Richard A. Slayden - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This Proposal is in response to PA-01-113, "Therapeutics Research on AIDS-Associated Opportunistic Infections and Malignancies" and specifically addresses the study of Mycobacterium tuberculosis (MTB). The thrust of this proposal is the development of novel drug targets to counteract multiple drug resistant organisms. The long-term goal of this research program is to develop novel classes of chemotherapeutics that target the regulation, and coordination of chromosomal segregation and cellular division in MTB. Toward this objective we have identified in the MTB genome, gene products that are homologous to proteins associated with these processes in other prokaryotes. Moreover, our preliminary results provide strong evidence that some of these gene products (FtsZ and Ftsl homologues) actively participate in the cellular division of MTB. However, a more extensive analysis of these gene products and global assessment of the potential regulatory networks involved in the division of MTB cells are required. Similar to work with Caulobacter crescentus we hypothesize that DNA microarray analysis with synchronized cultures of MTB will allow us to develop a detailed pattern of gene expression profiles across the entire cell division cycle of this bacterium. Additionally, the use of known inhibitors of early (FtsZ activity) and late (Ftsl activity) events of cell division along with global gene expression studies will further elucidate the regulatory networks that are activated during different stages of cell division. A final analysis of putative regulatory genes already identified and new ones elucidated through gene expression profiling will enable us to develop a detailed picture of the regulatory networks and temporal gene expression responsible for MTB cellular division. Such studies will ensure that future resources are well directed at appropriate chemotherapeutic targets and developing suitable drug discovery strategies. Thus, the studies proposed in this application are designed to examine the replication dynamics of MTB, specifically focusing on cell cycle-regulated genes that are involved in cell division. [unreadable] [unreadable] [unreadable]

Introduction: The Genomic/Proteomics core facility will provide support to the entire Region VIII RCE. This core will function in three broad ways: (1) the core will provide specific services that are deemed universal in terms of the development and execution of post-genomic studies. To include the development and printing of genome DMA-based microarrays, array data acquisition and analysis, DMA sequencing and proteomic support such as tandem mass spectrometry and 2-D PAGE image analysis. (2) the Genomics/Proteomics Core will provide technical assistance to investigators as needed with the design of oligonucleotides for real-time-PCR and their application, proteomic analysis via 2-D-PAGE and mass spectrometry, and high throughput production of recombinant proteins and development of purification techniques and Bioinformatics. (3) Develop new postgenomic platforms for drug screening The Genomics/Proteomics Core will support the projects of the RCE in obtaining, analyzing and interpreting post-genomic information. A central theme of this RCE proposal is to develop new vaccines, diagnostics and novel chemotherapeutics against pathogens that are bioweapon agents. This core will develop post-genomic tools and resources, and accordingly provide support to each of the research projects so that these projects may exploit current and future genomic data. Project interactions: The Post Genomics Bioinformatics Core has direct connections to proposed research plans of several RCE Nodes: II.A. Bacterial Zoonoses Disease Control and Biodefense: The Post Genomics Bioinformatics Core will support this research node by providing DMA microarrays, and performing proteomic-related mass spectrometry. The core will also assist in the validation of post-genomic data via RT-PCR and the production of recombinant products. In addition Aim 3. of the core will be integral with the development of novel inhibitors by providing HT screening on compound libraries (such as M.A.2., II.A.4.) II.B. Arboviral Disease Control and Biodefense: The major contribution to this project by the Post Genomics Bioinformatics Core is envisioned to be primarily in providing support for use of the mouse DMA microarrays, immunoregulatory DMA microarray and RT-PCR technologies. The Viral Zoonoses Program will also benefit from the production of recombinant products and mass spectrometry support (section D.1.f.). II.C. UCHSC Molecular Pathogenesis of Burkholderia Select Agents: The University of Colorado Health Sciences Project encompassing Projects II.C.1.-II.C.6. will be supported by the Post Genomics Bioinformatics Core in a similar fashion to the other projects. The core will support post-genomic studies involving DMA microarrays, molecular identification of proteins by mass spectrometry and high throughput screening. II.D. Development of Bacteriophage Amplification Reagents and Immuno-detectors for Y. pestis and F. tularensis (CSM). The Post Genomics Bioinformatics Core will provide bioinformatics support for comparative genomics and genome mining. II.G. Antiviral therapies for potential Bioterrorism Viruses (Utah State University Research Project): The Utah State University Research Project (Project II.G.2.) will obtain assistance making a VEE construct (TC-83 vaccine strain) expressing Bcl-2 gene. Other support could include whole mouse array and aspects of recombinant protein production. The cores in this RCE proposal have an integral relationship to each other. The Post Genomics Bioinformatics Core will support the Animal Core (I.B.S.a) by providing tools and reagents to help characterize the animal models of infection on the genomic scale. This support will primarily come in the form of the whole genome mouse array, the immunoregulatory direct array and the real time PCR technologies for sequence detection. In addition, work done in the Product Development and Manufacturing Core (I.B.S.b.) will benefit from the Post Genomics Bioinformatics Core facility because of the recombinant products support and mass spectrometry support.

