In silico SNP analysis and bioinformatics tools: a review of the state of the art to aid drug discovery.

SNPs can alter protein function and phenotype, leading to altered pharmacogenomic drug profiles. The exponential number of SNPs makes it impossible to perform wet laboratory experiments to determine the biological significance of each one. However, bioinformatics tools can be used to screen for potentially deleterious SNPs that might affect important drug targets before further investigation by wet laboratory techniques. As there are numerous web-based bioinformatics tools, much time and effort is needed to select the most appropriate tool to use. Here, we review state-of-the-art bioinformatics tools to help researchers analyze and select the most promising SNPs for drug discovery in the shortest time possible.

UASIS: Universal Automatic SNP Identification System.

BACKGROUND: SNP (Single Nucleotide Polymorphism), the most common genetic variations between human beings, is believed to be a promising way towards personalized medicine. As more and more research on SNPs are being conducted, non-standard nomenclatures may generate potential problems. The most serious issue is that researchers cannot perform cross referencing among different SNP databases. This will result in more resources and time required to track SNPs. It could be detrimental to the entire academic community. RESULTS: UASIS (Universal Automated SNP Identification System) is a web-based server for SNP nomenclature standardization and translation at DNA level. Three utilities are available. They are UASIS Aligner, Universal SNP Name Generator and SNP Name Mapper. UASIS maps SNPs from different databases, including dbSNP, GWAS, HapMap and JSNP etc., into an uniform view efficiently using a proposed universal nomenclature and state-of-art alignment algorithms. UASIS is freely available at http://www.uasis.tk with no requirement of log-in. CONCLUSIONS: UASIS is a helpful platform for SNP cross referencing and tracking. By providing an informative, unique and unambiguous nomenclature, which utilizes unique position of a SNP, we aim to resolve the ambiguity of SNP nomenclatures currently practised. Our universal nomenclature is a good complement to mainstream SNP notations such as rs# and HGVS guidelines. UASIS acts as a bridge to connect heterogeneous representations of SNPs.

Antioxidant effect of ethyl pyruvate in respiring neonatal cerebrocortical slices after H(2)O(2) stress.

Administration of ethyl pyruvate, which is formed from pyruvate and ethanol, has been found capable of rescuing cells injured by oxidative stress. In one perspective the rescue has been postulated to be metabolic, with the resulting intracellular delivery of pyruvate seen as providing substrate for the TCA Cycle, making it possible to counteract sequela of poly(ADP-ribose)ribosylation, such as depletion of cytosolic NAD(+), glycolytic arrest, and mitochondrial deprivation of pyruvate. The rescue has also been attributed to radical scavenging via the carbonyl groups in ethyl pyruvate and pyruvate. In a previous study we exposed superfused neonatal (P7) brain slices for 60min to 2mM H(2)O(2) and found evidence for both rescue mechanisms. To see if ethyl pyruvate's actions stemmed more from being an antioxidant than from being a nutrient we conducted six new experiments using the same H(2)O(2) protocol, but with two new rescue solutions: [10mM] glucose (glc) plus one of the following: ethyl pyruvate [20mM], or the nonmetabolizable radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1mM). Final ATP values compared to initial, measured in 14.1T (31)P NMR spectra of PCA extracts, were the same: 0.70+/-0.08 for the former (N=3), and 0.64+/-0.08 for the latter (N=3). Quantifications of this study's (1)H NMR metabolites, also measured at 14.1T, exhibited separate clustering when pooled with data from the previous study and compared in a metabolomic multivariate analyses. Because the addition of ethyl pyruvate provided the same ATP protection as the addition of a nonmetabolizable antioxidant, antioxidant protection was its prominent protective mechanism in the chosen, high glucose protocol. Having distinct clusters in the Scores Plot of a Partial Least Squares-Discriminant Analysis suggests the feasibility of constructing statistical models that are predictive.

Photoluminescent characterization of atomic diffusion in core-shell nanoparticles.

Eu3+ doped LaF3 nanoparticles with core/shell morphologies were synthesized and selected spectroscopic properties were measured as a function of heat treatment times and temperatures. More specifically, the relative intensity of photoluminescence spectra, both through direct excitation of the lanthanide as well as phonon sideband spectra were evaluated with increasing amounts of time held at specific temperatures. A one dimensional approximation was used to compute an effective diffusion coefficient for the rare earth dopants in LaF3. Despite the simplicity of the model employed, the calculated diffusion coefficients based on the spectroscopic results are accurate within an order of magnitude in comparison to other fluoride crystals yielding a simplified approach to estimating kinetic and diffusion effects in optical materials.

