Structural Fluctuations of Aromatic Residues in an Apo-Form Reveal Cryptic Binding Sites: Implications for Fragment-Based Drug Design.

Cryptic sites are binding pockets that are transiently formed in an apo form or that are induced by ligand binding. The investigation of cryptic sites is crucial for drug discovery, since these sites are ubiquitous in disease-related human proteins, and targeting them expands the number of drug targets greatly. However, although many computational studies have attempted to identify cryptic sites, the detection remains challenging. Here, we aimed to characterize and detect cryptic sites in terms of structural fluctuations in an apo form, investigating proteins each of which possesses a cryptic site. From their X-ray structures, we saw that aromatic residues tended to be found in cryptic sites. To examine structural fluctuations of the apo forms, we performed molecular dynamics (MD) simulations, producing probability distributions of the solvent-accessible surface area per aromatic residue. To detect aromatic residues in cryptic sites, we have proposed a "cryptic-site index" based on the distribution, demonstrating the performance via several measures, such as recall and specificity. Besides, we found that high-ranking aromatic residues were likely to probe concaves in a cryptic site. This implies that such fluctuations provide a profile of scaffolds of compounds with the potential to bind to a particular cryptic site.

Prediction of Passive Membrane Permeability by Semi-Empirical Method Considering Viscous and Inertial Resistances and Different Rates of Conformational Change and Diffusion.

Membrane permeability is an important property of drugs in adsorption. Many prediction methods work well for small molecules, but the prediction of middle-molecule permeability is still difficult. In the present study, we modified a classical permeability model based on Fick's law to study passive membrane permeability. The model consisted of the distribution of solute from water to membrane and the diffusion of solute in each solvent. The diffusion coefficient is the inverse of the resistance, and we examined the inertial resistance in addition to the viscous resistance, the latter of which has been widely used in permeability prediction. Also, we examined three models changing the balance between the diffusion of solute in membrane and the conformational change of solute. The inertial resistance improved the prediction results in addition to the viscous resistance. The models worked well not only for small molecules but also for middle molecules, whose structures have more conformational freedom.

Peripheral blood metabolome predicts mood change-related activity in mouse model of bipolar disorder.

Bipolar disorder is a major mental illness characterized by severe swings in mood and activity levels which occur with variable amplitude and frequency. Attempts have been made to identify mood states and biological features associated with mood changes to compensate for current clinical diagnosis, which is mainly based on patients' subjective reports. Here, we used infradian (a cycle > 24 h) cyclic locomotor activity in a mouse model useful for the study of bipolar disorder as a proxy for mood changes. We show that metabolome patterns in peripheral blood could retrospectively predict the locomotor activity levels. We longitudinally monitored locomotor activity in the home cage, and subsequently collected peripheral blood and performed metabolomic analyses. We then constructed cross-validated linear regression models based on blood metabolome patterns to predict locomotor activity levels of individual mice. Our analysis revealed a significant correlation between actual and predicted activity levels, indicative of successful predictions. Pathway analysis of metabolites used for successful predictions showed enrichment in mitochondria metabolism-related terms, such as "Warburg effect" and "citric acid cycle." In addition, we found that peripheral blood metabolome patterns predicted expression levels of genes implicated in bipolar disorder in the hippocampus, a brain region responsible for mood regulation, suggesting that the brain-periphery axis is related to mood-change-associated behaviors. Our results may serve as a basis for predicting individual mood states through blood metabolomics in bipolar disorder and other mood disorders and may provide potential insight into systemic metabolic activity in relation to mood changes.

A prospective compound screening contest identified broader inhibitors for Sirtuin 1.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Circadian Gene Circuitry Predicts Hyperactive Behavior in a Mood Disorder Mouse Model.

Bipolar disorder, also known as manic-depressive illness, causes swings in mood and activity levels at irregular intervals. Such changes are difficult to predict, and their molecular basis remains unknown. Here, we use infradian (longer than a day) cyclic activity levels in αCaMKII (Camk2a) mutant mice as a proxy for such mood-associated changes. We report that gene-expression patterns in the hippocampal dentate gyrus could retrospectively predict whether the mice were in a state of high or low locomotor activity (LA). Expression of a subset of circadian genes, as well as levels of cAMP and pCREB, possible upstream regulators of circadian genes, were correlated with LA states, suggesting that the intrinsic molecular circuitry changes concomitant with infradian oscillatory LA. Taken together, these findings shed light onto the molecular basis of how irregular biological rhythms and behavior are controlled by the brain.

