Conformations of trypsin-bound amidine inhibitors of blood coagulant factor Xa by double REDOR NMR and MD simulations.

Double rotational-echo double resonance (double REDOR) has been used to investigate the bound conformations of (13)C,(15)N,(19)F-labeled factor Xa inhibitors to bovine trypsin. Carbon-fluorine dipolar couplings were measured by (13)C{(19)F} REDOR with natural-abundance background interferences removed by (13)C{(15)N} REDOR. The conformations of the bound inhibitors were characterized by molecular dynamics (MD) simulations of binding restrained by double REDOR-determined intramolecular C-F distances. A symmetrical bisamidine inhibitor and an asymmetrical monoamidine-monoamine inhibitor of the same general shape had distinctly different conformations in the bound state. According to the MD models, these differences arise from specific interactions of the amidine and amine groups with the active-site residues of trypsin and nearby water molecules.

Predicting relative binding affinities of non-peptide HIV protease inhibitors with free energy perturbation calculations.

The relative binding free energies in HIV protease of haloperidol thioketal (THK) and three of its derivatives were examined with free energy calculations. THK is a weak inhibitor (IC50 = 15 microM) for which two cocrystal structures with HIV type 1 proteases have been solved [Rutenber, E. et al., J. Biol. Chem., 268 (1993) 15343]. A THK derivative with a phenyl group on C2 of the piperidine ring was expected to be a poor inhibitor based on experiments with haloperidol ketal and its 2-phenyl derivative (Caldera, P., personal communication). Our calculations predict that a 5-phenyl THK derivative, suggested based on examination of the crystal structure, will bind significantly better than THK. Although there are large error bars as estimated from hysteresis, the calculations predict that the 5-phenyl substituent is clearly favored over the 2-phenyl derivative as well as the parent compound. The unfavorable free energies of solvation of both phenyl THK derivatives relative to the parent compound contributed to their predicted binding free energies. In a third simulation, the change in binding free energy for 5-benzyl THK relative to THK was calculated. Although this derivative has a lower free energy in the protein, its decreased free energy of solvation increases the predicted delta delta G (bind) to the same range as that of the 2-phenyl derivative.

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ¿(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.

Stereoselectivity and enantioselectivity of glutathione S-transferase toward stilbene oxide substrates.

Isozyme 4-4 of rat liver glutathione S-transferase catalyzes the stereoselective addition of glutathione to the oxirane carbon of R-absolute configuration of cis-stilbene oxide, 2, to give 98 +/- 2% of the (1S,2S)-1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane product with a turnover number (kc) of 0.22 s-1. The two enantiomers of trans-stilbene oxide, 3, are somewhat poorer substrates for the enzyme. Enantioselective addition of glutathione to 3 proceeds with turnover numbers of 0.12 s-1 and 0.023 s-1 for the (R,R,)- and (S,S)-antipodes, respectively.