Thomas James - US grants

Purchase of a 31 cm bore nuclear magnetic resonance spectrometer/imager is proposed. The instrument will be used for a variety of in vivo studies with different animal organs, under normal and pathological conditions. The research should serve to elucidate some basic physiology as well as to enable improved medical therapy in various clinical situations.

A 500 MHz NMR spectrometer and computer system will be acquired for use in structural biology. New software will be developed for analyzing NMR data and utilizing that data to determine the structure of proteins in solution. Additional studies concern the relationship between the linear or primary sequence of proteins and the secondary and tertiary fording of proteins in three dimensional space. NMR data will be used to evaluate schemes for understanding the potential conformations of identical sequences of amino acids contained in different larger amino acid sequences. The structure and dynamics of short DNA oligomeric chains and of small molecules that interact with DNA and protein will also be studied. The information derived from the NMR experiments will be used in conjunction with distance geometry calculations, energy refinement calculations and computer graphics to generate molecular structures in solution in greater detail than is presently known. The information will be compared to our knowledge of the static structure of proteins and DNA known from X-ray diffraction data, and will lead to a better understanding of the relationship between architecture and the functional role of these macromolecules.

We propose the acquisition of a high-field NMR spectrometer. The spectrometer is necessary to provide structural information (e.g., from relaxation and two dimensional NMR experiments) about DNA fragments, proteins, and drugs in solution. The information derived from the NMR experiments will be used in conjunction with distance geometry calculations and computer graphics to generate molecular structures in solution in greater detail than presently exists.

The proposed project will continue development of the methodology for the determination of detailed molecular structures in non-crystalline environments. Principally, this entails two-dimensional nuclear magnetic resonance techniques analyzed in detail with a complete relaxation matrix analysis and used in conjunction with distance geometry, molecular mechanics and molecular dynamics calculations. Specifically, the methodology will be applied to the investigation of nucleic acid fragments, with an emphasis on those containing adenine (A) or thymine (T). Although related DNA fragments will constitute the majority of studies, analogous RNA and RNA-DNA hybrids will also be subjects for study. Further studies will include modified bases. Finally, binding of drugs which show preference to AT moieties of DNA will be investigated.

We propose the acquisition of a console capable of both magnetic resonance spectroscopy and imaging, using an existing magnet, for in vivo studies. The instrument will be used for studies of heart, brain, gut, and tumors in small animals (principally rats) as well as for development of new magnetic resonance methodologies.

We propose the acquisition of a set of self-shielded gradient coils which will be useful for both magnetic resonance spectroscopy and imaging, using an existing magnetic resonance instrument, for in vivo studies. The instrument will be used for studies of heart, brain, kidney and tumors in animals as well as for development of new magnetic resonance methodologies.

We propose the acquisition of an upgraded console for a 500 MHz NMR spectrometer. The research proposed for the new instrument will improve our capability for determining high-resolution protein and nucleic acid structures in solution from two-dimensional NMR experiments. The most effective means of deriving nucleic acid and protein solution structure by using the complete relaxation matrix analysis of 2D NOE spectra in conjunction with the computational techniques of distance geometry, molecular mechanics and restrained molecular dynamics will be explored. Reverse-detection heteronuclear 2D NMR experiments will enable resonance assignments and structural insights. Some studies necessitate the use of H2O solutions, thus requiring efforts to overcome the dynamic range problem due to water protons. The distance information obtained via 2D NOE spectra will be augmented by dihedral angle constraints derived from scaler coupling-based 2D NMR experiments. The improved capabilities should also aid in our studies of ligand binding to biopolymers, thus leading to greater mechanistic insights.

This study will examine the interaction of three major forces in the evolution of state policies governing management of coastal barrier systems: 1) short-term and long-term changes to the physical environment of coastal barriers; 2) substantive knowledge and expertise about environmental change processes; and 3) the implementation of alternative policy innovations in response to environmental change. Case studies of coastal states that have experienced environmental changes will be conducted to provide qualitative empirical validation of a conceptual model of institutional response to chronic environmental change. The work will also improve understanding of key relationships that stimulate environmental policy innovation. Increasing concern about global environmental change has focused attention on the detrimental effects that future climate change might exert on natural environments and society. This research will provide valuable theory and data for aiding societal decision making regarding environmental policies to plan for and protect against adverse effects of climate change.