A gentle introduction to SNP analysis: resources and tools.

Bioinformatics is the use of informatics tools and techniques in the study of molecular biology, genetic, or clinical data. The field of bioinformatics has expanded tremendously to cope with the large expansion of information generated by the mouse and human genome projects, as newer generations of computers that are much more powerful have emerged in the commercial market. It is now possible to employ the computing hardware and software at hand to generate novel methodologies in order to link data across the different databanks generated by these international projects and derive clinical and biological relevance from all of the information gathered. The ultimate goal would be to develop a computer program that can provide information correlating genes, their single nucleotide polymorphisms (SNPs), and the possible structural and functional effects on the encoded proteins with relation to known information on complex diseases with great ease and speed. Here, the recent developments of available software methods to analyze SNPs in relation to complex diseases are reviewed with emphasis on the type of predictions on protein structure and functions that can be made. The need for further development of comprehensive bioinformatics tools that can cope with information generated by the genomics communities is emphasized.

Incidence and survival of mucinous adenocarcinoma of the colorectum: a population-based study from an Asian country.

PURPOSE: Previous studies have shown conflicting results on the prognosis of mucinous adenocarcinoma of the colorectum. This could be because of heavy bias on patient selection. Furthermore, little data are available from Asian populations. This study was designed to examine incident and prognostic characteristics of mucinous adenocarcinoma of the colorectum based on data obtained from a population-based, Asian, cancer registry. METHODS: A total of 627 of 15,762 were mucinous adenocarcinoma cases from invasive colorectal cancer patients registered in the Singapore Registry from 1968 to 1997. Age-standardized incidence rate was used to describe the incident pattern of mucinous adenocarcinoma of colon and rectum during a period of time. Survival of patients with mucinous adenocarcinoma or ordinary adenocarcinoma was compared using relative survival and proportional hazards model. RESULTS: Age-standardized incidence rate of mucinous adenocarcinoma of the colon and rectum were almost unchanged in males, rising slightly in females during the study periods from 1968 to 1972 to 1993 to 1997. The proportion of mucinous adenocarcinoma cases was similar among genders and calendar-year periods but was higher in younger age groups, Malays and Indians, in advanced stages of the disease, and proximal colon. Five-year relative survival rate of patients with mucinous adenocarcinoma were similar in the colon but were lower in the rectum. CONCLUSIONS: Colorectal mucinous adenocarcinoma as a different etiologic entity from other histologic types of colorectal cancer was suggested. Possibly greater aggressiveness of mucinous adenocarcinoma occurring in the rectum requires confirmation but suggests that mucin is important in the pathogenesis of mucinous adenocarcinoma.

Akt phosphorylation and cell survival after hypoxia-induced cytochrome c release in superfused respiring neonatal rat cerebrocortical slices.

Phosphorylation of Akt before hypoxia (30 min) and during reoxygenation (4 h) was evaluated in superperfused neonatal rat cerebrocortical slices (350 microm, P7, Sprague-Dawley). Cytosolic cytochrome c intensities in Western blots, which were increased at the end of hypoxia. were decreased during reoxygenation. Western blot intensities of phosphorylated Akt (phospho-Akt), nearly undetectable at the end of hypoxia, recovered quickly during reoxygenation, in a trend opposite that for cytochrome c. At 1.5 h and 4 h after hypoxia they became larger or the same as before hypoxia. Total Akt was unchanged by hypoxia and reoxygenation. Phosphocreatine (PCr) and nucleotide triphosphates (NTP) were measured in parallel 14.1 Tesla ex vivo 31P NMR superfused brain slice studies. PCr and alpha-NTP were nearly undetectable at the end of hypoxia. Although they recovered quickly after hypoxia, they were lower than before hypoxia. Reductions in phospho-Akt during hypoxia were consistent with the general unavailability of basic high energy phosphates. Preferential phosphorylation of Akt after hypoxia suggested that substantial reductions in intracellular energy, as indicated by PCr and NTP, might be tolerated by processes important for generating phospho-Akt. Additionally, the post-hypoxia increase in phospho-Akt might have contributed to concomitant reductions in cytosolic cytochrome c.

NTP and PCr responses to hypoxia by hypothermic and normothermic respiring, superfused, neonatal rat cerebrocortical slices: an NMR spectroscopy study at 14.1 Tesla.

