Lead optimization of 3-indoleacetonitrile identifies candidate compounds with improved anti-influenza activity and safety profiles.

INTRODUCTION: Influenza A virus (IAV) remains a major respiratory pathogen that causes substantial morbidity, mortality, and socioeconomic burden worldwide. Although several antiviral agents are available for clinical use, their effectiveness is often compromised by the emergence of drug-resistant variants, limited therapeutic windows, and potential adverse effects. Small-molecule antivirals represent an important strategy for influenza treatment; however, improving antiviral efficacy while minimizing toxicity remains a major challenge in drug development. In this study, we sought to identify candidate low-toxicity derivatives structurally related to 3-indoleacetonitrile, a previously characterized compound with anti-influenza activity. METHODS: Candidate compounds were selected through a computational screening strategy integrating structural similarity analysis, toxicity prediction, and drug-likeness assessment. Cytotoxicity was evaluated by CCK-8 assays and morphological observation in A549 cells. Antiviral activity was assessed using a recombinant luciferase-expressing PR8 influenza virus. Promising candidates were subsequently evaluated in a lethal influenza mouse model challenged with 10 × LD50 PR8 virus. Body weight changes, survival rates, lung pathology, viral burden, and serum AST/ALT levels were analyzed to determine antiviral efficacy and safety in vivo. RESULTS: Four candidates-Indole-3-ethanol, Indole-3-acetamide, 3-Indoleacetic acid, and Acetohydroxamic acid-were identified. Cytotoxicity analyses revealed that Indole-3-ethanol and Indole-3-acetamide exhibited substantially lower toxicity than the parent compound 3-indoleacetonitrile, maintaining high cell viability at concentrations exceeding 640 μM. Morphological examination further confirmed their improved safety profiles. In antiviral assays, Indole-3-ethanol and Indole-3-acetamide demonstrated potent inhibition of IAV replication, while 3-Indoleacetic acid and Acetohydroxamic acid showed little or no detectable antiviral activity. In vivo, Indole-3-ethanol treatment alleviated body weight loss, delayed mortality, reduced pulmonary pathological injury, and significantly decreased viral loads in the lungs of PR8-infected mice. In addition, AST/ALT analysis indicated lower toxicity of Indole-3-ethanol and Indole-3-acetamide compared with 3-indoleacetonitrile. CONCLUSION: Structural optimization of the lead compound 3-indoleacetonitrile led to the identification of derivatives with improved antiviral activity and reduced toxicity. Among the compounds evaluated, Indole-3-ethanol exhibited the most favorable balance between efficacy and safety, demonstrating potent anti-influenza activity in both cell-based and animal models. These findings support the utility of computationally guided lead optimization and identify Indole-3-ethanol as a promising candidate for further development as an anti-influenza therapeutic.