Comprehensive Identification and Mechanistic Evaluation of Novel DHODH Inhibitors as Potent Broad-Spectrum Antiviral Agents

<b>Background/Objectives</b>: This study identifies novel dihydroorotate dehydrogenase (DHODH) inhibitors exhibiting potent broad-spectrum antiviral agents, particularly against influenza A virus (A/PR/8/34(H1N1)) and SARS-CoV-2. <b>Methods</b>: Structure-based virtual screening of 1.6 million compounds (ChemDiv and TargetMol databases) yielded 10 candidates, with compounds <b>6</b>, <b>9</b>, and <b>10</b> demonstrating significant anti-influenza activity (IC₅₀ = 4.85 ± 0.58, 7.35 ± 1.65, and 1.75 ± 0.28 μM, respectively). Building on these, molecular hybridization principles and scaffold hopping principles were applied to design and synthesize six novel compounds (<b>11</b>-<b>16</b>) through cyclization, coupling, and carboxylate deprotection. Prior to subsequent biological assays, the molecular structures of each compound were elucidated by NMR spectroscopy and MS. Their antiviral activities were subsequently assessed against both influenza virus and SARS-CoV-2. The compound <b>11</b>, demonstrating the most potent antiviral activity, was further subjected to surface plasmon resonance (SPR) analysis to assess its binding affinity for human DHODH. <b>Results</b>: Compound <b>11</b> emerged as the most potent DHODH inhibitor (<i>K</i>_D = 6.06 μM), exhibiting superior broad-spectrum antiviral activities (IC₅₀ = 0.85 ± 0.05 μM, A/PR/8/34(H1N1); IC₅₀ = 3.60 ± 0.67 μM, SARS-CoV-2) to the reported DHODH inhibitor (Teriflunomide<b>,</b> IC₅₀ = 35.02 ± 3.33 μM, A/PR/8/34(H1N1); IC₅₀ = 26.06 ± 4.32 μM, SARS-CoV-2). Mechanistic evaluations via 100 ns MD simulations and QM/MM calculations revealed stable binding interactions, particularly hydrogen bonds with GLN47 and ARG136, while alanine scanning mutagenesis confirmed these residues' critical roles in binding stability. <b>Conclusions</b>: This work identifies compound <b>11</b> as a potent broad-spectrum antiviral compound, offering a promising strategy for broad-spectrum antiviral therapy against RNA viruses by depleting pyrimidine pools essential for viral replication.