In the green quadrant of the image above, we focus on Basic Strategy 2: cases where the protein structure is known, but no known ligand structure is available. Here, we need to conduct fragment or compound library screening at the start of a structure-based drug design. Since most proteins currently have AlphaFold-generated 3D structures, virtual screening can also be applied to these models after constructing QSAR and pharmacophore models. In addition, as in the Strategy 1 case, we can use biophysical screening methods like NMR spectroscopy or weak-affinity chromatography (WAC). Once we identify suitable binders, we can enhance our QSAR model and also clone, express, purify, and crystallize the protein in complex with the best ligands, moving us toward Basic Strategy 1. This approach validates the binding mode, offers direct insights into stabilizing interactions within the protein-ligand complex, and aids structure-based discovery and design. We should also note that having a ligand at the protein’s binding site often makes crystallization of the complex easier, which might otherwise be difficult. If we cannot obtain a crystal, we can continue with our QSAR and pharmacophore models, use, e.g., NMR spectroscopy to verify binding modes, and perform biochemical and cell-based assays to confirm activity. In other words, we continue to use a pure ligand-based drug design strategy (Basic Strategy 3).