The proposed project will continue development of the methodology for the determination of detailed molecular structures in non-crystalline environments. Principally, this entails two-dimensional nuclear magnetic resonance techniques analyzed in detail with a complete relaxation matrix analysis and used in conjunction with distance geometry, molecular mechanics and molecular dynamics calculations. Specifically, the methodology will be applied to the investigation of nucleic acid fragments, with an emphasis on those containing adenine (A) or thymine (T). Although related DNA fragments will constitute the majority of studies, analogous RNA and RNA-DNA hybrids will also be subjects for study. Further studies will include modified bases. Finally, binding of drugs which show preference to AT moieties of DNA will be investigated.

The proposed research will improve the capability for determining high- resolution protein structures in solution from two-dimensional NMR experiments. Part of this advance will come via implementation of methodology developed in this lab for the complete relaxation matrix analysis (CORMA) of two-dimensional nuclear Overhauser effect (2D NOE) spectra, enabling the determination of a large number of internuclear distances at considerably greater accuracy than previously possible. The new methodology should also enable somewhat longer distances to be determined-up to 5angstrom or possibly 6angstrom. The most effective means (as well as the influence of plausible errors and assumptions) of deriving protein solution structure by using the complete relaxation matrix analysis in conjunction with the computational techniques of distance geometry, molecular mechanics and restrained molecular dynamics will be explored. The distance information obtained via 2D NOE spectra will be augmented by dihedral angle constraints derived from scalar coupling-based 2D NMR experiments. The effects of molecular motions and multiple conformations will also be examined. Programs of merit which are developed by this project will be disseminated to other research labs. The improved methodology will be especially useful in protein moieties where improved structural knowledge is highly desirable, i.e., in ligand-binding sites. The improved structures should lead to greater mechanistic insights.

We propose the acquisition of a 600 Mhz NMR spectrometer. The research proposed for the new instrument will improve our capability for determining high-resolution protein and nucleic acid structures in solution from two- dimensional NMR experiments. The most effective means of deriving nucleic acid and protein solution structure by using the complete relaxation matrix analysis of 2D NOE spectra to obtain numerous internuclear distances and scalar-coupling-based 2D NMR experiments to yield torsion angle constraints, in conjunction with the computational techniques of distance geometry, molecular mechanics and restrained molecular dynamics will be utilized. Reverse-detection heteronuclear 2D NMR experiments will enable resonance assignments and structural insights. Some studies necessitate the use of H2O solutions, thus requiring efforts to overcome the dynamic range problem due to water protons. The improved capabilities should also aid in our studies of ligand binding to biopolymers, thus leading to greater mechanistic insights. The major user group (with NIH support) is composed of Vladimir J. Basus (RR01695), Thomas L. James (GM39247, GM41639), George L. Kenyon (GM39552, AR17323, GM45070), Irwin D. Kuntz (RR01695, GM19267), Norman J. Oppenheimer (GM22982, CA47764), and Richard H.Shafer (CA27343).

The three-dimensional structure of DNA is quite dependent on sequence. This should be intuitively obvious from the sequence specificity required by many proteins for recognition. The few available x-ray crystal structures and NMR solution structures attest to this structural flexibility as well. The location and orientation of potential ligand binding functions on the DNA such as charges, hydrogen bonding and hydrophobic sites can be modified substantially from that which one might expect on the basis of assuming a canonical B-DNA structure. Consequently, we intend to continue development of the methodology for determination of high-resolution nucleic acid structures in solution and apply this methodology to some oligonucleotides and oligonucleotide complexes. This entails methods designed to improve the accuracy and resolution of the structures determined. Improved structures can be obtained with more accurate and more numerous experimental distance and torsion angle constraints, as well as improvements in calculating structure from these constraints. Enhancements will result from improvements in our iterative complete relaxation matrix program MARDIGRAS, development of a more encompassing density matrix approach for analysis of spectra derived from any pulse sequences (even those not yet invented), development of tailored excitation pulses, inclusion of experimental molecular motion information, and development of alternative methods of reducing experimental structural constraint data to structures. The latter includes (a) for restrained molecular dynamics simulations, use of improved force fields, empirical development of improved force fields, and use of constraint terms permitting a more realistic picture of conformational flexibility, and (b) development of an alternative restrained Monte Carlo method in torsion angle and helical parameter space, which is quite promising especially for structure refinement directly against NOE intensities. Applications will include oligonucleotides of interest, in particular sequences recognized by transcription factors or regulators, genome targets, antisense oligonucleotides, and a DNA microcircle duplex. Structures of proteins (including nucleic acid complexes) which are important for initiation or regulation of transcription will be determined. In particular, the 72-residue protein GerE which is a regulatory protein that binds specifically to a target site in promoter DNA will be the subject of study. Other proteins will be evaluated as possible candidates for study.