Synaptosomal-associated protein 25 mutation induces immaturity of the dentate granule cells of adult mice.

BACKGROUND: Synaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown. RESULTS: In this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the "immature DG" (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants. CONCLUSIONS: These findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders.

Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia.

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Identification of a novel compound with antiviral activity against influenza A virus depending on PA subunit of viral RNA polymerase.

Influenza viruses have developed resistance to current drugs, creating a need for new antiviral targets and new drugs to treat influenza virus infections. In this study, computational and experimental screening of an extensive compound library identified THC19, which was able to suppress influenza virus replication. This compound had no cytotoxic effects and did not disrupt cell cycle progression or induce apoptosis in MDCK cells as confirmed by WST-1 assays, flow cytometry analysis, and caspase-3 assays. Time-of-addition experiments showed that THC19 acts at a relatively early stage of the viral lifecycle. Subsequent mini-genome assays revealed that THC19 inhibited viral genome replication and/or transcription, suggesting that it interferes with one or more of the viral components that form the ribonucleoprotein complexes, namely polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP) and viral RNA. Finally, mini-genome assays where PB2, PB1, PA or NP from A/WSN/33 (H1N1) virus were replaced with those from A/Udorn/307/1972 (H3N2) virus effectively demonstrated that THC19 inhibited viral multiplication in a manner dependent upon the PA subunit. Taken together, these results suggest that influenza virus PA protein is a potential target for, and may aid the development of, novel compounds that inhibit influenza A virus replication.

Deciphering the molecular details for the binding of the prion protein to main ganglioside GM1 of neuronal membranes.

The prion protein (PrP) resides in lipid rafts in vivo, and lipids modulate misfolding of the protein to infectious isoforms. Here we demonstrate that binding of recombinant PrP to model raft membranes requires the presence of ganglioside GM1. A combination of liquid- and solid-state NMR revealed the binding sites of PrP to the saccharide head group of GM1. The binding epitope for GM1 was mapped to the folded C-terminal domain of PrP, and docking simulations identified key residues in the C-terminal region of helix C and the loop between strand S2 and helix B. Crucially, this region of PrP is linked to prion resistance in vivo, and structural changes caused by lipid binding in this region may explain the requirement for lipids in the generation of infectious prions in vitro.

The autoimmune TCR-Ob.2F3 can bind to MBP85-99/HLA-DR2 having an unconventional mode as in TCR-Ob.1A12.

The generation of T cell receptor (TCR) sequence diversity can produce 'forbidden' clones able to recognize self-antigens. Here, the structure of the complex between a myelin basic protein peptide (MBP85-99), human leukocyte antigen (HLA)-DR2 (DRB1*1501/DRA) and TCR-Ob.2F3, the dominant autoimmune clone obtained from a multiple sclerosis (MS) patient, has been determined using structural docking simulation and dynamics in silico and compared to the structure of TCR-Ob.1A12 complexes with the same MHC/peptide determined by X-ray crystallography. The two TCRs differ by three amino acids in the CDR3 α and ß loops. As the result different hydrogen bonds are formed between the two CDR3ß loops and the peptide in the complexes of the simulated structures, with three hydrogen bonds seen in the TCR-Ob.2F3 complex and five in the TCR-Ob.1A12 complex. The two TCRs, each located near the N-terminal end of the HLA-DR2 binding groove and both had an orthogonal binding axis but they deviated by about 10°. Simulation methods, such as structural docking and molecular dynamics as used here, provide an avenue to understand molecular binding mode efficiently and more rapidly than obtaining multiple crystal structures when a large structural database is already available.

Novel anti-cancer compounds: structure-based discovery of chemical chaperons for p53.

Thermal stability of p53 is crucial in preventing cancer proliferation. Critical mutations which significantly destabilize p53 conformation prevent normal interaction between p53 and DNA and consequently interfere with its inhibitory function against cancer proliferation. The purpose of this study was to discover the small compounds called 'chemical chaperons' that can efficiently stabilize the functional p53 conformation and restore the anti-cancer activity. To search for such compounds, we performed a docking simulation using the AutoDock program and the ZINC database. Simply based on the docking energy, we extracted 70 compounds (GJC1-GJC70) and examined their anti-cancer activity using the MTT assay of the human colon cancer cells, HCT116. We found that two compounds, GJC29 and GJC30, significantly inhibited the proliferation of cancer cells compared to the positive control staurosporine. Interaction between p53 and novel anti-cancer compounds were confirmed using SPR measurements. Intriguingly, in the simulated binding mode, both compounds bind to the pocket in the vicinity of the residue V143, one of the mutation hot-spots in p53. Finally, we injected each compound subcutaneously into the nude mice implanted with HCT116 and found that GJC29 has a strong suppressive effect against cancer proliferation in vivo. In conclusion, p53 is an appropriate target for the rational design of the chemical chaperon for cancer treatment.

