A Marine Anticancer Cinnamyloxyl Derivative with Unique Binding Sites at Carbonic Anhydrase IX (CAIX) Inhibits Adenocarcinomic A549 Cells

<b>Background:</b> 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 <i>Cymodocea serrulata</i> and has binding sites at CAIX that are distinct from those of current inhibitors. <b>Methods:</b> Compound and reference drug treatment for cell lines; <b>Cell viability:</b> MTT; <b>Staining</b>: Ao/PI/DAPI; <b>MMP shifts and cell cycle</b>: FACS; <b>Gene and protein expression</b> of CAIX, <i>BAX, BAD</i>: qPCR and Western blotting. <b>Results:</b> 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, <i>BAX</i> and <i>BAD</i> 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]. <b>Conclusions:</b> 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.