The activities of the new compounds identified in screening campaigns need to be verified using biochemical and cell-based assays. The best compounds are then taken to the next optimization cycle. The process of optimization, often designated as the design-make-test-analyze cycle, can involve many cycles of synthesis-activity assessment, crystallization, and structure determination, followed by the use of structural data to guide the synthesis of new compounds. During these cycles, multiple protein-ligand complex structures are determined, e.g., by X-ray crystallography, NMR spectroscopy, or cryo-EM.
