The inhibitory effects of squalene-derived triterpenes on protein phosphatase PP2A

This paper reports testing 15 polyether triterpenes with a squalene carbon skeleton for inhibitory effects on type 2A protein phosphatase. Two compounds, 16-hydroxydehydrothyrsiferol 10 and thyrsenol B 14, exhibited significant inhibitory action at a concentration of 10 microM. Comparison with thyrsiferyl-23-acetate 1 showed that a similar spatial disposition for the hydroxy group around C-15 or C-16 was the structural feature shared by these metabolites.     

Squalene cyclase and oxidosqualene cyclase from a fern

Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.     

Leishmanicidal and Anticandidal Activity of Constituents of Indian Edible Mushroom Astraeus hygrometricus

Two new lanostane-type triterpenes, 1 and 2, were isolated from Astraeus hygrometricus. The structures were established by IR, (1) H- and (13) C-NMR, MS, and X-ray crystallographic experiments. The triterpenes exhibited excellent in vitro toxicities against Candida albicans, comparable to standard antifungal antibiotics. The triterpene 2 significantly inhibited the growth of Leishmania donovani promastigotes in vitro. The triterpene skeleton may be considered a template structure in search for new compounds with anticandidal and leishmanicidal activity.     

Further study on synthesis and evaluation of 3,16,20-polyoxygenated steroids of marine origin and their analogs as potent cytotoxic agents

A series of new polyoxygenated steroid derivatives with various steroid skeleton moieties were synthesized. Antitumor activity of the compounds against three tumor cell lines (Breast cancer MCF7, lung cancer NCI and oral cancer KB) were evaluated. Compounds with aromatic A ring of this series exhibited the most potent cytotoxicities in all tested cells. The absence of OH at C-16 or lack of cholesterol like side chain at C-20 in the steroid skeleton apparently result in decreased cytotoxicity. The compound became inactive when the side chain contains double bond at C-24-C-25. When hydroxyl group at C-3 was protected no cytotoxicities against MCF7 and NCI and considerable low cytotoxicity against KB cell lines were observed.     

Structural basis of hierarchical multiple substates of a protein. III: Side chain and main chain local conformations

An analysis is carried out of differences in the minimum energy conformations obtained in the previous paper by energy minimization starting from conformations sampled by a Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatic trypsin inhibitor. Main conformational differences in each pair of energy minima are found usually localized in several side chains and in a few local main chain segments. Such side chains and local main chain segments are found to take a few distinct local conformations in the minimum energy conformations. Energy minimum conformations can thus be described in terms of combinations of these multiple local conformations.     

Molecular dynamics studies of the conformation of sorbitol

Molecular dynamics simulations of a 3 molal aqueous solution of d-sorbitol (also called d-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2–C-3 torsion (spanned by the O-2–O-4 hydrogen bond), where the NMR data support a more bent structure.     

Differentiation-inducing activity of lupane triterpenes on a mouse melanoma cell line

Lupane triterpenes were found to promote melanogenesis, a hallmark of B16 2F2 mouse melanoma cell differentiation. Studies of the structure-activity relationships demonstrated that the keto function at C-3 of the lupane skeleton played important roles in the melanogenic activities of lupane triterpenes on melanoma cells. The carbonyl group at C-17 of lupane triterpenes was essential against their apoptosis-inducing activity against human cancer cells via the inhibition of topoisomerase I. We investigated whether signaling mechanisms were involved in the stimulative effects of lupane triterpenes on the melanogenesis of B16 2F2 cells. In experiments using selective inhibitors against various signal transduction molecules and Western blotting analysis, it was suggested that p38 MAPK was involved in melanoma cell differentiation as a downstream effector of PKA. Lupeol (compound 1), a lupane triterpene, induced dendrite formations, a morphological hallmark of B16 2F2 cell differentiation by rearrangement of the actin cytoskeleton. The activation of cofilin, an actin depolymerizing factor, by compound 1 caused actin fiber disassembly in B16 2F2 cells. Furthermore, compound 1 was shown to inhibit the cell motilities of human melanoma and neuroblastoma in vitro.     

Synthesis and cytotoxicity of lupane-type triterpenoid glyceryl esters

A new series of betulinic acid and betulin derivatives were synthesized by introducing a D-glycerol moiety at the C-3 and/or C-28 positions of the lupane skeleton. The resulting glyceryl esters were evaluated in vitro for their cytotoxic activity against A549, DLD-1 and WS1 human cell lines. The structure-activity relationships study revealed that the incorporation of a glycerol unit at the C-3 or C-28 position of the lupane core resulted in compounds exhibiting potent cytotoxic activity together with decreased liposolubility.     

