Medicinal Chemistry Strategies Targeting Viral Proteases: From Classical Design to Next-Generation Therapeutics

Viral proteases are central targets in antiviral drug discovery and development because they play essential roles in viral replication and maturation. Although protease inhibitors have achieved major clinical success, traditional design strategies face challenges, including resistance development, poor oral exposure of early peptidomimetics, and off-target toxicity of highly reactive covalent warheads. Classical approaches, such as peptidomimetics, macrocyclization, and covalent warhead engineering, are discussed alongside contemporary strategies, including allosteric modulation and targeted protease degradation via proteolysis-targeting chimeras (PROTAC) technology. Particular emphasis is placed on how these strategies address key obstacles, such as resistance evolution, selectivity, metabolic stability, and oral bioavailability. Several quantitative case studies have also demonstrated the growing significance of computational tools in contemporary antiviral discovery. For SARS-CoV-2 main protease (Mpro), these workflows were enabled by the rapid availability of high-resolution experimental crystal structures of the target protein. The evolution of a weak fragment (Kd ≈ 1.7 mM; ΔG ≈ −3.6 kcal/mol) into a covalent inhibitor (QUB-00006-Int-07) with enzymatic inhibition (IC 50 ≈ 830 nM) was successfully guided by molecular dynamics (MD) simulations and absolute binding free energy calculations. This was subsequently confirmed experimentally using NMR, ESI-MS, and FRET assays. Furthermore, out of 25 computationally prioritized candidates with Ki values less than 4 μM, 15 active Mpro inhibitors were identified using accelerated free-energy perturbation-based repurposing campaigns. Long-range allosteric pathways connecting the catalytic site to resistance-associated regions and experimentally verified allosteric pockets have also been discovered using dynamic nonequilibrium MD. Together, these integrated in silico approaches enable the early prioritization of high-affinity ligands, mechanistic understanding of resistance, and significant reduction of late-stage attrition in antiviral drug discovery. Through detailed case studies on SARS-CoV-2 main protease (Mpro), Zika virus NS2B–NS3 protease, and Dengue virus NS2B–NS3 protease, the review illustrates how medicinal chemistry principles translate molecular insights into clinically relevant antivirals. Finally, a forward-looking development roadmap is proposed that integrates potency, selectivity, pharmacokinetics, manufacturability, and resistance management toward the goal of broad-spectrum, durable, and adaptable protease-targeted therapeutics development. • Protease inhibitors remain the most clinically validated antiviral class, from HIV therapy to oral SARS-CoV-2 drugs • Macrocyclization, warhead tuning, and non-peptidic scaffolds improve metabolic stability, permeability, and oral bioavailability • Structure-guided optimization enabled conversion of weak fragments (Kd ≈ 1.7 mM) into potent SARS-CoV-2 Mpro inhibitors with enzymatic activity around IC 50 ≈ 830 nM. • Computational prioritization campaigns identified 15 active inhibitors from 25 candidates with Ki < 4 μM, demonstrating the impact of free-energy–guided screening. • Medicinal-chemistry optimization produced clinical inhibitors such as nirmatrelvir (Ki ≈ 3.11 nM) through warhead tuning and scaffold rigidification.