Although mechanisms of hypothermic neuroprotection during oxygen deprivation have long been investigated, further characterizations of various molecular mechanisms are appropriate. Anticipating future studies of hypothermia and hypoxia/ischemia, we investigated the extent to which our ex vivo, NMR-based, superfused brain slice model might be helpful. (Slices are approximately 350 microm thick, with 18 slices per 8 mm NMR tube.) 31P NMR spectroscopic measurements were made of hypothermia-induced changes in high energy phosphates, while simultaneously monitoring and controlling tissue temperature, using 1H NMR, the high spectroscopic resolution available at 14.1 Tesla (600 MHz for protons), and a recently published protocol. NTP and PCr concentrations in healthy, well-oxygenated slices decreased to (55 +/- 15)% and (66 +/- 30)% of their respective values at 28.0 degrees C when warmed to 38.0 degrees C, in approximate agreement with earlier in vivo studies by others. During 30 min hypoxia NTP and PCr decreased to non-observable values, regardless of temperature. After reoxygenation, NTP and PCr recoveries as percentages of respective prehypoxia values were (63% +/- 16%; 70%) +/- 5%) for hypothermic slices (28.0 degrees C), and (46% +/- 13%; 41% +/- hypothermic neuroprotection during oxygen deprivation in this model, which appears suitable for use in further studies.

Preservation of the morphological and molecular stability of embryonic tissues.

There is presently great interest in using early embryonic tissues, particularly human tissue, for studies of protein and gene expression. Embryonic human tissue is very fragile, and delays often occur before it can be properly prepared for scientific study. Using chick embryos, we have studied the effects of delaying fixation or biochemical isolation on the preservation of cytological characteristics and biochemical molecules. Our study shows that by 60 min post-harvest, tissue morphology and immunofluorescence staining degrades, but the total mRNA profile remains stable. This study suggests that the time between removal of the tissue and fixation is critical to the results and that the critical time is much shorter for embryonic tissues than for more developed tissues. Our results have implications for all research where embryonic tissues are harvested but not processed immediately.

Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR.

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.

Molten-globule structure and membrane binding of the N-terminal protease-resistant domain (63-193) of the steroidogenic acute regulatory protein (StAR).

The first step in steroidogenesis is the movement of cholesterol from the outer to inner mitochondrial membrane; this movement is facilitated by the steroidogenic acute regulatory protein (StAR). StAR has molten-globule properties at low pH and a protease-resistant N-terminal domain at pH 4 and pH 8 comprising residues 63-193. To explore the mechanism of action of StAR we investigated the structural properties of the bacterially expressed N-terminal domain (63-193 StAR) using CD, limited proteolysis and NMR. Far- and near-UV CD showed that the amount of secondary structure was greater at acidic than at neutral pH, but there was little tertiary structure at any pH. Unlike 63-193 StAR liberated from N-62 StAR by proteolysis, biosynthetic 63-193 StAR was no longer resistant to trypsin or proteinase K at pH 7, or to pepsin at pH 4. Addition of trifluoroethanol and SDS increased secondary structure at pH 7, and dodecylphosphocholine and CHAPS increased secondary structure at pH 2, pH 4 and pH 7. However, none of these conditions induced tertiary structure, as monitored by near-UV CD or NMR. Liposomes of phosphatidylcholine, phosphatidylserine and their mixture increased secondary structure of 63-193 StAR at pH 7, as monitored by far-UV CD, and stable protein-liposome complexes were identified by gel-permeation chromatography. These results provide further evidence that the N-terminal domain of StAR is a molten globule, and provide evidence that this conformation facilitates the interaction of the N-terminal domain of StAR with membranes. We suggest that this interaction is the key to understanding the mechanism of StAR's action.

NMR determination of oligonucleotide structure.

This unit provides an overview of the use of NMR to determine oligonucleotide structure. It covers basic NMR spectral properties, acquisition of interproton distance restraints and torsion angle restraints, structure refinement, assessment of the quality of the structure obtained. Software programs used in the process are also described.

The solution structure of the viral binding domain of Tva, the cellular receptor for subgroup A avian leukosis and sarcoma virus.

The cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A) is Tva, which contains a motif related to repeats in the low density lipoprotein receptor (LDLR) ligand binding repeat (LBr) and which is necessary for viral entry. As observed with LBr repeats of LDLR, the 47 residue LBr domain of Tva (sTva47) requires calcium during oxidative folding to form the correct disulfide bonds, and calcium enhances the structure of correctly oxidized sTva47, as well as its ability to bind the viral envelope protein (Env). However, solution nuclear magnetic resonance studies indicate that, even in the presence of excess calcium, sTva47 exists in an ensemble of conformations. Nonetheless, as reported here, the structure of the predominant sTva47 solution conformer closely resembles that of other LBr repeats, with identical S-S binding topology and octahedral calcium coordination. The location of W48 and other critical residues on the surface suggests a region of the molecule necessary for Env binding and to mediate post-binding events important for ALSV-A cell entry.