Coil-globule Coexistence and Compaction of DNA Chains

In this work we discuss different factors governing coil-globule coexistence in the compaction process of DNA. We initially analyse the role played by fluctuations in the degree of binding of an external compacting agent in the conformational behavior of the chain backbone. The analysis relies both on Monte Carlo simulation results and simple statistical approaches. Compacting agents of various binding characteristics are taken into consideration and the degree of charge neutralization upon the chain is related to conformational indicators. Selected model systems comprising stiff chains in the presence of multivalent ions are employed to assess intrinsic single-chain conformational fluctuation, in the presence of external agents but not resulting from differences in binding. It is shown that trends found for a variety of compacting agents, including the extension of the coil-globule coexistence regions, can be ratio-nalised on the basis of this analysis.     

A linear sesterterpene,two squalene derivatives and two peptide derivatives from Croton hieronymi

Aerial parts of Croton hieronymi furnished in addition to a large number of plant sterols and triterpenes the C-25 analog of trans-phytol, the squalene derivatives all-trans-2,6,15,19,23-pentamethyltetracosa-2,6,10,(28),14,22,28-hexaene-11-ol and all-trans-10-methylene-2,6,10,14,18,22-pentamethyltetracosa-1,6,10,14,18,22-hexaen-3-ol, the sesquiterpenes epicubenol and T-cadinol, the acetophenone derivative xanthoxylin and the peptide derivatives aurentiamide acetate and N-benzoylphenylalanyl-N-benzoylphenylalaninate.     

Role of proximal His93 in nitric oxide binding to metmyoglobin. Application of continuum solvation in Monte Carlo protein simulations.

Monte Carlo protein simulations with continuum solvation were used to explore the conformational mobility of NO within the active site of metmyoglobin. To the best of our knowledge this is the first application of a continuum solvation model for exploring protein binding sites. The usefulness of the Monte Carlo conformational analysis was demonstrated in comparative molecular dynamics simulations. Analysis of conformer populations revealed that Monte Carlo conformational analysis is more effective in mapping the relevant region of the potential surface. Distribution of low-energy conformers obtained for the metmyoglobin-NO complex was found to depend on the orientation of proximal His93. Different charge distributions corresponding to the two experimentally verified possible torsions of this proximal residue result in strong binding of NO or its release to a nearby hydrophobic trap. Conformer populations obtained by Monte Carlo conformational analysis were grouped into three main families: one, with the NO directly bound to the iron, appears when the CA-CB-CG-CD2 torsion of His93 is at its ligand binding value (-113 degrees); and two conformers exist where NO is trapped in a nearby hydrophobic pocket, the same cavity that was determined to be the geminate trap of CO in ferrous Mb as a result of the torsional flip of His93 to its ligand releasing state (-125 degrees). Based on this analysis, we suggest that the electrostatic rearrangement coupled to the conformational fluctuation of the proximal His leads to the geminate trapping of the ligand. Conformational rearrangement of the proximal side would provide the possibility of rebinding of the ligand to Fe.     

Sequence specific resonance assignment via Multicanonical Monte Carlo search using an ABACUS approach

ABACUS [Grishaev et al. (2005) Proteins 61:36-43] is a novel protocol for automated protein structure determination via NMR. ABACUS starts from molecular fragments defined by unassigned J-coupled spin-systems and involves a Monte Carlo stochastic search in assignment space, probabilistic sequence selection, and assembly of fragments into structures that are used to guide the stochastic search. Here, we report further development of the two main algorithms that increase the flexibility and robustness of the method. Performance of the BACUS [Grishaev and Llinás (2004) J Biomol NMR 28:1-101] algorithm was significantly improved through use of sequential connectivities available from through-bond correlated 3D-NMR experiments, and a new set of likelihood probabilities derived from a database of 56 ultra high resolution X-ray structures. A Multicanonical Monte Carlo procedure, Fragment Monte Carlo (FMC), was developed for sequence-specific assignment of spin-systems. It relies on an enhanced assignment sampling and provides the uncertainty of assignments in a quantitative manner. The efficiency of the protocol was validated on data from four proteins of between 68-116 residues, yielding 100% accuracy in sequence specific assignment of backbone and side chain resonances.     

Bayesian analysis of matrix normal graphical models

We present Bayesian analyses of matrix-variate normal data with conditional independencies induced by graphical model structuring of the characterizing covariance matrix parameters. This framework of matrix normal graphical models includes prior specifications, posterior computation using Markov chain Monte Carlo methods, evaluation of graphical model uncertainty and model structure search. Extensions to matrix-variate time series embed matrix normal graphs in dynamic models. Examples highlight questions of graphical model uncertainty, search and comparison in matrix data contexts. These models may be applied in a number of areas of multivariate analysis, time series and also spatial modelling.     