The behavior of individuals with respect to risks to their health or to the environment may be significantly affected by how they perceive the risks. This has implications for public health and environmental policy designed to remedy or manage these risks. Further, differences in risk perception may increase confrontations between various groups in society or may render public policy aimed at health protection and risk management ineffective. Careful, in-depth studies of distinctive social groups and their interaction with health and environmental risks can inform risk theory and promote the development and implementation of more effective public policies. This study contributes to the understanding of risk perception formation and the influences of psychological, social and cultural factors in Native American populations, specifically, the Mvskoke of Oklahoma. Ethnographic methods and a survey instrument will be developed that integrate psychometric and cultural factors in risk perception to capture a richer framework for addressing the risk responses of diverse cultural groups.

We propose a collaborative project between UCSF and the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, in the area of nucleic acid conformations. Specifically we plan to investigate a novel type of polynucleotide folding, a Slipped Loop Structure (SLS), proposed recently by the moscow group. The aim of this project is to supplement our existing evidence in order to prove the existence of the SLS in solution and to build its three-dimensional molecular model. We will study specifically designed deoxyoligonucleotides of ca. 55 bases in length, which should favor SLS formation but not eh usual stem-and -loop conformations. In this project, the Moscow group will obtain a various information about the single- and double-stranded regions and stability for these oligonucleotides using a unique set of fluorescence techniques (some of which have been originally developed by the Moscow group), as well as chemical and enzymatic probing of the DNA conformation, while the UCSF group will perform the high resolution proton NMR studies. We have already obtained data for certain sequences, which strongly support the concept of SLS formation in solution under physiological conditions. In the later stages of this project, we will perform conformational calculations of the SLS conformation with restraints obtained from all experimental methods. The SLS folding, when found in genomic DNA or RNA, may have a role in the regulation of gene expression, in both transcription and translation, similar to that proposed for other unusual structures (cruciform, H-form, RNA pseudoknot). This could have future potential for the rational interference in the activity of selected genes, e.g. those responsible for a malignant transformation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In the past few years, three-dimensional structures of about 200 small proteins and a DNA oligomers in solution have been determined. Multi-dimensional NMR, in particular two-dimensional nuclear Overhauser effect(2D NOE) spectra, when used in conjunction with distance geometry and energy refinement calculations can be used to determine the high-resolution structure of DNA fragments, small proteins and complexes. A major goal of our research is to improve the capability for determining high-resolution protein and nucleic acid structures in solution, including a depiction of their dynamic nature. To this end, we have been developing methods to obtain more accurate structural restraints(in the form of internuclear distances and torsion angles) and a greater number of structural restraints. We are currently working on new refinement protocols using restrained molecular dynamics and the particle mesh Ewald technique as implemented in the sander module of AMBER 5.0. In addition, as we deal mostly with conformationally flexible molecules in solution, we have been exploring computational methodologies for ascertaining the dynamic nature of these molecules. Publications - Konerding DE, Cheatham TE 3rd, Kollman PA, James TL.:"Restrained molecular dynamics of solvated duplex DNA using the particle mesh Ewald method.":Journal of Biomolecular Nmr:13:119-31:1999;Achievements - We used the T3E at PSC to run a 2 nanosecond unrestrained MD simulation of DNA in explict solvent using AMBER 5.0. See publication #1 below for more details. Publications - None that have not previously been reported.