1H-benzimidazol-2-ylcarbamic Acid Methyl Ester; 2-(Methoxycarbonylamino)benzimidazole; 2-Benzimidazolecarcamic Acid Methyl Ester; Affinity; Animal Model; Animal Models and Related Studies; Animals; Anti-Bacterial Agents; Antibacterial Agents; B. pseudomallei; BCM; BMC; BMK; Bacteria; Bioavailability; Biologic Availability; Biological Availability; Bp50; Burkholderia pseudomallei; CD40; CDW40; Carbendazim; Carbendazole; Chemicals; Development; Enzymes; F. tularensis; Francisella tularensis; Generations; Goals; IC50; In Vitro; Infection; Inhibitory Concentration 50; Lead; MBC; MGC9013; Melioidosis; Methyl Benzimidazol-2-ylcarbamate; Methyl-1H-benzimidazol-2-yl Carbamate; Methyl-2-benzimidazole Carbamate; Methyl-2-benzimidazolecarbamate; Methyl-alpha-benzimidazole Carbamate; Nucleic Acid Biochemistry, Molecular Modeling; P. pseudomallei; P.pseudomallei; Pasteurella pestis; Pasteurella tularensis; Pathway interactions; Pb element; Physiologic Availability; Plague; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein/Amino Acid Biochemistry, Molecular Modeling; Pseudomonas pseudomallei; R01 Mechanism; R01 Program; RPG; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Resources; Series; TNFRSF5; TNFRSF5 gene; Testing; Therapeutic; Toxic effect; Toxicities; Tularemia; Tumor Necrosis Factor Receptor Superfamily Member 5 Gene; Y. pestis; Y.pestis; Yersinia pestis; Yersinia pestis disease; anti-bacterial; antibacterial; base; benzimidazolecarbamate methyl ester; bioavailability of drug; carbendazin; carbendazine; carbendazyme; design; designing; diphenyl ether; efflux pump; enoyl reductase; fatty acid biosynthesis; heavy metal Pb; heavy metal lead; improved; in vivo; inhibitor; inhibitor/antagonist; mecarzole; medamine; mekarzole; methoxybenzimidazole-2-carbamic acid; methyl 2-benzimidazil carbamate; methyl-N-(2-benzimidazolyl)carbamate; methylbenzimidazole-2-ylcarbamate; model organism; molecular modeling; mutant; novel; p50; pathogen; pathway; phenyl ether; pre-clinical; preclinical; preclinical study; programs

DESCRIPTION (provided by applicant): The long term goal of our program is to develop novel broad spectrum chemotherapeutics that can be used for Phase 1 clinical trials for treating the diseases caused by priority pathogens such as Francisella tularensis, Burkholderia pseudomallei and Yersinia pestis. We have developed a series of high affinity inhibitors of the fatty acid biosynthesis enoyl reductase (Fabl) enzyme that demonstrate in vitro potency against F. tularensis, M. tuberculosis and B. pseudomallei, and are active in an animal model of tularemia, confirming the validity of the target and of our approach. The goal of the present project is to dramatically improve the therapeutic dose by determining how factors such as formulation and modes of delivery, and structural modifications impact the in vivo antibacterial activity of the compounds. Studies will initially focus on the category A pathogen F. tularensis and the tularemia model of infection as this is the system in which we have had the most success. As the project evolves the information gained will be used to drive additional studies on B. pseudomallei and the melioidosis animal model of infection and Y. pestis. We will perform lead optimization studies on the Fabl inhibitors to: (i) improve pharmacology and reduce metabolic liability, (ii) facilitate deliverability/bioavailability, (iii) improve release kinetics, and (iv) assess alternative routes of delivery to optimize candidates for preclinical studies and performing required benchmarks for submission of an IND application. Our goal of developing inhibitors with enhanced in vivo efficacy will be achieved with the following Specific Aims: Aim 1: Effect of formulation and method of delivery on PK and in vivo antibacterial activity. Aim 2: Introduction of structural modifications to improve PK/PD. Aim 3: Extension to Other Priority Pathogens. RELEVANCE (See instructions): This proposal is to enhance the therapeutic effect of broad spectrum chemotherapeutics with efficacy against F. tularensis and B. pseudomallei and Y. pestis infections. In addition, such chemotherapeutics can be used to treat bacterial agents with significant health relevance, particularly Gram-positive pathogens, including MRSA and Gram-negative pathogens including B. cenocepacia, A. baumannii, and P. aeruginosa

