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| DESCRIPTION: In order to make predictions about potential chemical inhibitors, the structure of the target molecule in question is critical. Current technologies used to determine protein structure, including X-ray crystallography or NMR, are slow and limited only to proteins that can be stably expressed and highly purified in significant amounts. The generation and subsequent analyses of these structural models are limiting factors in structure-based drug design, and new technology aimed at circumventing these limitations is required to enhance current drug discovery efforts.
The focus of this effort is to gain molecular information that will advance current candidate drug screening efforts by improving the selection criteria of compounds analyzed. In order to avoid the limitations associated with current structure-based drug design methodologies, specifically determining protein structure by physical analysis, we seek the development of new technology to conduct such analyses using a bioinformatics approach. Requirements include the generation and analysis of surface structure models from primary DNA or amino acid sequence with resolutions at or near that of crystallographic resolution, to include any accessory molecules required for protein function. Further requirements include the ability to analyze these models and provide structures of potential inhibitory molecules that can be synthesized and transitioned into current drug discovery programs. Key aspects of this analysis should consider: 1) the identification of compounds that are specific for the target protein and are not predicted to inhibit any homologous human proteins, and 2) exploit regions outside of the active site of a target protein that may be suitable of chemical inhibition. The ability to synthesize these novel compounds in quantities sufficient for biological characterization (milligram) will be positively considered but is not absolutely required. Rational drug design will focus our current screening efforts to allow for the better utilization of limited funding . This will increase our capacity to characterize critical inhibitors and identify potential lead compounds, thus expediting the drug discovery process. This technology will provide critical data for and transition into existing drug discovery programs and may be further exploited to encompass additional, potentially new drug targets in P. falciparum as well as other organisms central to infectious disease research of both DoD and civilian importance. PHASE I: Propose a system to use computational methodologies to: 1) generate molecular models of various target proteins, and 2) use these models to design/identify novel chemical inhibitors. Validate the system using the sequence of P. falciparum MRK or PK5 for which inhibitory data for specific compounds have been previously been determined. PHASE II: Utilize the system validated in Phase I to generate molecular models of additional target proteins for which there is limited inhibitor data available. Provide molecular models and structures of compounds, or families of compounds, that could serve as novel chemical inhibitors to be used in conjunction with high-throughput drug screening efforts. PHASE III DUAL USE APPLICATIONS: The exploitation of bioinformatically-derived information has the potential to yield significant advances of military and civilian importance both in drug discovery as well as other areas of biomedical research. In addition to serving as the foundation for structure-based drug design, the ability to structurally analyze proteins in silico will enhance any project where molecular analysis is vital. Potential areas of research that could benefit include examining the effects of mutations on the emergence of drug resistance, enhancing our understanding of the molecular interactions of chemical and biological agents with target proteins, and the more effective design of antigens/antibodies for vaccine research. ACCESS TO GOVERNMENT SUPPLIES: The design of chemical inhibitors indicated in Phase II may be faciliated by support from the Walter Reed Army Institute of Research. The candidate contractor should coordinate with the COR for any required support prior to the submission of the proposal. REFERENCES: 1) Clore G M, Gronenborn, A M. Determining the structures of large proteins and protein complexes by NMR. 1998. Trends in Biotechnology. 16(1) 22-32. 2) Gane P J, Dean P M. Recent Advances in Structure-Based Rational Drug Design. 2000. Current Opinion in Stuructural Biology. 10(4) 401-1. 3) Smyth M S, Martin J H. X-ray Crystallography. 2000. Molecular Pathology. 53(1) 8-14. 4) Xiao Z, Waters N C, Woodard C L, Li Z, Li P. Design and synthesis of pfmrk inhibitors as potential antimalarial agents. 2001. Bioorg Med Chem Lett 11:21 2875-8.
