventional sulphonamide CAIX inhibitors. This naturally occurring compound may be a potent CAIX inhibitor with newer binding sites, which could help treat hypoxic lung cancers.

Insights

A novel compound from seagrass effectively inhibits carbonic anhydrase IX (CAIX) in lung cancer cells, showing distinct binding sites and potential as a new therapeutic agent. This compound induces cell death and apoptosis, offering a promising alternative to existing treatments.

Area of Science:

Biochemistry
Molecular Biology
Pharmacology

Background:

Carbonic anhydrase IX (CAIX) is a key target in hypoxia-driven lung cancers.
Existing CAIX inhibitors have limitations, necessitating novel therapeutic strategies.
Seagrasses are a source of unique bioactive compounds with potential pharmaceutical applications.

Purpose of the Study:

To discover and characterize a novel CAIX inhibitor from the seagrass *Cymodocea serrulata*.
To investigate the compound's mechanism of action and efficacy against lung cancer cells.
To explore its potential as a therapeutic agent for hypoxic lung cancers.

Main Methods:

Cell viability assays (MTT) and apoptosis staining (Ao/PI/DAPI).
Flow cytometry (FACS) for mitochondrial membrane potential (MMP) and cell cycle analysis.
Quantitative PCR (qPCR) and Western blotting for gene and protein expression (CAIX, BAX, BAD).
Molecular dynamics (MD) simulations and in silico docking for binding affinity analysis.

Main Results:

The novel compound demonstrated potent CAIX inhibition and cytotoxicity against A549 lung cancer cells (IC50: 11.61 µM).
It induced mitochondrial dysfunction, S-phase cell cycle arrest, and DNA fragmentation, leading to apoptosis.
The compound upregulated pro-apoptotic genes (BAX, BAD) and downregulated CAIX expression.
Molecular simulations confirmed stable binding to CAIX at unique sites (Gln242, Ala392), distinct from sulfonamide inhibitors.

Conclusions:

The seagrass-derived compound acts as a novel CAIX inhibitor with a unique binding mechanism.
It effectively induces apoptosis and inhibits proliferation in lung cancer cells.
This compound represents a promising lead for developing new treatments for hypoxic lung cancers.

Read full analysis

Keywords:

Cymodocea serrulata anticancer apoptosis carbonic anhydrase IX (CAIX inhibitor)extracellular pH (ΔpHe)marine compound

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