Fig. 7.9 In the “SAR by NMR” method, ligands with weak affinity to a protein, in this case stromelysin, are sought from a large complex mixture. 15N-labeled protein is used and so-called 1H-15N HSQC spectra are measured. If a ligand such as acetohydroxamic acid 7.1 becomes apparent through a shift in the resonance of specific amino acids that protrude into the binding pocket, the binding geometry can be deduced (a, d). Later the binding site is saturated with these ligands. Further NMR measurements are carried out to identify ligands for neighboring binding positions. These are revealed by the shift in the resonances of neighboring amino acids. That is how 4-cyano-4′-hydroxybiphenyl 7.2 was discovered (b, d). A chemical coupling of both hits 7.1 and 7.2 with a –CH2CH2O– linker produced 7.3, which is a nanomolar inhibitor of the protease stromelysin (c, e).