Variety of antiprion compounds discovered through an in silico screen based on cellular-form prion protein structure: Correlation between antiprion activity and binding affinity.

Transmissible spongiform encephalopathies are associated with the conformational conversion of the prion protein from the cellular form (PrP(C)) to the scrapie form. This process could be disrupted by stabilizing the PrP(C) conformation, using a specific ligand identified as a chemical chaperone. To discover such compounds, we employed an in silico screen that was based on the nuclear magnetic resonance structure of PrP(C). In combination, we performed ex vivo screening using the Fukuoka-1 strain-infected neuronal mouse cell line at a compound concentration of 10 microM and surface plasmon resonance. Initially, we selected 590 compounds according to the calculated docked energy and finally discovered 24 efficient antiprion compounds, whose chemical structures are quite diverse. Surface plasmon resonance studies showed that the binding affinities of compounds for PrP(C) roughly correlated with the compounds' antiprion activities, indicating that the identification of chemical chaperones that bind to the PrP(C) structure and stabilize it is one efficient strategy for antiprion drug discovery. However, some compounds possessed antiprion activities with low affinities for PrP(C), indicating a mechanism involving additional modulation factors. We classified the compounds roughly into five categories: (i) binding and effective, (ii) low binding and effective, (iii) binding and not effective, (iv) low binding and not effective, and (v) acceleration. In conclusion, we found a spectrum of compounds, many of which are able to modulate the pathogenic conversion reaction. The appropriate categorization of these diverse compounds would facilitate antiprion drug discovery and help to elucidate the pathogenic conversion mechanism.

Positioning of autoimmune TCR-Ob.2F3 and TCR-Ob.3D1 on the MBP85-99/HLA-DR2 complex.

Since the first determination of structure of the HLA-A2 complex, >200 MHC/peptide structures have been recorded, whereas the available T cell receptor (TCR)/peptide/MHC complex structures now are <20. Among these structures, only six are TCR/peptide/MHC Class II (MHCII) structures. The most recent of these structures, obtained by using TCR-Ob.1A12 from a multiple sclerosis patient and the MBP85-99/HLA-DR2 complex, was very unusual in that the TCR was located near the N-terminal end of the peptide-binding cleft of the MHCII protein and had an orthogonal angle on the peptide/MHC complex. The unusual structure suggested the possibility of a disturbance of its signaling capability that could be related to autoimmunity. Here, homology modeling and a new simulation method developed for TCR/peptide/MHC docking have been used to examine the positioning of the complex of two additional TCRs obtained from the same patient (TCR-Ob.2F3 or TCR-Ob.3D1 with MBP85-99/HLA-DR2). The structures obtained by this simulation are compatible with available data on peptide specificity of the TCR epitope. All three TCRs from patient Ob including that from the previously determined crystal structure show a counterclockwise rotation. Two of them are located near the N terminus of the peptide-binding cleft, whereas the third is near the center. These data are compatible with the hypothesis that the rotation of the TCRs may alter the downstream signaling.

Strange kinetic phase in the extremely early folding process of beta-lactoglobulin.

A continuous-wave probed laser-induced temperature jump system was constructed and applied to monitor the changes in tryptophan fluorescence of the beta-lactoglobulin during its folding; the kinetic phases were traced from 300 ns to 10 ms after a temperature jump. Notably, an early phase with typical squeezed-exponential characteristics, [exp[-(kt)(beta)], beta>1.0], was observed around several tens of microseconds after the temperature jump, which is actually the earliest phase ever observed for beta-lactoglobulin. This process can be explained by conformational shift occurring within the unfolded ensemble (U-->U'), which is followed by the non-native intermediate (I) formation of this protein.

Modeling of a propagation mechanism of infectious prion protein; a hexamer as the minimum infectious unit.

