Vincent J. Davisson - US grants

The overall goal of this project is to establish a detailed understanding of the enzymatic steps involved in the histidine biosynthetic pathway, leading ultimately to a knowledge of the organization of enzyme active sites in vivo. Because of the unique substrates and chemistry of the pathway, these steps are potential targets for selective inhibitors or alternate substrates with direct applications in the development of antimicrobial, antifungal or herbicidal agents. This knowledge maybe coupled with the wealth of genetic information about this system, to create new in vivo metabolic pathways, and for this reason, the research will take on a multifaceted approach. The theme of this proposal is to establish the major mechanistic steps involving the three central enzymes preceding the metabolic branch point linking specific aims. 1. Recombinant DNA methods will be used for the subcloning and over-expression of five target enzymes involved in the de novo biosynthesis of histidine in Escherichia coli. Protocols for the isolation and characterization of three of these enzymes will be developed. 2. The catalytic mechanism of the 5'- phosphoribosylformimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'ProFAR) isomerase will be investigated using several isotopically labeled and fluorinated substrate analogs. A general method will be developed for the synthesis of the key substrate 5'-Pro FAR from D-ribose that will allow for the synthesis of 2H, 3H, 13C labeled analogs from commercially available materials. The synthetic method will be extended to a preparation of four fluorinated ribose derivatives of 5'-ProFAR that will serve as substrate analogs and/or mechanism based inhibitors of the 5'- ProFAR isomerase enzyme. 3. A structure will be determined for the product of the N1-(5'-Phosphoribulosyl)-formimino-5-aminoimidazole-4- carboxamide ribonucleotide glutamine amidotransferase reaction, in order to establish a mechanism for its formation, and the nature of the enzyme activity for the protein encoded by the hisF gene. As a direct comparison to other glutamine amidotransferases, the catalytic properties of the glutamine binding site in this enzyme will be analyzed for a covalent adduct via a gamma-glutamyl ester and the amino acid residue involved in this bond will be identified by affinity labeling and peptide sequence analysis. Efforts toward a three dimensional structure for this enzyme will be initiated exploring a variety of conditions to arrive at crystals of the glutamine amidotransferase that are suitable for x-ray diffraction studies. 4. Plasmid DNA clones for the HIS6 and HIS7 genes of Saccharomyces cerevisiae will be isolated by genetic complementation tests in deficient strains of this organism. These target genes code for the analogous genes in the E. coli histidine biosynthetic pathway which are the subject to this proposal. The DNA sequence of these clones will be analyzed and the deduced amino acid sequence will be used for a comparison with the corresponding proteins in eubacteria. Because of the assumed bifunctional nature of the enzyme encoded by the HIS7 gene, a DNA vector will be developed that allows for expression of this protein in E. coli.

