Targeting Metabolic and pH Regulatory Pathways in Cancer via Dual Inhibition of Nicotinamide Phosphoribosyltransferase and Carbonic Anhydrases IX and XII

We report the first dual inhibitors of the tumor-associated carbonic anhydrases (hCA IX and XII) and nicotinamide phosphoribosyltransferase (NAMPT), designed to target metabolic and pH-regulatory mechanisms critical for tumor growth and resistance. Enzymatic assays identified hits showing potent nanomolar inhibition of both targets and superior efficacy compared to reference inhibitors. Computational studies elucidated key ligand–target interactions and structure–activity relationship. In high-throughput screening cellular assays, several compounds outperformed monotherapies with strong antiproliferative effects against renal carcinoma, glioblastoma, and colorectal cancer cells under normoxic and hypoxic conditions, and limited toxicity toward nonmalignant cells. In three-dimensional (3D) spheroids, compound 45 reduced the cumulative spheroid area approximately 10-fold more than the single-target inhibitors FK866 or SLC-0111 and induced apoptosis through NAD depletion, mitochondrial dysfunction, and suppression of ERK/mTOR signaling. These results support dual hCA IX/XII–NAMPT inhibition as an effective strategy to impair tumor growth and survival under hypoxic stress.