Large-scale comparison of four binding site detection algorithms.

A large-scale evaluation and comparison of four cavity detection algorithms was carried out. The algorithms SiteFinder, fpocket, PocketFinder, and SiteMap were evaluated on a protein test set containing 5416 protein-ligand complexes and 9900 apo forms, corresponding to a subset of the set used earlier for benchmarking the PocketFinder algorithm. For the holo structures, all four algorithms correctly identified a similar amount of pockets (around 95%). SiteFinder, using optimized parameters, SiteMap, and fpocket showed similar pocket ranking performance, which was defined by ranking the correct binding site on rank 1 of the predictions or within the first 5 ranks of the predictions. On the apo structures, PocketFinder especially and also SiteFinder (optimized parameters) performed best, identifying 96% and 84% of all binding sites, respectively. The fpocket program predicts binding sites most accurately among the algorithms evaluated here. SiteFinder needed an average calculation time of 1.6 s compared with 2 min for SiteMap and around 2 s for fpocket.

Solvent interactions and conformational choice in a core N-glycan segment: gas phase conformation of the central, branching trimannose unit and its singly hydrated complex.

The intrinsic conformational preferences and structures of the branched trimannoside, alpha-phenyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (which contains the same carbohydrates found in a key subunit of the core pentasaccharide in N-glycans) and its singly hydrated complex, have been investigated in the gas phase isolated at low temperature in a molecular beam expansion. Conformational assignments of their infrared ion dip spectra, based on comparisons between experiment and ONIOM (B3LYP/6-31+G(d):HF/6-31G(d)) and single-point MP2 calculations have identified their preferred structures and relative energies. The unhydrated trimannoside populates a unique structure supported by two strong, central hydrogen bonds linking the central mannose unit (CM), and its two branches (3M and 6M) closely together, through a cooperative hydrogen-bonding network: OH4(CM)-->OH6(3M)-->OH6(6M). A closely bound structure is also retained in the singly hydrated oligosaccharide, with the water molecule bridging across the 3M and 6M branches to provide additional bonding. This structure contrasts sharply with the more open, entropically favored trimannoside structure determined in aqueous solution at 298 K. In principle this structure can be accessed from the isolated trimannoside structure by a simple conformational change, a twist about the alpha(1,3) glycosidic linkage, increasing the dihedral angle psi[C1(3M)-O3(3M)-C3(CM)-C2(CM)] from approximately 74 degrees to approximately 146 degrees to enable accommodation of a water molecule at the centrally bound site occupied by the hydroxymethyl group on the 3M ring and mediation of the water-linked hydrogen-bonded network: OH4(CM) -->OH(W)-->OH6(6M). The creation of a "water pocket" motif localized at the bisecting axis of the trimannoside is strikingly similar to the structure of more complex N-glycans in water, suggesting perhaps a general role for the "bisecting" OH4 group in the central (CM) mannose unit.

Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor.

Inhibitors of HIV protease have been shown to have antiapoptotic effects in vitro, yet whether these effects are seen in vivo remains controversial. In this study, we have evaluated the impact of the HIV protease inhibitor (PI) nelfinavir, boosted with ritonavir, in models of nonviral disease associated with excessive apoptosis. In mice with Fas-induced fatal hepatitis, Staphylococcal enterotoxin B-induced shock, and middle cerebral artery occlusion-induced stroke, we demonstrate that PIs significantly reduce apoptosis and improve histology, function, and/or behavioral recovery in each of these models. Further, we demonstrate that both in vitro and in vivo, PIs block apoptosis through the preservation of mitochondrial integrity and that in vitro PIs act to prevent pore function of the adenine nucleotide translocator (ANT) subunit of the mitochondrial permeability transition pore complex.

Structure-based drug design: docking and scoring.

