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Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three-dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid-binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP-cholylglycine complex and with related lipid-binding proteins. Protein structures were determined in solution by using two-dimensional (2D)- and 3D-homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine-binding site. The bile acid is deeply buried within ILBP with its flexible side-chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein-binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system.

A series of 138 nonchiral 3-amidinobenzyl-1H-indole-2-carboxamides and analogues as inhibitors of the blood coagulation enzyme factor Xa (fXa) were designed, synthesized, and investigated by X-ray structure analysis and 3D quantitative structure-activity relationship (QSAR) studies (CoMFA, CoMSIA) in order to identify important protein-ligand interactions responsible for biological affinity and selectivity. Several compounds from this series are highly potent and selective inhibitors of this important enzyme linking extrinsic and intrinsic coagulation pathways. To rationalize biological affinity and to provide guidelines for further design, all compounds were docked into the factor Xa binding site. Those docking studies were based on X-ray structures of factor Xa in complex with literature-known inhibitors. It was possible to validate those binding modes by four X-ray crystal structures of representative ligands in factor Xa, while one ligand was additionally crystallized in trypsin to rationalize requirements for selective factor Xa inhibition. The 3D-QSAR models based on a superposition rule derived from these docking studies were validated using conventional and cross-validated r(2) values using the leave-one-out method and repeated analyses using two randomly chosen cross-validation groups plus randomization of biological activities. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which were found to correspond to experimentally determined factor Xa binding site topology in terms of steric, electrostatic, and hydrophobic complementarity. Subsets selected as smaller training sets using 2D fingerprints and maximum dissimilarity methods resulted in 3D-QSAR models with remarkable correlation coefficients and a high predictive power. The final quantitative SAR information agrees with all experimental data for the binding topology and thus provides reasonable activity predictions for novel factor Xa inhibitors.

A set of 90 novel 2-(arylsulfonyl)-1,2,3, 4-tetrahydroisoquinoline-3-carboxylates and -hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) was designed, synthesized, and investigated by 3D-QSAR techniques (CoMFA, CoMSIA) and X-ray structure analysis. Docking studies of a reference compound are based on crystal structures of MMP-8 complexed with peptidic inhibitors to propose a model of its bioactive conformation. This model was validated by a 1. 7 A X-ray structure of the catalytic domain of MMP-8. The 3D-QSAR models based on a superposition rule derived from these docking studies were validated using conventional and cross-validated r2 values using the leave-one-out method, repeated analyses using two randomly chosen cross-validation groups plus randomization of biological activities. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which were found to correspond to experimentally determined MMP-8 catalytic site topology in terms of steric, electrostatic, and hydrophobic complementarity. Subsets selected as smaller training sets using 2D fingerprints and maximum dissimilarity methods resulted in 3D-QSAR models with remarkable correlation coefficients and a high predictive power. This allowed to compensate the weaker zinc binding properties of carboxylates by introducing optimal fitting P1' residues. The final QSAR information agrees with all experimental data for the binding topology and thus provides clear guidelines and accurate activity predictions for novel MMP-8 inhibitors.

Rab proteins are a family of approximately 25kD ras-related GTPases which are associated with distinct intracellular membranes where they control vesicle traffic between intracellular compartments. The late-endosomal rab protein rab7(1-207), (lacking only the C-terminal lipids of the native molecule) and three C-terminal truncated constructs rab7(1-202), rab7(1-197) and rab7(1-182) were purified using an E. coli expression system. The C-terminal tail region of rab proteins is of special interest because it is thought to target rab proteins to particular intracellular membranes. A comparison of TOCSY-NMR spectra from intact rab7(1-207) and the tail-less construct rab7(1-182) suggested that much of the C-terminal tail is flexible in solution. The GTP hydrolysis, and GDP association and dissociation rates for all the truncated and intact constructs were similar, showing that the tail region of rab7(1-207) has little influence on the hydrolysis and exchange rates of the nucleotide.

Using a focused screening approach, acyl ureas have been discovered as a new class of inhibitors of human liver glycogen phosphorylase (hlGPa). The X-ray structure of screening hit 1 (IC50 = 2 microM) in a complex with rabbit muscle glycogen phosphorylase b reveals that 1 binds at the AMP site, the main allosteric effector site of the dimeric enzyme. A first cycle of chemical optimization supported by X-ray structural data yielded derivative 21, which inhibited hlGPa with an IC50 of 23 +/- 1 nM, but showed only moderate cellular activity in isolated rat hepatocytes (IC50 = 6.2 microM). Further optimization was guided by (i) a 3D pharmacophore model that was derived from a training set of 24 compounds and revealed the key chemical features for the biological activity and (ii) the 1.9 angstroms crystal structure of 21 in complex with hlGPa. A second set of compounds was synthesized and led to 42 with improved cellular activity (hlGPa IC50 = 53 +/- 1 nM; hepatocyte IC50 = 380 nM). Administration of 42 to anaesthetized Wistar rats caused a significant reduction of the glucagon-induced hyperglycemic peak. These findings are consistent with the inhibition of hepatic glycogenolysis and support the use of acyl ureas for the treatment of type 2 diabetes.

The GTP/GDP conformational switch of members of the rab family of ras-related GTP-ases control specific intracellular vesicle transport pathways. We report the crystallization of the late-endosomal rab protein rab7, in both GTP and GDP conformations. X-ray data from crystals of rab7(1-207) GppNHp (i.e., intact rab7, without C-terminal bound lipid, complexed with a non-hydrolysable GTP analog), rab7(1-197)GppNHp and rab7(1-197)GDP were collected to 1.9A (0 degree C), 1.76A (100 degrees K) and 1.75A (100 degrees K) respectively. Rab7-GDP crystals diffract to at least 1.35A.

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5·9. The crystals diffract to a resolution of 3·0 A and have space group P212121 with unit cell dimensions a = 74·8 A , b = 76·0 A , c = 157·7 A . There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0·014 A and 3·2 °, respectively, and a crystallographic `R factor of 17%.