THE SIR SUPPLIED DR. JAMES WITH 1G OF D, L-ARGININE-GUANIDO 13C, 15N2. ARGININE WITH 13C AND 15N LABELED IN THE GUANIDO GROUP IS OBTAINED FROM THE NATIONAL STABLE ISOTOPE RESOURCE AT LOS ALAMOS NATIONAL LABORATORY. THIS LABELED ARGININE IS INCORPORATED INTO A 25 AMINO ACYL PEPTIDE WHICH CONTAINS THE RNA BINDING DOMAIN OF THE TAT PROTEIN FROM HIV-1 AND HAS BEEN SHOWN TO PROMOTE HIV-1 REPLICATION CELLS. WHILE THE 25-MER HAS HAD ITS SOLUTION STRUCTURE DETERMINED VIA HOMONUCLEAR PROTON NMR STUDIES, THE SIDE-CHAIN PROTON RESONANCES OF THE SEVERAL ARGININES CANNOT BE RESOLVED IN THE SPECTRA. PLACING THE LABELED ARGININE AT THE POSITION CORRESPONDING TO R52 IN HIV-1 TAT IS IMPORTANT IS SUBSEQUENT STUDIES OF THE BIOLOGICALLY ACTIVE TAT PEPTIDE WITH TAR, THE RNA TO WHICH IT BINDS IN VIRAL REPLICATION. R52 HAS BEEN DEMONSTRATED TO BE ESSENTIAL FOR TAT ACTIVITY. THE STRUCTURE OF THIS SELECTIVELY LABELED PEPTIDE BOUND TO TAR SHOULD BE MUCH BETTER DEFINED BY USE OF THE LABELED ARG. CONSEQUENTLY, THE STRUCTURE DETERMINED SHOULD PROVIDE A BETTER TARGET FOR DEVELOPMENT OF A DRUG TO COMBAT AIDS.

virus; proteins; nucleic acids; AIDS; immunology; communicable diseases; lymphatic system; biomedical resource; biological products;

DESCRIPTION: Sequence-dependent structural variations in nucleic acids are important for protein-nucleic acid and RNA-DNA interactions essential for life as well as for nucleic acid packaging. It is proposed that methodological developments be continued which have the aim of determining the dynamic nature of the structure of nucleic acids. The principal tool to be utilized is NMR. Computational methods of analyzing the NMR data are proposed. Primary emphasis, however, is on applying the methodology to determine structure and dynamics in several systems of nucleic acids. Systems include DNA promoter sequences, RNA-DNA bybrids including a hybrid with a mismatch, slipped loop and pseudosquare knot DNA and slipped loop RNA, nicked DNA, and duplexes formed between the target RNA and antisense DNA oligonucleotides. With the increasing numbers of high-definition structures becoming available, statistical analyses of solution structures of DNA duplexes and RNA-DNA hybrids will be carried out to discern sequence-dependent structural features which may have useful predictive value. Molecular motions, or conformational fluctuations, will be investigated by measuring imino proton exchange kinetics and heteronuclear relaxation parameters, using DNA labeled either selectively or uniformly with 13C, 15N, or 2H. Structural and stability studies of sequences composed of RNA complexed by complementary antisense DNA strands containing chirally pure methylophonates at alternating backbone positions, as well as chirally pure phosphorothioates or phosphorodithioates at each position, and 2'-O-alkoxy derivatives, will be compared with the RNA bound by the normal complementary DNA. To date, there has been little structural information available on these antisense nucleotides, in spite of their growing use as therapeutic and diagnostic agents. Slipped loop and the related pseudosquare knot DNA structures are potentially formed with short direct repeats; while some trinucleotide repeats have been associated with genomic instability leading to diseases such as myotonic dystrophy, somewhat longer direct repeats often occur in gene regulatory regions and have been postulated as significant for regulation.

This proposal requests funds for the purchase of a new electrospray tandem quadrupole orthogonal-acceleration time-of flight mass spectrometer (Q- TOF). This shared instrument will be used for structural studies of bio- macromolecules and their myriad covalent modifications that are of critical importance to on-going research in the biological and medical sciences in the San Francisco Bay Area. It will be housed in the NIH NCRR- supported Mass Spectrometry Resources at UCSF. This environment ensures the availability of the scientific, technical and management expertise required to optimize its productive in solving problems at the forefront of macromolecular biology and medicine for a large number of investigators. The proposed instrumentation will provide previously unavailable absolute sensitively for on-line capillary separation-ES1 MS/MS to project investigators with electrospray experience and established needs. The major users are a group of 20 scientists and clinicians involved in over 50 NIH-supported research programs. These research projects are at the forefront of biomedical problems concerning the identification of native and covalently modified peptides, proteins, carbohydrates and nucleic acids required in deciphering cell function and dysfunction. Most of these studies require the high absolute sensitivity and performance of the instrument requested. Prof. George L. Kenyon, Dean of the School of Pharmacy, will chair the Advisory Committee responsible for the overall guidance of the shared instrument's use. The committee will assure that the time is shared equitably and that some 10% of its time will be made available to other projects requiring its unique capability. The School will contribute $160,820.00 toward this purchase. Mr. David Maltby, who has extensive experience in electrospray and other types of biological mass spectrometry, will oversee scheduling, training and maintenance and will provide daily supervision of usage. Long term productive usage and maintenance will be assured by the close association with a major Mass Spectrometry Facility and financial support of the major users.