The hypothesis of this program is that Fabl, the conserved enoyl reductase enzyme in the bacterial fatty acid biosynthesis pathway, is a target for the development of preclinical lead compounds with broad spectrum activity against priority pathogens, including F. tularensis, B. pseudomallei, and Y. pestis. Based on this approach, we have developed inhibitors with potent activity against the Fabl enzyme from F. tularensis and 8. pseudomallei. Significantly, we have demonstrated efficacy in an animal model of tularemia. Encouraged by this progress and due to the need to develop chemotherapeutics against other priority pathogens, we will extend our studies to include the development of potent in vivo antibacterial agents against 6. pseudomallei and Y. pestis. Our overall goal is to rapidly progress lead compounds into animal models of infection for efficacy testing with the following Specific Aims: Aim 1: Rational Optimization of Lead Compounds Against F. tularensis. We will design and synthesize subsequent generations of our lead compounds using SAR information derived from molecular modeling, activity against whole bacteria and efficacy in animals and bioavailability studies. Aim 2: In Vitro and In Vivo Antibacterial Activity against B. pseudomallei. The in vitro activity of the current diphenyl ether compounds against 8. pseudomallei will be assessed by determining (i) the IC50 for inhibition of the 8. pseudomallei Fabl enzyme (FablBpm), (ii) antibacterial activity (MIC and MBC) (iii) toxicity, PK/PD and deliverability. Selected compounds will be progressed to efficacy testing in the 8. pseudomallei animal model of infection. Aim 3: Extension to Y. pestis. We will extend our antibacterial discovery efforts to include the pathogen Y. pestis. Testing will be conducted using the established approach and compounds with appropriate activity will be evaluated in animal models of infection. This research project fits within the RMRCE Integrated Research Focus on Bacterial Therapeutics, and will interact directly with RP 2.1, RP 2.2, RP 2.5 and RP 2.6, and utilize the resources of Core C and Core E.

The goal of the Genomics Proteomics Core (Core E) of the Rocky Mountain Regional Center of Excellence in Biodefense (RMRCE) is to provide post-genomic resources and materials, to provide access to state-ofthe- art post-genomic instrumentation, and to provide expert assistance in performing post-genomic experiments, and bioinformatics, resulting in enhanced research opportunities for investigators at reduced costs. The GP-Core accomplishes this goal by producing whole genome mouse and pathogen DMA arrays and other molecular detection tools and platforms, by providing and maintaining post-genomic instrumentation in a centralized facility adjacent to the BL-3 laboratories, and by performing experimentation for investigators who would otherwise not be able to perform experiments because of a lack of manpower or expertise, and providing access to bioinformatics resources, staff, and support. The GP-Core also develops technology and custom platforms for investigators for focused or more advanced post-genomic studies. Accordingly, the GP-Core (1) produces and develops post-genomic resources for investigators, (2) provides access to post-genomic equipment (3) provides genomics, proteomics, and bioinformatics assistance and technical expertise, and (4) provides bioinformatics and Computer-Aided Drug Design (CADD) services to advance diagnostic and therapeutic drug discovery, design, and development for RMRCE investigators. Together, the activities of the GP-Core support research objectives on infectious diseases important to public health by providing resources and equipment that are required for post-genomic studies, technical assistance for experimental design, data analysis and other bioinformatics-related analyses, and postgenomic services on a fee-for-services basis. Core E will support all three of the RMRCE Integrated Research Foci on Immunomodulation, Adjuvants and Vaccines (IRF 1), Bacterial Therapeutics (IRF 2), and Viral Therapeutics (IRF 3). Its resources will be utilized by RPs 1.4, 1.6, 1.7, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7 and 3.4

This investigation concerns the development of mathematical algorithms to provide rapid diagnostic tools for the early detection of infection by the Ebola virus. Unlike current methods, the proposed approach will not require that the subject be symptomatic for detection of the virus. The proposed methodology exploits the observation that the immune system behaves like a canary in a coal mine, providing an early warning system that, if quantitatively understood, could be used to identify infection, accelerate treatment and improve outcomes. The detection technique consists of building an array of mathematical models for, e.g., gene expression data, that characterize the nominal state of the healthy immune system. These models are then applied to detect novel, or anomalous behavior of the immune system in infected subjects. The initial model building phase will employ non-human primate and mouse data to establish viability of the approach.

Transcriptional analysis has been widely applied to identify markers for disease classification, diagnosis, and prognosis. Many methods have been developed to identify the signaling pathways that respond to the changes between varying biological states, i.e., healthy and disease states, from a static viewpoint. However, the transition between biological states is a complex dynamic process that is information rich. In preliminary work on influenza, a nonlinear model of gene expression was built for over 400 pathways using data from healthy individuals that were experimentally infected with influenza virus. Of these pathways, the cytosolic DNA/RNA sensing pathway (a system for detecting pathogen-associated nucleic acids) was the first to exhibit changes in gene expression in the majority of subjects who became symptomatic, reflecting the immune system's initial response to an invading pathogen. Moreover, as the immune system response progressed, there was a cascade of anomalous pathway signaling, reflected by changes in gene expression, which could provide an early warning signature for detection of a pathogen well before externally observable symptoms of the disease appear. In this project, the pathway cascade of the mammalian cell response to Ebola virus will be investigated with the goal of characterizing the features of its disease-specific evolution, which will be used to identify molecular signatures for diagnosis prior to observable symptoms. The sensitivity and robustness of the modeling procedure will also be explored.

This contract provides in vitro testing of potential anti-bacterial and anti-fungal agents, maintenance of bacterial and fungal stocks, and the development of related assays.