The Department of Medicinal Chemistry and Pharmacognosy at Purdue University requests funds for the acquisition of a Varian Gemini-300 NMR spectrometer system, with broad-band probes and electronics, variable-temperature capability, and an Ethernet link to a SUN Microsystems SPARC workstation (also requested). The Gemini-300 will replace older and less capable 200 MHz instruments and will allow multinuclear one-dimensional survey spectra (e.g., 1H, 13C, 19F, and 31p) and various two-dimensional experiments to be run routinely, in a "walk-on" mode. The SPARC workstation will also be networked to an existing Varian VXR-500, thus allowing data processing and hard copy production to be accomplished off-line, and maximizing the use of both spectrometers for the acquisition of spectral data. The Major Users' needs for multinuclear observation, variable temperature capability, and an off-line workstation are documented, and a justification for the purchase of a computer-switchable, four-nucleus VT probe is presented. The Major User group comprises 12 faculty members from the Department of Medicinal Chemistry and Pharmacognosy. Dr. Schwab's project concerns the structures and catalytic mechanisms of several bacterial enzymes, and the synthesis of isotopically labeled enzyme substrates and inactivators. Dr. Bergstrom will synthesize and characterize modified oligonucleotides and study their interactions with nucleic acids. Dr. Byrn will synthesize and characterize oligonucleotides tethered to acridines and anthracyclines as potential antiviral agents. Dr. Chang's research concerns the discovery of novel antitumor agents from higher plants, guided by ii variety of mechanism-based bioassays. Dr. Cushman describes the synthesis of a wide range of potential antiviral, antitumor, and antibacterial agents. Dr. Davisson will study enzyme mechanisms in the bacterial pathway for histidine biosynthesis. Dr. Green will synthesize radiopharmaceuticals labeled with metal radionuclides, for use in positron emission tomography (PET). Dr. Loudon is developing new chemical techniques for the identification of peptide hormones in crude biological preparations. Dr. McLaughlin's research centers about the isolation and characterization of novel bioactive principles from higher plants, using several simple and inexpensive, yet effective, bioassays. Dr. Nichols conducts structure-activity studies of dopamine and serotonin agonists and antagonists. Dr. Pidgeon is synthesizing and using NMR spectroscopy to study the structures and dynamics of phospholipids that are covalently linked to silica (immobilized artificial membranes). Dr. Post will use NMR relaxation measurements to probe the active site metal ion environment of phosphoglucomutase. The need for an updated "walk-on" NMR spectrometer is highlighted by the recent growth of the Department's faculty (including five new faculty members over the past two years) as well as the strongly chemical nature of the research being conducted in the Department.

Further studies on enzymes catalyzing allylic rearrangments are proposed. Beta-Hydroxydecanoyl thiol ester dehydrase (E. coli), which equilibrates thiol esters of (R)-3hydroxydecanoic acid, E-2-decenoic acid, and Z-3-decenoic acid, is the key enzyme in the biosynthesis of bacterial unsaturated fatty acids. Dehydrase is rapidly inactivated by the mechanism-based inactivator 3decynoyl-NAC (NAC =N-acetylcysteamine thiol ester), via dehydrase-catalyzed isomerization to 2,3decadienoyl-NAC. The amino acid sequence of dehydrase has recently been determined, and the residue that is modified by 3-decynoyl-NAC (and is therefore implicated as the active site base) was shown to be His-70. The long-term objective is to further knowledge of enzymw structure and function. Such knowledge is critical for the rational design of drup that act at the enzyme level. The specific aims of this proposal include (a) determination (by 15N NMR) of which of dehydrase's imidazole nitrogens is modified by 3-decynoyl-NAC; (b) synthesis and application of novel enzyme inactivators, with analysis of protein by peptide mapping and multinuclear NMR, in order to identify catalytically important residues other than His-70; (c) synthesis and evaluation of ketone analogs of dehydrase's thiol ester substrates as potential nonhydrolyzable substrates and inhibitors; (d) determinnation of the three-dimensional crystal structures of dehydrase that has been inactivated with novel 3-decynoic acid thiol esters, including the ACP thiol ester; (e) complete characterization of dehydrase by multinuclear NMR, allowing comparison of crystal- and solution-state samples and enabling the identification of dehydrase-ACP contact points; (f) generation of mutant dehydrases, with modified active site residues and modified acyl chain binding regions, to test the roles of amino acid residues that are suspected to be important in catalysis and to generate E. coli strains with altered unsaturated fatty acids; (g) reevaluation of the stereochemical course of the allylic rearrangement catalyzed by the Betahydroxydodecanoyl thiol ester dehydrase component of Brevibacterium ammoniagenes fatty acid synthetase (by 2H-decoupled 1H,13C chemical shift correlation spectroscopy), in order to test for mechanistic unifomity among enzymes catalyzing allylic rearrangements.