To construct a new model of the propagation mechanism of infectious scrapie-type prion protein (PrP(Sc)), here we conducted a disruption simulation of a PrP(Sc) nonamer using structure-based molecular dynamics simulation method based on a hypothetical PrP(Sc) model structure. The simulation results showed that the nonamer disrupted in cooperative manners into monomers via two significant intermediate states: (1) a nonamer with a partially unfolded surface trimer and (2) a hexamer and three monomers. Dimers and trimers were rarely observed. Then, we propose a new PrP(Sc) propagation mechanism where a hexamer plays an essential role as a minimum infectious unit.

Hot spots in prion protein for pathogenic conversion.

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP(C)) to the scrapie form (PrP(Sc)) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP(C) conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a "pocket" created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP(Sc). Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP(C), will open the way to the development of novel anti-prion drugs.

Transition state of a SH3 domain detected with principle component analysis and a charge-neutralized all-atom protein model.

The src SH3 domain has been known to be a two-state folder near room temperature. However, in a previous study with an all-atom model simulation near room temperature, the transition state of this protein was not successfully detected on a free-energy profile using two axes: the radius of gyration (R(g)) and native contact reproduction ratio (Q value). In this study, we focused on an atom packing effect to characterize the transition state and tried another analysis to detect it. To explore the atom packing effect more efficiently, we introduced a charge-neutralized all-atom model, where all of the atoms in the protein and water molecules were treated explicitly, but their partial atomic charges were set to zero. Ten molecular dynamics simulations were performed starting from the native structure at 300 K, where the simulation length of each run was 90 ns, and the protein unfolded in all runs. The integrated trajectories (10 x 90 = 900 ns) were analyzed by a principal component analysis (PCA) and showed a clear free-energy barrier between folded- and unfolded-state conformational clusters in a conformational space generated by PCA. There were segments that largely deformed when the conformation passed through the free-energy barrier. These segments correlated well with the structural core regions characterized by large phi-values, and the atom-packing changes correlated with the conformational deformations. Interestingly, using the same simulation data, no significant barrier was found in a free-energy profile using the R(g) and Q values for the coordinate axes. These results suggest that the atom packing effect may be one of the most important determinants of the transition state.

Temperature dependence and counter effect of the correlations of folding rate with chain length and with native topology.

There is a controversy about the major determinants of the folding rate of small single-domain proteins. To shed light on this issue, we examined a possibility that the major determinants may change depending on temperature by conducting molecular dynamics simulations for 17 small single-domain proteins using an off-lattice Go-like model over a wide range of temperature. It was shown that the rank order of the folding rates is temperature dependent, which indicates that the major determinants are dependent on temperature. It was also found that as temperature is decreased, the correlation of the folding rate with the chain length becomes weakened, whereas that with the native topology becomes enhanced. Our simulation results, therefore, may provide a clue to reconcile the apparent controversy between the study by Plaxco based on experimental data and the previous theoretical and subsequent simulation studies: the former showed that the folding rate of two-state folders does not correlate with the chain length but correlates well with the native topology, whereas the latter showed that the folding rate does correlate with the chain length. We propose a possible scenario reconciling the controversy, explaining the reason why the correlation of the folding rate with the chain length became weakened and that with the native topology became enhanced with decreasing temperature.

Squeezed exponential kinetics to describe a nonglassy downhill folding as observed in a lattice protein model.

We previously studied the so-called strange kinetics in the two-dimensional lattice HP model. To further study the strange kinetics, folding processes of a 27-mer cubic lattice protein model with Go potential were investigated by simulating how the bundle of folding trajectories, consisting of a number of independent Monte Carlo simulations, evolves as the folding reaction proceeds, covering a wide range of temperature. Three realms of folding kinetics were observed depending on temperature. Although at temperatures where folding was two-state-like, the kinetics was conventional single exponential, we found that the time course data were well represented by a squeezed (or "shrunken") exponential function, exp [-(t/tau)beta] with beta > 1, at temperatures lower than the folding temperature, where folding was fastest and of a nonglassy downhill type. The squeezed exponential kinetics was found to pertain to the subdiffusion on the nonglassy downhill free energy surface and presents a marked contrast both to the single exponential kinetics and to the stretched exponential kinetics that was observed at lower temperatures where folding was also downhill but topological frustration came into effect. The observed temperature dependence of the folding kinetics suggests that some small single-domain proteins may follow the squeezed exponential kinetics at about the room temperature.