Identification of a Small Molecule Inhibitor Targeting T Cell Immunoglobulin and Mucin Domain-containing Protein-3 (TIM-3)

TIM-3 (T cell immunoglobulin and mucin-domain containing-3) is an immune checkpoint receptor and a key negative regulator of immune responses. Its interaction with one of its ligands, known as Galectin-9, results in immune suppression in case of cancer and chronic viral infections; however, during autoimmune diseases, impaired TIM-3 signaling can result in loss of immune tolerance and disease exacerbation. This makes the TIM-3/Galectin-9 axis an important therapeutic target for immunotherapy. Although various TIM-3 monoclonal antibodies (mAbs) are under clinical trials, they have several limitations. These include high manufacturing costs and reduced tumor penetration capabilities. However, small molecules can serve as an alternative therapeutic option. They may have oral bioavailability, less harmful effects, and are less expensive. Currently, there are no small molecules under clinical trial. The objective of this study was to identify a novel small-molecule inhibitor that can block the binding between TIM-3 and Galectin-9. Virtual screening was conducted on 10,769 compounds to identify potent small-molecule inhibitors. Their solubility was predicted using various computational tools. The solubility was also experimentally measured in a 0.5% DMSO/PBS buffer, using fluorescence or UV3Vis absorbance-based detection methods. ELISA-based binding and inhibition assays were also conducted. These assays determined the binding affinity between TIM-3 and Galectin-9 and assessed the potential of various small molecules in inhibiting the interaction between Galectin-9 and TIM-3. A total of 5 screened potent compounds were selected for wet-lab experiments. A prominent difference was observed between predicted and experimentally measured solubilities. Galectin-9 showed the strongest binding to TIM-3, and it was selected for further studies. Three independent inhibition experiments were conducted under identical conditions each performed in triplicate. Despite several attempts and various strategies, including preincubation and co-incubation, the previously reported TIM-3/Galectin-9 inhibitor A-41 consistently failed to demonstrate inhibitory activity. Lactose was used as a positive control. It showed reproducible 40-60% inhibition under static in vitro conditions. These results underscore the importance of combining biochemical validation and solubility profiling when screening small molecule immune checkpoint inhibitors in their early stages of development. Additionally, they highlight the limitations of ELISA as a static assay platform and emphasize the need for kinetic binding or the complementation of cell-based techniques for a more precise assessment of inhibitor efficacy.