DESCRIPTION: (provided by applicant) Many drugs from nature present complex molecular architectures that result in varied degrees of specific interactions with cellular targets. The rational selection and implementation of new cancer therapies would be improved if a basis for assessing all the drug target interactions were available. This proposal will address the importance of multiple drug targets for the ansamycin class of antitumor agents. Our efforts will focus on the molecular target(s) for the experimental breast cancer drug l7-allylamino-17- demethoxygeldanamycin (17-AAG). The benzoquinoid ansamycins geldanamycin (GA) and herbimycin A and the nonapolyketide radicicol have been investigated for their antiproliferative activity showing both cytostatic and cytotoxic activities. Recent studies predict the mode of action for the cytotoxicity is associated with inhibition of the cellular chaperone Hsp90 or its homolog Grp94. This proposal challenges the assumptions that Hsp90 is a primary mode for induction of tumor cell death by GA. We have recently identified a high-affinity binding site for the drug geldanamycin on an enzyme in the de novo purine biosynthetic pathway called ADE2. A thorough analysis of this interaction in tumor cells and its consequences for drug action will provide the scientific basis for using this information in future applications of this drug class. A detailed biochemical characterization of the human ADE2 protein from tumor cells and an investigation of the molecular details dictating its inhibition by GA are proposed. These studies will assess if ADE2 is a suitable target for cancer chemotherapy and if purine depletion is a critical step in the induction of apoptosis. The studies constitute a biorational approach toward a new perspective on targets for anticancer drugs in de novo purine metabolism. A mechanistic understanding of the biochemical role and regulation of the human ADE2 protein in tumor cells will be a fundamental basis for this analysis.

With support from the Major Research Instrumentation (MRI) Program, Prof. R. Graham Cooks of Purdue University will develop a state-of-the-art Kingdon trap mass spectrometer for high performance chemical and biochemical analysis. This instrument represents a fundamentally new approach to high accuracy/precision mass measurements. In addition, its performance should be equivalent to a Fourier Transform-Ion Cyclotron Resonance (FT-ICR) instrument, which is considerably more expensive.

The project will impact the education of a number of graduate students and postdoctoral associates at Purdue, who will work directly to develop the instrument or will be involved in its testing. A set of short courses in biological instrumentation will also be offered.

DESCRIPTION (provided by applicant): The goals of this application are to develop the concepts of degenerate recognition in nucleic acid enzymology as a general strategy for understanding or manipulating biological systems. The hypothesis under consideration is that the electronic tuning of a set of isosteric and conformationally ambiguous nucleobases can result in useful probes of selectivity in protein-nucleic acid interactions. In the course of testing this hypothesis, we will explore opportunities to refine chemical probes of DNA polymerases and expand the tools for basic biochemical studies as well as enhance biotechnology. The opportunities for applying the principle of degenerate recognition will be also explored in the context of RNA dependent viruses that pose serious threats to human health. A team of investigators are assembled that will enable an effective combination of chemical, biochemical, and biophysical methods to address the molecular, structural and dynamic properties of the RNA and DNA polymerases. By focusing on the application of novel biochemical tools to investigate important biological systems, we aim to advance both our understanding of molecular biology, and the characterization of potential therapeutic targets. The goals of this proposal are: 1. To further develop azole carboxamide nucleobases as mechanistic probes of Pol I DNA polymerases. 2. To expand the technology base for azole carboxamide nucleotide triphosphate incorporation and strand extension using DNA polymerases. 3. To implement azole nucleobases as random mutagens to study the molecular evolution of 5- aminoimidazole ribonucleotide carboxylase (AIRC), an enzyme in de novo purine metabolism. 4. To develop chemoenzymatic methodologies for creating RNA templates containing azole carboxamide nucleobases and to analyze these templates in their recognition by RNA dependent polymerases.

