The substrate envelope strategy: Structural basis for resistance mitigation in antiviral drug design

Drug resistance remains a major hurdle in antiviral drug development. This review highlights the "substrate envelope hypothesis", a structure-based drug design (SBDD) paradigm that constrains the spatial boundaries and binding footprint of inhibitors within the conserved active site volume defined by natural viral substrates. By restricting inhibitor interactions to evolutionarily conserved residues, this approach ensures that mutations abrogating drug binding simultaneously impair natural substrate processing. This imposes an insurmountable fitness cost on the virus, thereby mitigating resistance. Through the analysis of viral enzyme-substrate co-crystal structures, we systematically review the application of this framework in human immunodeficiency.virus (HIV) protease, hepatitis C virus (HCV) NS3/4A protease, influenza virus (IFV) neuraminidase (NA), and severe acute respiratory Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) main protease, illustrating how substrate envelope-guided design achieves high potency while circumventing resistance. Additionally, we extend this strategy from traditional protease targets to non-protease enzymes. By integrating molecular dynamics and structural biology, this framework offers a rational approach for developing next-generation antivirals.