hCAIX-Targeted Inhibitor–Photosensitizer Conjugates for Enhanced Photodynamic Therapy of Hypoxic Tumors

Human carbonic anhydrase IX (hCAIX) is a zinc metalloenzyme that catalyzes CO₂ hydration and plays a crucial role in tumor progression, including proliferation, metastasis, and adhesion. Its overexpression in hypoxic tumors and extracellular localization make it an attractive therapeutic target.1 In a previous study, compound 1, containing a benzenesulfonamide moiety, showed low-nanomolar inhibition of hCAIX.2 Here, we report two conjugates (2 and 3) designed for hCAIX-targeted photodynamic therapy (PDT). These molecules link the scaffold of compound 1 to a benzophenothiazinium (EtNBS) photosensitizer via different linkers, enabling near-infrared (NIR)-activated type-I Photo Dynamic Therapy while simultaneously binding to the enzyme active site, thus potentially limiting tumor compensation mechanisms associated with hCAIX activity. Compounds 2 and 3, along with controls 4 and 5 lacking the sulfonamide group or containing the chromophore Rhodamine B, were synthesized and characterized by NMR, MS, UV–vis, and fluorescence. Emission fluorescence and fluorescence polarization binding studies showed nanomolar affinity of 2, 3, and 5 toward hCAIX. These results were further supported by CO₂ hydrase stopped-flow assays using acetazolamide as reference inhibitor. ROS production was evaluated using a combination of ROS-specific assays. EtNBS-containing compounds showed efficient superoxide anion (O₂•⁻) generation, consistent with a type-I PDT mechanism, whereas negligible ROS production was observed for compound 5. In vitro, compounds 2 and 3 selectively targeted CAIX-expressing MDA-MB-231 cells over MCF-7 controls, exhibiting strong PDT efficacy at low-nanomolar concentrations with negligible dark cytotoxicity.