DESCRIPTION (provided by applicant): Scientific Focus The long-term goals of this project are to establish a molecular basis for the interdomain communication in glutamine amidotransferases that relates their dynamic properties to the control of their catalytic efficiencies. This investigation aims to delineate the major mechanistic and structural aspects of two enzymes, imidazole glycerol phosphate synthase (IGPS), and guanosine monophosphate synthetase (GMPS), which catalyze the transfer of ammonia from glutamine to a purine nucleotide acceptor substrate. The general hypothesis under consideration for the glutamine amidotransferase sub-family is that the functional link between the two active sites involves the transduction of binding and chemical energy from the acceptor active site to the glutaminase site. Furthermore, the glutamine substrate specificity and catalytic efficiency is controlled by dynamic protein-protein interactions that modulate a molecular tunnel for ammonia transfer. The proposed studies will focus upon structural and mechanistic information to define the domain interactions that control the timing of the nucleotide substrate activation and glutarnine hydrolysis steps. Significance of objectives Because of the unique chemical features present in the metabolic pathway under investigation, and the detailed comparative structural information that will emerge, this project has the potential to impact the discovery and development of new therapeutic agents. In addition, these enzymes represent important examples of convergent evolution that hold many mechanistic details of how proteins transmit specific chemical information over large distances. Overall, the combination of biochemical and biophysical approaches proposed will: a) contribute to a comprehensive knowledge regarding nitrogen metabolism and b) provide critical information for the design of new selective agents of potential pharmacological importance that target essential enzymes in ammonia metabolism.

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to identify a series of novel small molecule modulators of Hsp90-centered biological networks. Hsp90 is an important oncological target and several compound inhibitors are undergoing clinical trials. A growing interest in exploiting the use of Hsp90 inhibitors for other disease states is emerging including the use of sub-cytotoxic concentrations to increase expression levels of Hsp90 to provide neuroprotection. These inhibitors and others have had an invaluable role in establishing our current knowledge of Hsp90 biology. However, the exact mode of action of Hsp90 inhibitors and their effect on the surrounding cellular networks is still unclear. This problem is not trivial due to the intricate and extensive roles Hsp90 plays in myriad biological processes. Identification of additional pharmacological modulators will have a substantial impact in our ability to modulate Hsp90 pathways. We will exploit the unparalleled knowledge base [unreadable] in yeast genetics and the tractability of the organism to high throughput screening (HTS) to develop an assay capable of identifying chemical modulators, including both antagonists and agonists of Hsp90 pathways. Our genetic-based and multifactorial approach determines growth parameters of a set of yeast strains in the presence of compounds. The growth profiles of these signature strains will aid in identifying compound classes and allow functional linkage with stages of the Hsp90 chaperone cycle and specific subsets of Hsp90 pathways. Solutions to traditional problems with using yeast as a drug-screening platform will be outlined and incorporated in our assay development. The assay we are developing will be in a simple and robust high density 384-well experimental format that will allow screening of a large chemical library matrix such as the Molecular Libraries Small Molecule Repository. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are to develop new metabolomic methodologies to identify pathways involved with metabolism and biosynthesis of endogenous cannabinoid signaling molecules such as anandamide and 2-arachidonylglycerol (2-AG). Ultimately, these lipidomic strategies can be employed in in vivo or in vitro systems to better understand the profile of endogenous cannabinoids produced under a variety of stimuli. The endogenous cannabinoids are a family of lipid signaling molecules that are typically amides or esters derived from long-chain fatty acids. These compounds have neuromodulatory activity and appear to play a role in multiple physiological processes. The two major endocannabinoids identified in the brain are anandamide (N-arachidonyl- ethanolamide) and 2-AG. The proposed studies are designed to refine the approaches used to study production and recycling of anandamide as well as other putative endogenous cannabinoids. The specific metabolic and biosynthetic pathways linked to certain calcium channels as well as neurotransmitter receptor activation will be explored. The Specific Aims of this project are: 1) To develop and apply specific metabolite fingerprinting and profiling methodology for identifying the lipidomic profile for endogenous cannabinoids, and 2) To determine stimulus-dependent differences in the release of endogenous cannabinoids. The studies will use molecular, biochemical, analytical (thin layer chromatography, mass spectrometry), and lipidomic/ metabolomic approaches. These innovative comparative lipidomic studies will examine in detail the recycling products derived from metabolized anandamide. A better understanding of the mechanisms of endogenous cannabinoid production and release will provide new insight into the system that is acted upon by marijuana. The Public Relevance: This project has direct relevance to human health because the endocannabinoid system is implicated in modulation of memory, mood, cognition, pain, fever, and the immune system. As such, pharmacological manipulation of this system has potentially therapeutic effects in such conditions as convulsions, glaucoma, movement disorders, anxiety, obesity, and multiple sclerosis. Understanding the molecular basis for controlling the production of the endogenous cannabinoids is critical for future drug discovery efforts targeting this system.