The major goal of our research is to improve the capability for determining high-resolution protein and nucleic acid structures and dynamics in solution from multidimensional NMR experiments and to apply that methodology to interesting subjects. A topic of especial interest is to develop the means of ascertaining the dynamic structure of biopolymers, since real molecules undergo conformational fluctuations in solution. At present, we continue to improve methodology, and applications include DNA gene targets, RNA, and proteins which regulate gene transcription. The subjects for structure determination are often chosen to be targets for subsequent drug design. Part of the core research of the Computer Graphics Laboratory is development of Sparky, the NMR analysis tool. We make extensive use of Sparky. Members of my lab provide most of the user input for development of Sparky. CGL facilities are utilized to display the three-dimensional structure and analyze structural features. We also use the graphics lab during the course of structure refinement, as the MidasPlus delegates, NOEshow and AMBERshow, enable us to readily examine any inconsistencies of interim structures with experimental data. In our most recent work, we are generating conformational ensembles. We are working on ways of depicting the large amount of information on the ensembles in an effective graphical representation.

The overall goal of this project is to develop promising drug candidate leads using a new drug design paradigm based on initial three-dimensional RNA-structure-based computational screening of about 255,000 commercially available compounds for binding to selected RNA targets using the three-dimensional structure of portions of the HIV-1 genome. Specifically, the transactivation response element (TAR) and the dimer linkage site (DLS) are proposed as targets. The latter is a novel target for drug design. Good computational "hits" will be tested for inhibition in functional assays. The best of these will be utilized in NMR studies to verify the mode of binding to the RNA target. The best candidates will be water-soluble, nonpeptide, nonnucleotide organic compounds generally with molecular weight less than 500 daltons, but it may be necessary to consider slightly larger compounds as leads. The NMR structural studies of complexes formed with potential leads and their RNA targets should yield insight into features governing affinity and specificity. Promising leads will be selected for further development via computational and experimental combinatorial chemistry. Some further development of the methodology is proposed.

We propose the acquisition of an 800 MHz NMR spectrometer that will serve the research community of UCSF primarily with some service for researchers on other University of California campuses in Northern California. The research proposed for the new instrument will improve our capability to use multidimensional NMR experiments for determining high-resolution structures in solution primarily of proteins, nucleic acids, and complexes entailing proteins and nucleic acids, as a means of elucidating biological function. The additional sensitivity and resolution, as well as enhanced utilization of residual dipolar coupling measurements and pulse sequences incorporating transverse relaxation optimized spectroscopy (TROSY), afforded by the new instrument will not only increase through-put in structure determination, but it will permit determination of resonance assignments in regions which are currently ambiguous, enable additional structural restraints to be determined leading to higher quality structures, enable some structure determinations which have not been possible so far due to aggregation problems, and extend our ability to determine structures of biopolymers with molecular mass >30 kilodaltons.

A collaborative project is proposed between UCSF and the Engelhardt Institute of Molecular Biology, Russian Academy of Science, Moscow, in the area of RNA conformations. The parent grant for the present proposal is GM39247 (7/1/98-6/30/02). Alternative conformations of RNA will be studied for functionally important sequences. Specifically, two cases will be investigated where conformation switching of RNA may play an important functional role: (1) structures that are formed during the dimerization of genomic RNA of avian retroviruses. (2) a hypothetical transient RNA psuedoknot at the ribosomal peptidyl transferase cancer, proposed recently by the Moscow group based on phylogenetic analysis of ribosomal RNA sequences. A set of RNA oligonucleotides (40 to 50 nucleotides in length) will be studied in solution, including sequences from a number of avian retroviruses, human and bacterial rRNA sequences, a number of mutated sequences. In this project, the Moscow group will characterize the RNA oligonucleotides by physicochemical methods and map the single- and double-stranded regions using chemicals and enzymatic probing. Based on these data, three-dimensional models will be calculated for the structures involved, and suitable sequences will be studied by UCSF groups using high resolution NMR. A reversible formation of a pseudoknot structure at certain stages of the ribosome operation may be responsible for the large-scale dynamics during protein synthesis. Understanding the detailed mechanisms of this process as well as that of the retroviral dimerization and knowledge of the structures involved may open new perspectives in designing new bacterial and antiviral therapeutic agents.