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR.

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds L-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 microM.

Determination of the populations and structures of multiple conformers in an ensemble from NMR data: multiple-copy refinement of nucleic acid structures using floating weights.

A new algorithm is presented for determination of structural conformers and their populations based on NMR data. Restrained Metropolis Monte Carlo simulations or restrained energy minimizations are performed for several copies of a molecule simultaneously. The calculations are restrained with dipolar relaxation rates derived from measured NOE intensities via complete relaxation matrix analysis. The novel feature of the algorithm is that the weights of individual conformers are determined in every refinement step, by the quadratic programming algorithm, in such a way that the restraint energy is minimized. Its design ensures that the calculated populations of the individual conformers are based only on experimental restraints. Presence of internally inconsistent restraints is the driving force for determination of distinct multiple conformers. The method is applied to various simulated test systems. Conformational calculations on nucleic acids are carried out using generalized helical parameters with the program DNAminiCarlo. From different mixtures of A- and B-DNA, minor fractions as low as 10% could be determined with restrained energy minimization. For B-DNA with three local conformers (C2'-endo, O4'-exo, C3'-endo), the minor O4'-exo conformer could not be reliably determined using NOE data typically measured for DNA. The other two conformers, C2'-endo and C3'-endo, could be reproduced by Metropolis Monte Carlo simulated annealing. The behavior of the algorithm in various situations is analyzed, and a number of refinement protocols are discussed. Prior to application of this algorithm to each experimental system, it is suggested that the presence of internal inconsistencies in experimental data be ascertained. In addition, because the performance of the algorithm depends on the type of conformers involved and experimental data available, it is advisable to carry out test calculations with simulated data modeling each experimental system studied.

The novel immunosuppressant SDZ-RAD protects rat brain slices from cyclosporine-induced reduction of high-energy phosphates.

1. SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-effect of cyclosporine. 2. We studied the effect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31P-magnetic resonance spectroscopy (MRS). 3. Cyclosporine significantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 micrograms l-1: 93 +/- 3% of control (NTP), 91 +/- 3% (PCr); 500 micrograms l-1: 84 +/- 2% (NTP), 73 +/- 2 (PCr); 5000 micrograms l-1: 68 +/- 3% (NTP), 55 +/- 5% (PCr); n = 6; P < 0.02). 4. In contrast, after perfusion for 2 h, SDZ-RAD (500 micrograms l-1 and 5000 micrograms l-1) significantly increased high-energy phosphate concentrations in the brain slices (P < 0.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 micrograms l-1 SDZ-RAD + 5000 micrograms l-1 cyclosporine: 86 +/- 3% (NTP), 83 +/- 7% (PCr), n = 3, P < 0.03 compared to cyclosporine alone. 5. As evaluated using an algorithm based on Loewe isobolograms, the effects of SDZ-RAD/cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6. We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.

Three-dimensional RNA structure-based drug discovery.

Abstract We have initiated a program to develop promising drug candidate leads using a new drug discovery paradigm based on three-dimensional RNA-structure-based computational screening of about 200,000 commercially available compounds for binding to selected RNA targets. As our first endeavor, we are using the three-dimensional structure of portions of the HIV-1 genome. Candidate lead compounds we seek are water-soluble, nonpeptide, nonnucleotide organic compounds generally with molecular weight less than 500 daltons. Structural studies of complexes formed with potential leads and their RNA targets should eventually yield insight into features governing affinity and specificity. The promising leads identified by virtual screening are tested for inhibition in functional assays. Leads will be selected for further development via computational and experimental combinatorial chemistry.

Structure of the phylogenetically most conserved domain of SRP RNA.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA.

The two identical genomic RNA strands inside each HIV-1 viral particle are linked through homodimerization of an RNA stem-loop, termed SL1, near their 5' ends. SL1 first dimerizes through a palindromic sequence in its loop, forming a transient kissing-loop complex which then refolds to a mature, linear duplex. We previously reported the NMR structure of a 23-base truncate of SLI in kissing-dimer form, and here report the high-resolution structure of its linear isoform. This structure comprises three short duplex regions--derived from the central palindrome and two stem regions of each strand, respectively--separated by two bulges that each encompass three unpaired adenines flanking the palindromes. The stacking pattern of these adenines differs from that seen in the kissing-loop complex, and leads to greater colinear base stacking overall. Moreover, the mechanical distortion of the palindrome helix is reduced, and base pairs ruptured during formation of the kissing-loop complex are re-established, so that all potential Watson-Crick pairs are intact. These features together likely account for the greater thermodynamic stability of the mature dimer as compared to its kissing-loop precursor.