Developmental funds will be used to provide startup funds for the recruitment of outstanding scientists to Purdue and to support the development of a new drug discovery shared resource. Molecular Discovery and Evaluation (MDE). Support of faculty recruitment focuses on adding faculty whose research interests represent strategic additions to the Center. These funds are intended to assist in providing newly recruited faculty members with the resources to establish their research programs and attract independent research support. The Center is involved in joint recruitments with key Departments on campus to enhance cancerfocused research at Purdue University. Currently, four such recruitments are in progress. The Center strategic plan includes enhancing research and shared resource strengths of the Center through faculty recruitment. Thus, ample the recruitment of additional new faculty will be vigorously pursued over the next five years. Support for the development of the MDE share resource will provide needed expansion of drug development services. The MDE will expand and automate previous drug discovery services provided by the Purdue University Center for Cancer Research. The primary mission of the MDE developmental shared resource is to enhance the capacity of the Purdue University Center for Cancer Research to accelerate therapeutic development including targeted agents. Funds are sought to partially support the development of the shared resource.

DESCRIPTION (provided by applicant): Cervical cancer is second to breast cancer as the most common form of malignancy in both incidence and mortality for women worldwide. The population-wide utilization of screening cervical cytology (Pap tests) has been associated with a dramatic decrease in morbidity and mortality from cervical cancer in the United States and in other industrialized nations. Despite this success, the cytologic diagnosis of cervical lesions is plagued by a persistent problem of low specificity for clinically significant high-grade lesions in patients with low-grade cytologic abnormalities. As a result, over four million women each year receive a cytologic diagnosis that requires further evaluation to rule out the possibility of high-grade dysplasia or cancer. In most cases, further evaluation does not identify underlying high-grade lesions in patients with low-grade cytologic abnormalities. Although HPV testing can play an important role for the triage of some patients, it is not useful for several cytologic diagnoses. Complicating the situation is that simple detection of high risk HPVs does not predict an underlying high grade lesion, since infections do not indicate clinically significant cervical lesions. The long-term goal of this project is to apply emerging technology to develop a high-throughput cell-based analysis with suitable specificity to identify high grade premalignant and malignant lesions of the cervical mucosa. The methods to be used in this project will employ, test and validate the approach of cytometry-based molecular diagnostics to detect false negative cervical specimens. Under the guise of the previous grant phase, an application of protein expression of p16INK4a and Mcm5 (cervical cancer biomarkers) with high- throughput flow cytometry and cell sorting has been used to identify and capture the rare cancerous cells in cervical specimens. Furthermore, a multiplexed HPV genotyping assay has been implemented to analyze the rare cells isolated in this approach. Importantly, the work flow has been implemented using common sample preparation with current pathology testing protocols. The technology and methodology being applied in this application will be implemented using an integrated workflow, with substantial automation, to assess feasibility of further translation to accommodate clinical need and to improve the standard of care worldwide. The ultimate goal is to establish a primary assay with potential to supplant slide based cervical cytology with greater sensitivity, less subjectivity, and less labor intensiveness. PUBLIC HEALTH RELEVANCE: This proposed project will apply new technology, for detection of abnormal cervical cells, to clinical samples from previous Pap tests. We will use automated analysis of protein content of cells to isolate abnormal cells, after which automated DNA analysis will determine whether cancer-causing human papillomavirus types are present. These results will be compared to the Pap smear and biopsy results of the same samples in order to determine how well our test can detect early stages of cervical cancer.