The long term goal of this project is to improve computer based simulation methods that can be applied to study the structures, kinetics and thermodynamics of nucleic acids and their interaction with ligands. In the next period of grant support, we anticipate making major improvements in the energy function used to describe nucleic acids, and in the methodology to determine conformational free energies, free energies for mutations, and absolute free energies of association for nucleic acids and nucleic acid-ligand complexes using molecular dynamics/free energy approaches. We plan to use available X-ray and NMR data to evaluate our models. Applications to a number of the most interesting physical chemical phenomena in DNA and RNA structure and thermodynamics will be carried out including the Z phobicity of AT base pairs, the thermodynamics of RNA loops, the sequence selectivity and binding of DNA minor groove binding ligands, and the neighbor exclusion rule for ligand binding to DNA. Recent advances are allowing a most exciting synergy between computer based theoretical methods to study intermolecular inter-actions and experiments. These computer simulations are yielding more accurate insights than ever before and are often correctly predicting the results of subsequent experiments. The work proposed here has such a synergistic relationship to experiments and is aimed at a continued improvement of the predictive power of these computer based theoretical methods. The long term objective of our studies is to make the computer based approaches truly reliable and predictive and to use them in anti-cancer and anti-AIDS drug design.

The goal of this project is to develop and apply computational methods to reproduce, understand and predict structural and thermodynamical aspects of molecular recognition in the EcoRV enzyme-DNA and the small ligand-DNA complexes. We will use this methodology with a protein (EcoRV) interacting with DNA and small ligands: actinomycin, acridine, netropsin, furimidazoline. We will use the available experimental data on these systems as a critical guideline to evaluate the theory. Understanding recognition in the systems proposed here, especially in the EcoRV-DNA complex, will further the long term goal of the parent NIH grant, which is to make the computer approaches reliable and truly predictive in order to use them as an important tool in understanding molecular interactions as well as in the drug design process.