This review gives an introduction into ligand - receptor docking and illustrates the basic underlying concepts. An overview of different approaches and algorithms is provided. Although the application of docking and scoring has led to some remarkable successes, there are still some major challenges ahead, which are outlined here as well. Approaches to address some of these challenges and the latest developments in the area are presented. Some aspects of the assessment of docking program performance are discussed. A number of successful applications of structure-based virtual screening are described.

Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant.

Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.

AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis.

Cyclophilin A (CypA) was determined to interact with apoptosis-inducing factor (AIF) by mass spectroscopy, coimmunoprecipitation, pull-down assays, and molecular modeling. During the initial, caspase-independent stage of chromatin condensation that accompanies apoptosis, AIF and CypA were found to coimmunolocalize in the nucleus. Recombinant AIF and CypA proteins synergized in vitro in the degradation of plasmid DNA, as well as in the capacity to induce DNA loss in purified nuclei. The apoptogenic cooperation between AIF and CypA did not rely on the CypA peptidyl-prolyl cis-trans isomerase activity. In Cyp-expressing cells, AIF overexpression augmented apoptotic chromatinolysis. The AIF-dependent large-scale DNA fragmentation was less pronounced in CypA knockout cells as compared to controls. AIF mutants lacking the CypA-binding domain were inefficient apoptosis sensitizers in transfection experiments. Moreover, AIF failed to sensitize CypA knockout cells to apoptosis induction, and this defect in the AIF response was reversed by reintroduction of the CypA gene into CypA-deficient cells. In summary, AIF and CypA collaborate in chromatinolysis.

Virtual screening to enrich a compound collection with CDK2 inhibitors using docking, scoring, and composite scoring models.

Docking programs can generate subsets of a compound collection with an increased percentage of actives against a target (enrichment) by predicting their binding mode (pose) and affinity (score), and retrieving those with the highest scores. Using the QXP and GOLD programs, we compared the ability of six single scoring functions (PLP, Ligscore, Ludi, Jain, ChemScore, PMF) and four composite scoring models (Mean Rank: MR, Rank-by-Vote: Vt, Bayesian Statistics: BS and PLS Discriminant Analysis: DA) to separate compounds that are active against CDK2 from inactives. We determined the enrichment for the entire set of actives (IC50 < 10 microM) and for three activity subsets. In all cases, the enrichment for each subset was lower than for the entire set of actives. QXP outperformed GOLD at pose prediction, but yielded only moderately better enrichments. Five to six scoring functions yielded good enrichments with GOLD poses, while typically only two worked well with QXP poses. For each program, two scoring functions generally performed better than the others (Ligscore2 and Ludi for GOLD; QXP and Jain for QXP). Composite scoring functions yielded better results than single scoring functions. The consensus approaches MR and Vt worked best when separating micromolar inhibitors from inactives. The statistical approaches BS and DA, which require training data, performed best when distinguishing between low and high nanomolar inhibitors. The key observation that all hit rate profiles for all four activity intervals for all scoring schemes for both programs are significantly better than random, is evidence that docking can be successfully applied to enrich compound collections.

Entropy from state probabilities: hydration entropy of cations.

Entropy is an important energetic quantity determining the progression of chemical processes. We propose a new approach to obtain hydration entropy directly from probability density functions in state space. We demonstrate the validity of our approach for a series of cations in aqueous solution. Extensive validation of simulation results was performed. Our approach does not make prior assumptions about the shape of the potential energy landscape and is capable of calculating accurate hydration entropy values. Sampling times in the low nanosecond range are sufficient for the investigated ionic systems. Although the presented strategy is at the moment limited to systems for which a scalar order parameter can be derived, this is not a principal limitation of the method. The strategy presented is applicable to any chemical system where sufficient sampling of conformational space is accessible, for example, by computer simulations.

Building up key segments of N-glycans in the gas phase: intrinsic structural preferences of the alpha(1,3) and alpha(1,6) dimannosides.

