Discovery of triazole analogues as selective IDO1 inhibitors: Dual mechanistic effects on kynurenine pathway suppression and apoptosis in cancer cells

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key immunoregulatory enzyme and a prospective target for Cancer Immunotherapy, while triggering cell death through Apoptosis is a complementary Anticancer strategy. As a result, a number of triazole analogues were developed, synthesized, and tested as promising dual immunomodulatory and apoptosis-inducing agents. Among the compounds, 3b emerged as the most potent with a GI₅₀ of 0.72 μM against HepG2, compared to Imatinib (GI₅₀ = 1.92 μM). It also showed higher cytotoxicity against HepG2 (liver) than non-cancerous Vero cells, with a selectivity index of 121.58. In the enzymatic experiment, 3b demonstrated higher affinity against IDO1, with a submicromolar IC₅₀ of 0.093 μM. Further, it exhibited 199.13-fold and 313-fold selectivity against IDO1 compared to IDO2 and TDO. It also significantly and dose-dependently restored IDO1-mediated immune suppression by lowering kynurenine production and activating IL-2. In addition, 3b significantly and dose-dependently increased total Apoptosis in the Annexin V assay. Furthermore, the mechanism of cytotoxicity was studied through Apoptosis, as evidenced by increased levels of cytochrome c, Caspase-3, caspase-9, Caspase-8 (excluding), and PARP-1 in HepG2 cells. Consistent with this, higher expression of cleaved apoptotic markers (except Caspase-8) in Western blots at various concentrations validated the activation of the intrinsic mitochondrial apoptotic pathway. Moreover, docking, MD simulation, and DFT also suggest IDO1 as the molecular target responsible for the observed immunomodulatory effects. Collectively, these results suggest further biological investigation to explore the translational potential of compound 3b.

Apoptosis biomarkers; Enzymatic assay; IDO1 enzyme; Insilico study; SRB assay; Synthesis.