Effect of a cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) residue on mixed-helical folding of unnatural peptides.

The α/ß-peptide 11/9-helix and the ß-peptide 12/10-helix belong to "mixed" helices, in which two types of hydrogen bonds with opposite directionality alternate along the helical axis. cis-2-Aminocyclohexanecarboxylic acid (cis-ACHC) is known to promote these mixed helices and stabilize the helical propensity more than other acyclic ß-residues. Application of a mixed-helical backbone still requires sufficient solubility in aqueous solution. In this regard, we chose cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) as a foldamer building block that can provide both sufficient aqueous solubility and mixed-helical propensity. Conformational analyses of α/ß- and ß-peptides containing a cis-APiC residue by circular dichroism spectroscopy and single-crystal X-ray crystallography suggest that the incorporation of cis-APiC instead of cis-ACHC can enhance the aqueous solubility of the mixed-helical peptides without any adverse effect on helical folding. In addition, the ratio between right- and left-handed 12/10-helices of ß-peptides can be rationalized by relative energies between the local conformations of the cis-APiC residue.

Side chain-specific 11/9-helix propensity of α/ß-peptides with alternating residue types.

The 11/9-helix is among the most stable and non-traditional helical structures for α/ß-peptides with alternating residue types. The effect of side chain groups of α-residues and ß3-residues on the 11/9-helix propensity was examined under various solvent conditions. An α-amino acid residue with one of the four representative side chain groups was incorporated into the central position of an α/ß-pentapeptide backbone. A ß-branched valine residue did not show any destabilizing effect. α,α-Dimethylsubstituted Aib residue was tolerated under nonpolar conditions, but did not promote 11/9-helical folding. The oligomer with a glycine residue did not show 11/9-helical folding under polar solvent conditions. The single unmatched stereochemistry of d-alanine was deleterious to 11/9-helical folding. Replacement of a cyclic ß-residue with an acyclic ß3-residue in the 11/9-helical structure had a slight destabilizing effect, which could be compensated by a longer peptide sequence with more cyclic ß-residues. These results provide a guidance for incorporating functional groups into an 11/9-helical α/ß-peptide backbone to design functional oligomers.

[Difficulties in Caring for Psychiatric Patient as Experienced by Non-Psychiatric Nurses].

PURPOSE: The purpose of this study was to identify non-psychiatric nurses' difficulties in caring for patients with mental illness. METHODS: Data were collected from eighteen general medical-surgical nurses working at a university hospital in Seoul, Korea. This study involved two focus group discussions and three in-depth individual interviews. All interviews were recorded and transcribed as they were spoken, and data were analyzed using qualitative content analysis. RESULTS: General medical-surgical nurses experienced difficulties in 3 categories, 9 subcategories, 27 codes. The three categories were 'nurse' related factors, 'patient' related factors, 'resource' related factors. The nine categories were 'unpreparedness', 'nursing barriers due to stigma', 'undervaluing and avoidance of psychiatric nursing', 'eroding into the trap of a vicious cycle', 'facing unapproachable patients', 'dealing with unhelpful family members', 'burdening already overburdened staff', 'obstructive environment', and 'isolation of staff with heavy responsibilities'. CONCLUSION: The results of this study indicate the need to develop psychiatric mental health education programs for non-psychiatric nurses. Education about psychiatric mental health and support from institutions for non-psychiatric nurses can reduce their negative attitude toward psychiatric patients and difficulties in caring for psychiatric patients.

L-Methionine Production.

L-Methionine has been used in various industrial applications such as the production of feed and food additives and has been used as a raw material for medical supplies and drugs. It functions not only as an essential amino acid but also as a physiological effector, for example, by inhibiting fat accumulation and enhancing immune response. Producing methionine from fermentation is beneficial in that microorganisms can produce L-methionine selectively using eco-sustainable processes. Nevertheless, the fermentative method has not been used on an industrial scale because it is not competitive economically compared with chemical synthesis methods. Presented are efforts to develop suitable strains, engineered enzymes, and alternative process of producing L-methionine that overcomes problems of conventional fermentation methods. One of the alternative processes is a two-step process in which the L-methionine precursor is produced by fermentation and then converted to L-methionine by enzymes. Directed efforts toward strain development and enhanced enzyme engineering will advance industrial production of L-methionine based on fermentation.

Stabilization of 11/9-helical α/ß-peptide foldamers in protic solvents.