The intrinsic conformer specific vibrational spectra of two important subunits of the core pentasaccharide of N-linked glycans, the alpha(1,3) and alpha(1,6) dimannosides, have been recorded in the gas phase. Coupling these measurements with a computational exploration of their conformational landscapes has enabled their conformational assignment and has identified characteristic vibrational signatures associated with particular conformational families-including those that do or do not display inter-ring hydrogen bonding across the glycosidic linkage. In addition, the IR spectra of the monosaccharide moieties provide benchmarks, through which the conformational assignments can be refined. This introduces a general concept of modularity and secondary structure in oligosaccharides--essential for the success of similar studies on larger oligosaccharides in the future.

Hydrogen bonding and cooperativity in isolated and hydrated sugars: mannose, galactose, glucose, and lactose.

The conformation of phenyl-substituted monosaccharides (mannose, galactose, and glucose) and their singly hydrated complexes has been investigated in the gas phase by means of a combination of mass selected, conformer specific ultraviolet and infrared double resonance hole burning spectroscopy experiments, and ab initio quantum chemistry calculations. In each case, the water molecule inserts into the carbohydrate at a position where it can replace a weak intramolecular interaction by two stronger intermolecular hydrogen bonds. The insertion can produce significant changes in the conformational preferences of the carbohydrates, and there is a clear preference for structures where cooperative effects enhance the stability of the monosaccharide conformers to which the water molecule chooses to bind. The conclusions drawn from the study of monosaccharide-water complexes are extended to the disaccharide lactose and discussed in the light of the underlying mechanisms that may be involved in the binding of carbohydrate assemblies to proteins and the involvement, or not, of key structural water molecules.

A three-dimensional model of Suppressor Of Cytokine Signalling 1 (SOCS-1).

Suppressor Of Cytokine Signalling 1 (SOCS-1) is one of the proteins responsible for the negative regulation of the JAK-STAT pathway triggered by many cytokines. This important inhibition involves complex formation between SOCS-1 and JAK2, which requires particular structural domains (KIR, ESS and SH2) on SOCS-1. A three-dimensional theoretical model of SOCS-1 is presented here. The model was generated by the application of different modelling techniques, including threading, structure-based modelling, surface analysis and protein docking. The structure accounts for the interactions between SOCS-1 and two other key proteins in the JAK-STAT pathway, namely JAK2 and Elongin BC. The proposed model for the interaction between SOCS-1 and JAK2 suggests that the SOCS-1 suppress the kinase activity of JAK2 by obstructing the catalytic groove of the tyrosine kinase. Subsequent interaction of the JAK-SOCS complex with Elongin BC was also modelled. A sequence and structural comparison between the SH2 domain of SOCS-1 and the SH2 domains of other proteins highlights key residues that could be responsible for SOCS-1 specificity. Currently available mutational data are evaluated. The results are consistent with the experimental data and they provide deeper insights into the inhibitory function of SOCS-1 at a molecular level.

Prediction of the structure of human Janus kinase 2 (JAK2) comprising JAK homology domains 1 through 7.

A theoretical model of human Janus kinase 2 (JAK2) comprising all seven Janus homology domains is presented. The model was generated by application of homology modelling approaches. The three-dimensional structure contains, starting from the N-terminus, FERM (4.1, ezrin, radixin, moesin), SH2 (Src homology region 2), tyrosine kinase-like, and tyrosine kinase domains. The predicted inter-domain orientation in JAK2 is discussed and the currently existing mutational data for Janus kinases are evaluated. Structural details of the SH2 and the FERM domains are presented. The predictions indicate that the SH2 domain is not fully functional. A number of hydrophobic amino acids of the FERM domain that are predicted to be involved in the constitutive association with the cytokine receptors are highlighted. The model gives new insights into the structure-function relationship of this important protein, and areas that could be investigated by mutation studies are highlighted.

Inhibitors of vitamin D hydroxylases: structure-activity relationships.