A03-166 TITLE: Development of Bioassays for Prion Infectivity Using Human, Deer, or Elk Cells TECHNOLOGY AREAS: Biomedical ACQUISITION PROGRAM: DSA, MRMC OBJECTIVE: Develop a human, deer, or elk cell culture model for prion propagation that can be used as a bioassay for detecting prions. DESCRIPTION: There is significant need for better methods to detect prion-related diseases that cause transmissible spongiform encephalopathies (TSE). Current infectivity bioassays involve the use of mice or hamsters. A cell culture model for prion propagation could lead to the development of a reproducible model for studying the mechanism of prion infectivity and disease. Additionally, a cell culture model for prion propagation could be used as an assay for the screening of potential prion therapeutics and could replace the animal test with a more rapid and sensitive cell-based diagnostic assay. Research will be directed toward the development of novel human, deer, or elk cell-based models for prion propagation. The cell model can consist of primary and/or engineered cell lines or co-cultures that have been optimized to support the propagation of prions. The inclusion of secondary cell types and/or other biological factors that support the purpose of the model is encouraged. Methods that are both sensitive and specific for detecting prion infection in the cultured cells must also be identified. Collaborations to develop the cell model for prion propagation and the infectivity detection assay are encouraged. The test method developed must be sensitive, specific, reproducible, and timely. Innovative concepts are highly encouraged. PHASE I. Select one species, either human, deer, or elk, for the development of cell lines. Create several novel cell lines from the selected species that support prion propagation at detectable levels. A sensitive and specific method to detect prion propagation in the cultured cells is essential to demonstrate the proof-of-principle. A detailed and specific plan for access to cells and/or tissues for the selected species must be provided in the application. A detailed and specific plan for the tissue and types of cells that will be used, and how the cell lines will be developed must also be included. Alternative approaches should be discussed, and the selected approach justified. Potential technical problems should be identified and addressed. Collaboration/subaward with a lab that has access to and capabilities in prion research is encouraged for the development of the detection assay. The use of human cells and tissues and/or animal use will require approval by the appropriate USAMRMC regulatory board. PHASE II. The objective of Phase II is to develop and validate a bioassay for detecting prions in tissues and body fluids. This bioassay will include a single-cell or co-culture cell model and a detection assay. Using the species-specific cell culture(s) developed in Phase I, continue to optimize the cell model(s) and the prion detection assay. Evaluate the single-cell or co-culture model(s) for infectivity, prion propagation, and assay sensitivity. Concurrently, if more than one prion detection assay is being used/developed, then compare the assays using the different culture model(s). Select the most promising cell model and detection assay, and optimize the infectivity, sensitivity, and specificity of this bioassay. Validation of this bioassay will consist of benchmarking the results to a currently accepted animal model, and demonstrating the bioassay is sensitive, specific, and reproducible. PHASE III: The commercialization potential of the resulting cell model and bioassay is high and is an important consideration in both the military and civilian environments. The assay system for detecting prions should be applicable to assessing the food and blood supply. Analysis of animals at the time of slaughter, screening of live animals, and early detection of prions in humans and animals, and blood products are several applications of this technology. Moreover, this assay technology will be important to the research study of prions. Proof of principle in Phase II should be sufficient to facilitate marketing of this assay to pharmaceutical or biotech companies possessing the capability of completing assay kit development and any required FDA approvals. REFERENCES: 1. Ingrosso, L., Vetrugno, V., Cardone, F., and Pocchiari, M. Molecular diagnostics of transmissible spongiform encephalopathies. Trends in Molecular Medicine: 2002, 8:273-280. 2. Collinge, J. Prion diseases of humans and animals: Their causes and molecular basis. 2001, 24:519-50. 3. Prusiner, S. Prions. Proc. Natl. Acad. Sci USA. 1998, 95:13363-13383. KEYWORDS: Prion diagnostics, prion propagation, infectivity bioassay, diagnostics, cell culture model, neural cell model A03-167 TITLE: Innovative Manufacturing Techniques for Polysaccharide-Protein Conjugate Vaccines TECHNOLOGY AREAS: Biomedical ACQUISITION PROGRAM: DSA, MRMC OBJECTIVE: Develop an economical manufacturing process, compatible with current Good Manufacturing Procedures (cGMP), for production of conjugated Shigellla polysaccharide-protein vaccines. Chemically conjugated vaccines consisting of a bacterial surface polysaccharide and a protein carrier have been shown to protect Israeli soldiers from diarrhea caused by Shigella sonnei after a single intramuscular injection (Lancet 1997;72:155-9). S. sonnei is the most prevalent cause of bacillary dysentery effecting U.S.. S. soldiers deployed in the Middle East (New Engl. J. Med. 191;325:1423-8). Phase I trials of polysaccharide-protein conjugate vaccines prepared from S. flexneri and S. dysenteriae 1 have suggested that other etiologically important Shigella species could also be prevented using this technology (Infect. Immun. 