The broader impact/commercial potential of this I-Corps project involves a low cost, highly versatile platform technology for development of bioassay systems. The initial objective is to be able to evaluate biomarkers of health and disease using point-of-care and/or in-the-field-settings. A set of minimal requirements to make high impact applications include turn-key operations, ultra-sensitivity, and superior accuracy for internal standardization to allow for simultaneous analysis of multiple biomolecules. Using specialized nanomaterials as sensors for trace amounts is an arena ripe for development. The integration of molecular sensing technologies with biomolecular detection and separation on a simple, adsorbent sensor creates opportunity to pursue unmet medical needs for early detection and diagnosis.

This I-Corps project is based upon self-assembly of chemical components to create new organometallic composite materials with molecular signatures suitable for single molecule detection by Raman spectroscopy. A growing family of chemical sensors capable of quantitative analysis using surface-enhanced Raman opens avenues for both fundamental and applied inquiry. In complex mixtures, the special 'fingerprints' of specific chemical dyes become characteristics that are uniquely detectable by Raman. The use of stable isotope variants of these dyes provides a series of chemically-identical materials whose distinctions can be revealed by Raman. What is available now are a set of 'chemical barcodes' that stimulate intense interest in several fields ranging from theoretical studies of single molecule detection to the field of molecular diagnostics in medicine. The creative methods for making the new materials and the resulting physiochemical properties open distinctive new avenues for creation of nanoscale optical sensors. New substrates for embedding the resulting nanomaterials for practical assay development is integral to the next phase of technology development. The creation of materials with robust single-molecule signals, new substrates, and assay work-flows will also inspire new design and fabrication of instrumentation that can move applications directly to the field for consumers.

A research team from Purdue University and Texas Biomedical Research Institute is in pursuit of important new approaches to validate the host V-ATPase. The project goal to define antiviral drug leads for diseases caused by Ebola and Marburg filoviruses. Many viral pathogens bind to specific factors of host cells to penetrate the plasma member inside vesicles called endosomes. However, entry of the virus to the host cells requires escape from these vesicles. A majority of these viruses utilize the low pH of late endosomes to trigger the final steps in viral entry. Since the acidification of endosomes depends on vacuolar H(+)-ATPases (V-ATPases), inhibiting this pump interferes with entry of a wide range of viruses. Thus, the host V-ATPase represents a truly broad-spectrum target for antiviral drugs. While feasible, there are challenges for developing antiviral drugs that targets host factors like V-ATPase because of the potential risk of toxicity to non-infected cells and tissues. The approach used in this project directly addresses this challenge which will apply discovery and pharmaceutical technologies to achieve selectivity of drug action. The project team brings significant expertise to the challenges for discovery and development of host-targeted antiviral therapeutics. Efforts will integrate chemistry and biochemistry of natural-product V-ATPase inhibitors, viral-host entry pathways, Ebola and Marburg animal disease models in BSL4 facility, in vitro and in vivo ADME-pharmacokinetics, and toxicology. Currently, there is a lack of suitable in vivo chemical probes available to test safety and efficacy of pharmaceutical interventions targeting V-ATPase. The objectives for this five-year project are to: a) qualify a class of selective chemical inhibitors for V-ATPase suitable for in vivo antiviral efficacy testing, and b) identify a new drug lead candidate targeting V-ATPase function during the viral entry process for Ebola and Marburg filoviruses. These complementary objectives will impact the prospects and precise strategy for developing antiviral therapies to treat unmet needs.