[unreadable] DESCRIPTION (provided by applicant): Of all methods currently available for obtaining high resolution structures of biological macromolecules, NMR is the only one that can provide this information in solution under near physiological conditions. However, even NMR structures are still determined in vitro, and often buffer conditions are not selected for their closest match to the natural environment of the protein but to optimize experimental parameters such as solubility and sensitivity or to minimize NMR buffer signals that could interfere. Depending on the natural host cell and the exact cellular compartment, these NMR buffer conditions can be substantially different from a protein's natural environment and may influence its structure and dynamics. Furthermore, interactions with other cellular (macro-) molecules and post-translational modifications can alter the conformation. In principle, NMR spectroscopy, as a non-invasive spectroscopic technique, should be able to provide infonnation about the structure and dynamics of biological macromolecules inside living cells. Recently, we have demonstrated that the conformation and the dynamics of proteins can indeed be observed by NMR inside living E. coli bacteria. Clearly, developing these "in-cell" NMR experiments for eukaryotic cells would open new avenues to study the behavior of proteins and their interaction with other cellular components in their natural environment. The biggest advantage of these techniques would not be to determine structures, but to observe structural changes that can; for example, be caused by posttranslational modifications or binding to other cellular components. In addition, these techniques could be used to study the interaction of proteins inside the cell with potential drugs. While NMR-based drug screens are already a common tool in the pharmaceutical industry, an "in-cell" drug screen would, not only identify potentially interesting molecules, but could also show if these molecules can penetrate the cellular membrane and interact with their target inside a cell. Based on theoretical considerations, in-cell NMR experiments should be feasible in eukaryotic cells. In this grant application, we propose to extend our in-cell NMR techniques that we have developed for bacteria to eukaryotic model systems. In particular, we will take advantage of the high overexpression levels obtainable in yeast and in SF9 insect cells. In addition, we propose to explore the possibility to inject purified proteins into xenopus oocytes and to use these cells for in-cell NMR.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this project is to develop promising drug leads via three-dimensional RNA structure based computational screening of commercially available compounds for binding to selected RNA targets utilizing the 3D structure of portions of the HIV-1 genome. Candidates are small water-soluble, nonpeptide, nonnucleotide organic compounds. These compounds should also represent promising scaffolds for subsequent chemical modification to enhance their pharmaceutical properties. The project entails work on improving the methodology for efficient targeting of RNA. This includes working to make the well-known DOCK program more useful for targeting RNA. It also entails continued development of a new program that enables screening of a smaller database of compounds (up to 10,000) permitting induced fit with the receptor. However, we are quite interested in using the methodology even at its current stage to discover new lead compounds that bind to important HIV RNA structures and thus could be developed into drugs. The most promising computational "hits" are purchased and tested for binding to the target RNA via various assays we have developed, including NMR. Experiments will continue on compounds already identified to bind to TAR RNA and to the kissing loop duplex that is essential for assembly of the genomic RNA duplex in the budding virion. Among compounds to be examined are several phenothiazines that have been or are being synthesized to establish a structure-activity relationship for binding to TAR; phenothiazine is a promising scaffold for drug development due to low toxicity and good bioavailability. A library of low molecular weight compounds, with good bioavailability and low toxicity and the capability of being linked, that have been found to bind RNA will be developed. The mechanism of the transformation of the kissing loop dimer to the linear dimer, required for assembly of the budding virion, will be studied using a newly found small molecule agonist that promotes the transformation as does the nucleocapsid protein NCp7. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Women who consume large amounts of alcohol are also more likely to engage in risky reproductive behavior, increasing their likelihood of unplanned pregnancies, interpersonally transmitted infections (Leigh, 1999;O'Hare, 1998), and fetal alcohol exposure (Streissguth et al., 1994). The National Institute on Alcohol Abuse and Alcoholism recently reported an increase in heavy episodic drinking in college students (Hingson et al., 2009). Understanding appetitive decision-making in young women of reproductive age could inform appropriate intervention for this increasingly at-risk group. In particular, more specific knowledge is needed regarding the interaction between alcohol and the brain processes underlying motivated decision making that may bias alcohol-dependent women towards risky behavior. Young adulthood is a critical time during which not only reproduction most often occurs, but alcohol dependence and frontal neural systems underlying decision making develop, suggesting a possible interaction between the two developmental processes. The current study investigates the hypothesis that altered functional neuroanatomy of the prefrontal-limbic system is an important link between alcohol abuse and risky reproductive behavior. Furthermore, a woman's hormonal state is likely to be a relevant factor mediating alcohol and brain interactions, possibly creating windows of acute vulnerability for young women and their potential offspring. Recent evidence suggests that the prefrontal-limbic reward system differs functionally across a woman's menstrual cycle during appetitive decision-making (Rupp et al., 2009). Specifically, women evaluating novel male faces exhibited more neural activation in the orbitofrontal cortex when tested during ovulation as compared to the luteal phase of the cycle. We hypothesize that alcohol-dependent young women have a more sensitized reward brain circuitry in general, and that the hormonal state close to ovulation further enhances vulnerability to risk taking. Using functional magnetic resonance imaging (fMRI) we will examine the neural activation patterns of 20 alcohol-dependent and 20 nondependent young women in response to rewarding stimuli, including pictures of alcohol, male faces, and food. In order to test for effects of hormonal status, we will test naturally cycling women at two phases across their menstrual cycles, including during the follicular phase when a woman is most likely to conceive. We predict that alcohol-dependent women will demonstrate greater neural activation in brain regions associated with reward and less activation in brain regions related to behavioral inhibition, and that the difference will be especially pronounced around ovulation. The ability to balance between the risk and reward of potential appetitive situations and partners is imperative for the safety of young women and their potential offspring. An understanding of menstrual cycle interactions with neural activation and reproductive decision making in alcohol-dependent and non dependent women can enhance our ability to intervene to promote safer appetitive behavior in at-risk young women. PUBLIC HEALTH RELEVANCE: Young alcohol-dependent women demonstrate changes in brain function that may bias them towards risky reproductive decision-making. Unplanned pregnancies in women who consume large amounts of alcohol have long-term negative consequences for infants prenatally exposed to alcohol. An understanding of the functional neuroendocrinological changes that occur with alcohol-dependence, such as sensitization of neural systems associated with the appraisal of risk and reward, are critical to promote optimal appetitive decision-making in young women of reproductive age to protect them and their offspring from the adverse effects of alcohol exposure.