Aiming at new drugs to efficiently treat diseases, in which either increased or decreased levels of active vitamin D are desirable, we have designed some 400 structurally different azole-type inhibitors and examined their capacity to selectively block vitamin D metabolism by CYP24 or synthesis by CYP27B, in human keratinocytes. Based on resulting data, we built pharmacophore models of the active sites using commercial software. The overlay of potent selective compounds indicated similar docking modes in the two-substrate pockets and allowed for identification of bioactive conformations. Superimposing these bioactive conformations with low energy conformers of 25(OH)D(3) suggested that the substrate-mimicked by strong inhibitors in size, shape and lipophilic character-binds to both enzymes in 6s-trans configuration. Pharmacophoric models implied a similar geometry of the substrate sites, nevertheless specific features of CYP24 and CYP27B could be defined. Bulky substituents in alpha-position to the azole caused selectivity for CYP24, whereas bulky substituents in beta-position could result in selectivity for CYP27B. Moreover, studies with small sterically restricted inhibitors revealed a probable location of the 3-OH-group of 25(OH)D(3) in CYP27B. In the absence of crystal structures, our inhibitors are valuable tools to model and understand the active sites of vitamin D hydroxylases, resulting in the design of powerful, selective therapeutics.

Novel scoring functions comprising QXP, SASA, and protein side-chain entropy terms.

Novel scoring functions that predict the affinity of a ligand for its receptor have been developed. They were built with several statistical tools (partial least squares, genetic algorithms, neural networks) and trained on a data set of 100 crystal structures of receptor-ligand complexes, with affinities spanning 10 log units. The new scoring functions contain both descriptors generated by the QXP docking program and new descriptors that were developed in-house. These new descriptors are based on solvent accessible surface areas and account for conformational entropy changes and desolvation effects of both ligand and receptor upon binding. The predictive r(2) values for a test set of 24 complexes are in the 0.712-0.741 range and RMS prediction errors in the 1.09-1.16 log K(d) range. Inclusion of the new descriptors led to significant improvements in affinity prediction, compared to scoring functions based on QXP descriptors alone. However, the QXP descriptors by themselves perform better in binding mode prediction. The performance of the linear models is comparable to that of the neural networks. The new functions perform very well, but they still need to be validated as universal tools for the prediction of binding affinity.

Structure, modelling and dynamic behaviour of aza- and azaoxamacrocyclic ligands derived from (R,R)-1,2-diaminocyclohexane.

Investigations into the conformational behaviour of macrocyclic ligands 5 and 6 derived from (R,R)-1,2-diaminocyclohexane have been undertaken using molecular modelling, single crystal X-ray diffraction and variable temperature 1H NMR spectroscopy. These have revealed that the lowest energy conformers in both cases do not possess the expected C2-element of symmetry, which can only be accessed at higher temperatures. Instead both molecules exist as C1-conformers at room temperature and in the solid state. In solution a range of dynamic exchange processes is observed which result, in part from the inherent strain in these fused bicyclic systems. An unexpected but characteristic feature of the C1-symmetric conformers is highlighted by the presence of a signal at unexpectedly low field in their 1H NMR spectra due to the interaction of two of the sulfonyl oxygen atoms with one of the bridgehead hydrogen atoms.

Probing the glycosidic linkage: UV and IR ion-dip spectroscopy of a lactoside.

The beta(1-->4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.

Assessment of docking poses: interactions-based accuracy classification (IBAC) versus crystal structure deviations.

Six docking programs (FlexX, GOLD, ICM, LigandFit, the Northwestern University version of DOCK, and QXP) were evaluated in terms of their ability to reproduce experimentally observed binding modes (poses) of small-molecule ligands to macromolecular targets. The accuracy of a pose was assessed in two ways: First, the RMS deviation of the predicted pose from the crystal structure was calculated. Second, the predicted pose was compared to the experimentally observed one regarding the presence of key interactions with the protein. The latter assessment is referred to as interactions-based accuracy classification (IBAC). In a number of cases significant discrepancies were found between IBAC and RMSD-based classifications. Despite being more subjective, the IBAC proved to be a more meaningful measure of docking accuracy in all these cases.