Multipolar interactions in the D pocket of thrombin: large differences between tricyclic imide and lactam inhibitors

Abstract

Two series of tricyclic inhibitors of the serine protease thrombin, imides (±)-1–(±)-8 and lactams (±)-9–(±)-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the backbone CO moiety of Asn98 to the free enthalpy of protein–ligand complexation. The lactam derivatives are much more potent and more selective inhibitors (K_i values between 0.065 and 0.005 µM, selectivity for thrombin over trypsin between 361- and 1609-fold) than the imide compounds (K_i values between 0.057 and 23.7 µM, selectivity for thrombin over trypsin between 3- and 67-fold). The increase in potency and selectivity is explained by the favorable occupancy of the P-pocket of thrombin by the additional isopropyl substituent in the lactam derivatives. The nature of the substituent on the benzyl ring filling the D pocket strongly influences binding potency in the imide series, with K_i values increasing in the sequence: F < OCH₂O < Cl < H < OMe < OH < N_pyr ≪ Br. This sequence can be explained by both steric fit and the occurrence of orthogonal multipolar interactions with the backbone CO moiety of Asn98. In contrast, the substituent on the benzyl ring hardly affects the ligand potency in the lactam series. This discrepancy was clarified by the comparison of X-ray structures solved for co-crystals of thrombin with imide and lactam ligands. Whereas the benzyl substituents in the imide inhibitors are sufficiently close (≤3.5 Å) to the CO group of Asn98 to allow for attractive orthogonal multipolar interactions, the distances in the lactam series are too large (≥4 Å) for attractive dipolar contacts to be effective.