α/ß-Peptides with alternating α-amino acid and cis-2-aminocyclohexanecarboxylic acid (cis-ACHC) residues adopt 11/9-helical conformations, the folding propensity of which decreases as the solvent polarity increases. We report a new cis-ACHC analogue, cis-2-amino-cis-4-methylcyclohexanecarboxylic acid, which significantly stabilizes the 11/9-helix propensity in protic solvents.

The Relationship between Smoking Status and Suicidal Behavior in Korean Adults: The 4th Korea National Health and Nutrition Examination Survey (2007-2009).

BACKGROUND: This study was performed to evaluate the relationship between smoking status and suicidal ideations or suicide attempts in Korean adults. METHODS: The study used data from the 4th Korea National Health and Nutrition Examination Survey, and involved 17,065 participants. We used multiple logistic regression analysis to evaluate the relationship between smoking status and suicidal behavior. The results were adjusted for covariates including depression and physical disease. RESULTS: After adjusting for covariates, current smokers (odds ratio [OR], 1.66; 95% confidence interval [CI], 1.34 to 2.05) and former smokers (OR, 1.39; 95% CI, 1.13 to 1.72) proved more likely to have suicidal ideations than non-smokers. Among women, current smokers (OR, 2.00; 95% CI, 1.47 to 2.72) and former smokers (OR, 1.48; 95% CI, 1.11 to 1.99) were more likely to have suicidal ideations than non-smokers. However, among the men there was no significant relationship between smoking status and suicidal ideations. Among all the participants who had had suicidal ideations, there was a significant relationship between current smoking and suicide attempts (OR, 1.80; 95% CI, 1.05 to 3.07). There was also a relationship between current smoking and suicide attempts among women (OR, 2.40; 95% CI, 1.31 to 4.37), but no significant relationship among men. CONCLUSION: There is a relationship between smoking status and suicidal ideations and suicide attempts in the general population of Korea. This relationship is particularly clear in women, and is independent of comorbid chronic disease and depression. Current smokers and former smokers are more likely to have suicidal ideations than non-smokers; current smokers are also more likely to have attempted suicide.

Molecular details of the activation of the µ opioid receptor.

Molecular details of µ opioid receptor activations were obtained using molecular dynamics simulations of the receptor in the presence of three agonists, three antagonists, and a partial agonist and on the constitutively active T279K mutant. Agonists have a higher probability of direct interactions of their basic nitrogen (N) with Asp147 as compared with antagonists, indicating that direct ligand-Asp147 interactions modulate activation. Medium-size substituents on the basic N of antagonists lead to steric interactions that perturb N-Asp147 interactions, while additional favorable interactions occur with larger basic N substituents, such as in N-phenethylnormorphine, restoring N-Asp147 interactions, leading to agonism. With the orvinols, the increased size of the C19 substituent in buprenorphine over diprenorphine leads to increased interactions with residues adjacent to Asp147, partially overcoming the presence of the cyclopropyl N substituent, such that buprenorphine is a partial agonist. Results also indicate different conformational properties of the intracellular regions of the transmembrane helices in agonists versus antagonists.

Synthesis, modeling, and pharmacological evaluation of UMB 425, a mixed µ agonist/δ antagonist opioid analgesic with reduced tolerance liabilities.

Opioid narcotics are used for the treatment of moderate-to-severe pain and primarily exert their analgesic effects through µ receptors. Although traditional µ agonists can cause undesired side effects, including tolerance, addition of δ antagonists can attenuate said side effects. Herein, we report 4a,9-dihydroxy-7a-(hydroxymethyl)-3-methyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one (UMB 425) a 5,14-bridged morphinan-based orvinol precursor synthesized from thebaine. Although UMB 425 lacks δ-specific motifs, conformationally sampled pharmacophore models for µ and δ receptors predict it to have efficacy similar to morphine at µ receptors and similar to naltrexone at δ receptors, due to the compound sampling conformations in which the hydroxyl moiety interacts with the receptors similar to orvinols. As predicted, UMB 425 exhibits a mixed µ agonist/δ antagonist profile as determined in receptor binding and [(35)S]GTPγS functional assays in CHO cells. In vivo studies in mice show that UMB 425 displays potent antinociception in the hot plate and tail-flick assays. The antinociceptive effects of UMB 425 are blocked by naloxone, but not by the κ-selective antagonist norbinaltorphimine. During a 6-day tolerance paradigm, UMB 425 maintains significantly greater antinociception compared to morphine. These studies thus indicate that, even in the absence of δ-specific motifs fused to the C-ring, UMB 425 has mixed µ agonist/δ antagonist properties in vitro that translate to reduced tolerance liabilities in vivo.

