New peptidic non-covalent direct thrombin inhibitors (DTIs) were designed by generating peptide lead compounds derived from the substrate sequence Phe(P3)-Pro(P2)-Arg(P1). The free energy of interaction between each ligand and thrombin was calculated with the built-in molecular mechanics force field (MMFF) provided by the docking software SCULPT (Accelrys). During the original screening, the hexapeptides [D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-P2'-P3'-CONH2] and pentapeptides [D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-P2'-CONH2] were used as scaffolds for developing the optimized final tetrapeptide lead sequence, D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH2. Once the lead tetrapeptide scaffold was found to have higher affinity for thrombin than the hexa and pentapeptides, based on structure-activity relationship (SAR) studies on thrombin inhibition conducted in vitro, new peptide candidate inhibitors were further designed as derivatives of the tetrapeptide motif D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH2. New peptide sequences were developed by varying the P1' positions both with L and D natural or non-natural amino acids, covering a wide range of chemical structures. Trials for optimization of P3 position were further performed with un-natural amino-acids, such as D-3,3-di-Phenylalanine, trans and dihydrocinnamic acids, (L)/(D)-Tic [1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid], (L)/(D)-Thi [Thienylalanine] and D-Naphthylalanine (D-Nal). The protein template used in all molecular docking experiments was the structure of human a-thrombin in complex with the covalent inhibitor PPACK (PDB entry 1ABJ). The thrombin:peptide complex was minimized using the SCULPT built-in molecular mechanics force field (MMFF94). After each round of minimization, the free energy of interaction (scoring function) was assessed using both Van der Waals and electrostatic force fields.

Molecular docking and in silico structure-based design of D-(X)-Pro-D-Arg-derived direct thrombin inhibitors

244th National Meeting (2012)

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