Guiding probabilistic search of the protein conformational space with structural profiles

The roughness of the protein energy surface poses a significant challenge to search algorithms that seek to obtain a structural characterization of the native state. Recent research seeks to bias search toward near-native conformations through one-dimensional structural profiles of the protein native state. Here we investigate the effectiveness of such profiles in a structure prediction setting for proteins of various sizes and folds. We pursue two directions. We first investigate the contribution of structural profiles in comparison to or in conjunction with physics-based energy functions in providing an effective energy bias. We conduct this investigation in the context of Metropolis Monte Carlo with fragment-based assembly. Second, we explore the effectiveness of structural profiles in providing projection coordinates through which to organize the conformational space. We do so in the context of a robotics-inspired search framework proposed in our lab that employs projections of the conformational space to guide search. Our findings indicate that structural profiles are most effective in obtaining physically realistic near-native conformations when employed in conjunction with physics-based energy functions. Our findings also show that these profiles are very effective when employed instead as projection coordinates to guide probabilistic search toward undersampled regions of the conformational space.     

Biologically active metabolites from the basidiomycete Limacella illinita (Fr.) Murr

In the course of our search for new bioactive compounds from basidiomycetes, four new compounds were isolated from fermentations of Limacella illinita. Illinitone A (1) exhibited weak phytotoxic and moderate nematicidal activities against Caenorhabditis elegans, illinitone B (2) was moderately cytotoxic, while limacellone (3) exhibited weak cytotoxic and phytotoxic activities. The muurolane sesquiterpene 4a was found to be inactive in the assays performed here. Limacellone (3), which appeared to be related with the illinitones 1 and 2, has a new C15 carbon skeleton. It is possible that compounds 1, 2 and 3 are terpenoids/ secoterpenoids, but their biosyntheses were not investigated.     

Synthesis,in vitro and in vivo evaluation of 3-arylisoquinolinamines as potent antitumor agents

In the search for potent water-soluble 3-arylisoquinolines, several 3-arylisoquinolinamines were designed and synthesized. Various substituted 3-arylisoquinolinamines exhibited strong cytotoxic activity against eight different human cancer cell lines. In particular, C-6 or C-7 dimethylamino-substituted 3-arylisoquinolinamines displayed stronger potency than the lead compound 7a. Interestingly, compounds 7b and 7c showed more effective activity against paclitaxel-resistant HCT-15 human colorectal cancer cell lines when compared to the original cytotoxic cancer drug, paclitaxel. We analyzed the cell cycle dynamics by flow cytometry and found that treatment of human HCT-15 cells with 3-arylisoquinolinamine 7b blocked or delayed the progression of cells from G0/G1 phase into S phase, and induced cell death. Treatment with compound 7b also significantly inhibited the growth of tumors and enhanced tumor regression in a paclitaxel-resistant HCT-15 xenograft model.     

Linckosides C-E, three new neuritogenic steroid glycosides from the Okinawan starfish Linckia laevigata

Three new steroid glycosides, linckosides C-E, were isolated from the Okinawan starfish Linckia laevigata. Their structures and partial stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These metabolites are additional members of the linckosides that were previously discovered as a novel class of neuritogenic compounds. Each of them possesses two monosaccharide units at C-3 of a polyhydroxylated steroidal aglycon and at the side chain (C-28 or C-29). Linckosides C and D are the first steroids that possess a hydroxyisopropyl substituent at C-24 of the side chain. These compounds are not only potent inducers of neurite outgrowth on PC12 cells but also significant enhancers of nerve growth factor (NGF) to induce the neurite outgrowth. The structure-activity relationships within the linckosides revealed that the presence of xylopyranose at the side chain was important rather than arabinofuranose, but that the diversity of the side chain carbon skeleton was not.     

MCMC multilocus lod scores: application of a new approach

On extended pedigrees with extensive missing data, the calculation of multilocus likelihoods for linkage analysis is often beyond the computational bounds of exact methods. Growing interest therefore surrounds the implementation of Monte Carlo estimation methods. In this paper, we demonstrate the speed and accuracy of a new Markov chain Monte Carlo method for the estimation of linkage likelihoods through an analysis of real data from a study of early-onset Alzheimer's disease. For those data sets where comparison with exact analysis is possible, we achieved up to a 100-fold increase in speed. Our approach is implemented in the program lm_bayes within the framework of the freely available MORGAN 2.6 package for Monte Carlo genetic analysis (http://www.stat.washington.edu/thompson/Genepi/MORGAN/Morgan.shtml).