(Ala)(4)-X-(Ala)4 as a model system for the optimization of the χ1 and χ2 amino acid side-chain dihedral empirical force field parameters.

Amino acid side-chain fluctuations play an essential role in the structure and function of proteins. Accordingly, in theoretical studies of proteins, it is important to have an accurate description of their conformational properties. Recently, new side-chain torsion parameters were introduced into the CHARMM and Amber additive force fields and evaluated based on the conformational properties of the individual side-chains using protein simulations in explicit solvent. While effective for validation, molecular dynamics simulations of proteins must be extended into the microsecond regime to obtain full convergence of the side-chain conformations, limiting their use for force field optimization. To address this, we systematically test the utility of explicit solvent simulations of (Ala)(4)-X-(Ala)(4) peptides, where X represents the amino acids, as model systems for the optimization of χ(1) and χ(2) side-chain parameters. The effect of (Ala)(4)-X-(Ala)(4) backbone conformation was tested by constraining the backbone in the α-helical, C5, C7(eq), and PPII conformations and performing exhaustive sampling using Hamiltonian replica exchange simulations. Rotamer distributions from protein and the (Ala)(4)-X-(Ala)(4) simulations showed the highest correlation for the C7(eq) and PPII conformations, although agreement was the best for the α-helical conformation for Asn. Hydrogen bond analysis indicates the utility of the C7(eq) and PPII conformations to be due to specific side-chain-backbone hydrogen bonds not being oversampled, thereby allowing sampling of a range of side-chain conformations consistent with the distributions occurring in full proteins. It is anticipated that the (Ala)(4)-X-(Ala)(4) model system will allow for iterative force field optimization targeting condensed-phase conformational distributions of side-chains.

Force Field for Peptides and Proteins based on the Classical Drude Oscillator.

Presented is a polarizable force field based on a classical Drude oscillator framework, currently implemented in the programs CHARMM and NAMD, for modeling and molecular dynamics (MD) simulation studies of peptides and proteins. Building upon parameters for model compounds representative of the functional groups in proteins, the development of the force field focused on the optimization of the parameters for the polypeptide backbone and the connectivity between the backbone and side chains. Optimization of the backbone electrostatic parameters targeted quantum mechanical conformational energies, interactions with water, molecular dipole moments and polarizabilities and experimental condensed phase data for short polypeptides such as (Ala)5. Additional optimization of the backbone φ, ψ conformational preferences included adjustments of the tabulated two-dimensional spline function through the CMAP term. Validation of the model included simulations of a collection of peptides and proteins. This 1st generation polarizable model is shown to maintain the folded state of the studied systems on the 100 ns timescale in explicit solvent MD simulations. The Drude model typically yields larger RMS differences as compared to the additive CHARMM36 force field (C36) and shows additional flexibility as compared to the additive model. Comparison with NMR chemical shift data shows a small degradation of the polarizable model with respect to the additive, though the level of agreement may be considered satisfactory, while for residues shown to have significantly underestimated S2 order parameters in the additive model, improvements are calculated with the polarizable model. Analysis of dipole moments associated with the peptide backbone and tryptophan side chains show the Drude model to have significantly larger values than those present in C36, with the dipole moments of the peptide backbone enhanced to a greater extent in sheets versus helices and the dipoles of individual moieties observed to undergo significant variations during the MD simulations. Although there are still some limitations, the presented model, termed Drude-2013, is anticipated to yield a molecular picture of peptide and protein structure and function that will be of increased physical validity and internal consistency in a computationally accessible fashion.

Deconstructing 14-phenylpropyloxymetopon: minimal requirements for binding to mu opioid receptors.

A series of phenylpropyloxyethylamines and cinnamyloxyethylamines were synthesized as deconstructed analogs of 14-phenylpropyloxymetopon and analyzed for opioid receptor binding affinity. Using the Conformationally Sampled Pharmacophore modeling approach, we discovered a series of compounds lacking a tyrosine mimetic, historically considered essential for µ opioid binding. Based on the binding studies, we have identified the optimal analogs to be N-methyl-N-phenylpropyl-2-(3-phenylpropoxy)ethanamine, with 1520 nM, and 2-(cinnamyloxy)-N-methyl-N-phenethylethanamine with 1680 nM affinity for the µ opioid receptor. These partial opioid structure analogs will serve as the novel lead compounds for future optimization studies.

Intrinsic energy landscapes of amino acid side-chains.

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.

Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles.

While the quality of the current CHARMM22/CMAP additive force field for proteins has been demonstrated in a large number of applications, limitations in the model with respect to the equilibrium between the sampling of helical and extended conformations in folding simulations have been noted. To overcome this, as well as make other improvements in the model, we present a combination of refinements that should result in enhanced accuracy in simulations of proteins. The common (non Gly, Pro) backbone CMAP potential has been refined against experimental solution NMR data for weakly structured peptides, resulting in a rebalancing of the energies of the α-helix and extended regions of the Ramachandran map, correcting the α-helical bias of CHARMM22/CMAP. The Gly and Pro CMAPs have been refitted to more accurate quantum-mechanical energy surfaces. Side-chain torsion parameters have been optimized by fitting to backbone-dependent quantum-mechanical energy surfaces, followed by additional empirical optimization targeting NMR scalar couplings for unfolded proteins. A comprehensive validation of the revised force field was then performed against data not used to guide parametrization: (i) comparison of simulations of eight proteins in their crystal environments with crystal structures; (ii) comparison with backbone scalar couplings for weakly structured peptides; (iii) comparison with NMR residual dipolar couplings and scalar couplings for both backbone and side-chains in folded proteins; (iv) equilibrium folding of mini-proteins. The results indicate that the revised CHARMM 36 parameters represent an improved model for the modeling and simulation studies of proteins, including studies of protein folding, assembly and functionally relevant conformational changes.

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs.

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.

Computational ligand-based rational design: Role of conformational sampling and force fields in model development.

A significant number of drug discovery efforts are based on natural products or high throughput screens from which compounds showing potential therapeutic effects are identified without knowledge of the target molecule or its 3D structure. In such cases computational ligand-based drug design (LBDD) can accelerate the drug discovery processes. LBDD is a general approach to elucidate the relationship of a compound's structure and physicochemical attributes to its biological activity. The resulting structure-activity relationship (SAR) may then act as the basis for the prediction of compounds with improved biological attributes. LBDD methods range from pharmacophore models identifying essential features of ligands responsible for their activity, quantitative structure-activity relationships (QSAR) yielding quantitative estimates of activities based on physiochemical properties, and to similarity searching, which explores compounds with similar properties as well as various combinations of the above. A number of recent LBDD approaches involve the use of multiple conformations of the ligands being studied. One of the basic components to generate multiple conformations in LBDD is molecular mechanics (MM), which apply an empirical energy function to relate conformation to energies and forces. The collection of conformations for ligands is then combined with functional data using methods ranging from regression analysis to neural networks, from which the SAR is determined. Accordingly, for effective application of LBDD for SAR determinations it is important that the compounds be accurately modelled such that the appropriate range of conformations accessible to the ligands is identified. Such accurate modelling is largely based on use of the appropriate empirical force field for the molecules being investigated and the approaches used to generate the conformations. The present chapter includes a brief overview of currently used SAR methods in LBDD followed by a more detailed presentation of issues and limitations associated with empirical energy functions and conformational sampling methods.

Consensus 3D model of µ-opioid receptor ligand efficacy based on a quantitative Conformationally Sampled Pharmacophore.

Despite being studied for over 30 years, a consensus structure-activity relationship (SAR) that encompasses the full range peptidic and nonpeptidic µ-opioid receptor ligands is still not available. To achieve a consensus SAR the Conformationally Sampled Pharmacophore (CSP) method was applied to develop a predictive model of the efficacy of µ-opioid receptor ligands. Emphasis was placed on predicting the efficacy of a wide range of agonists, partial agonists, and antagonists as well as understanding their mode of interaction with the receptor. Inclusion of all accessible conformations of each ligand, a central feature of the CSP method, enabled structural features between diverse µ-opioid receptor ligands that dictate efficacy to be identified. The models were validated against a diverse collection of peptidic and nonpeptidic ligands, including benzomorphans, fentanyl (4-anilinopiperidine), methadone (3,3-diphenylpropylamines), etonitazene (benzimidazole derivatives), funaltrexamine (C6-substituted 4,5-epoxymorphinan), and herkinorin. The model predicts (1) that interactions of ligands with the B site, as with the 19-alkyl substituents of oripavines, modulate the extent of agonism; (2) that agonists with long N-substituents, as with fentanyl and N-phenethylnormorphine, can bind in an orientation such that the N substitutent interacts with the B site that also allows the basic N-receptor Asp interaction essential for agonism; and (3) that the µ agonist herkinorin, that lacks a basic nitrogen, binds to the receptor in a manner similar to the traditional opioids via interactions mediated by water or a ion. Importantly, the proposed CSP model can be reconciled with previously published SAR models for the µ receptor.