1993;61:3678-3687). DESCRIPTION: The main impediment to Shigella conjugate vaccine development is absence of an efficient, commercially viable manufacturing process yielding a product that could be licensed by the FDA. The polysaccharide component of conjugate vaccines is derived from Shigella lipopolysaccharide (LPS), which is tightly bound to the bacterial cell wall. LPS is extracted from bacteria using hot phenol – a laborious and expensive process with small product yield and high toxic waste output. The LPS must also be detoxified by removing the lipid component before conjugation with a protein carrier as the final step in vaccine manufacture. The DoD requires innovative approaches to the specific problems of bacterial LPS extraction, purification, detoxification, and chemical conjugation under cGMP. The manufacturing process should be economical and environmentally friendly (i.e. not employing caustic chemicals such as phenol). Injected Shigella polysaccharide-protein conjugate vaccine(s) would be widely used by deployed United States troops and by allied troops. In addition, civilian travelers to the developing world and children living in endemic areas constitute a large civilian market for these vaccines. PHASE I: Process development demonstrating economy, scalability and compatibility with cGMP conditions should also be accomplished in Phase I. The output ofa successful Phase I SBIR would be a research prototype S. sonnei or S. flexneri 2a conjugate vaccine with the chemical characteristics of a safe an immunogenic product. The manufacturing process should be cGMP compatible, but cGMP manufacture would not be expected in phase I. Animal testing demonstrating the safety and immunogenicity of the non-cGMP prototype conjugate vaccine would also occur in Phase II. PHASE II: This phase would begin with toxicity and immunogenicity studies on the research prototype conjugate vaccine product in animals. Demonstration of safety and immunogenicity of the prototype conjugate vaccine would support cGMP manufacture of a pilot lot of vaccine. The Phase II output would be an S. sonnei or S. flexneri 2a conjugate vaccine manufactured under cGMP for human use. Final chemical characterization, animal toxicity testing, and immunogenicity testing of the cGMP product should also be accomplished under the Phase II contract, and the product would be a successful Investigational New Drug (IND) Application to the FDA. PHASE III: The fully characterized cGMP conjugate vaccine product would be evaluated as an intramuscular vaccine in clinical trials under IND. Both safety and efficacy of the prototype vaccine in a Shigella challenge model should be assessed in these trials. Since the product would have commercial potential as a vaccine for deployed military personnel and for travelers in the developing world, commercial interests could fund early clinical trials. However, Shigella vaccines are needed for children of the developing world, and the public health sector (e.g., the Bill and Melinda Gates Foundation and/or the World Health Organization) are also interested in funding clinical development of economical conjugate vaccine(s). Ultimately a licensed vaccine against diarrheal disease for use in both the developed and in the developing world will require a combination of private and public funding. KEYWORDS: Shigella, conjugate vaccines, manufacturing process development A03-168 TITLE: Anti-Microbial Nanoparticles Composed of a Magnetic Core and Covered with Photocatalytic TiO2 TECHNOLOGY AREAS: Biomedical ACQUISITION PROGRAM: DSA, MRMC OBJECTIVE: The overall goal of this project is to develop a new generation of photocatalytic particles that can be applied topologically and irradiated with UV light for the purpose of disinfecting wounds. These nanoparticle photocatalysts have a magnetic core, which permits their complete removal after treatment. DESCRIPTION: TiO2 semiconducting photocatalysts, when irradiated with UV light, produce electron-hole pairs that can initiate reductive and oxidative reactions on the surface of these materials. An excellent review of photocatalysis in both the gas and liquid phase has been written by Mills and Lehunte. The holes, by virtue of producing OH radicals or through direct charge injection, are highly oxidizing and can be used as an anti-microbial agent. Nanoparticles of TiO2 can be routinely produced with dimensions of 3-8 nm suspended in aqueous-based solvents. Such materials can be directly sprayed on to wounds to decrease infection. Unfortunately, TiO2 particles are electrical insulators, and it is likely that it would be difficult to extract these nanoparticles from the wound after treatment! Therefore, this project is focused on producing a new class of removable nanoparticle photocatalysts that can be completely extracted from the treatment area by using a magnetic field. The successful completion of this project would produce a new generation of field-applied, sprayable, antimicrobial nanoparticle photocatalysts that would help to keep wounds clean of bacteria and could easily be removed after treatment. While nanoparticles of magnetite have been prepared there are no references to the coating of these magnetic core particles with photocatalysts. The first emphasis of this project is to produce these novel particles that have the potential of being applied topologically to reduce bacterial infections in wounds. Our desire is to create a sprayable form of this photocatalyst that can be easily applied and as easily removed with a magnet. Therefore, our first objective is to develop procedures for effectively coating magnetite nanoparticles with nanoparticulate TiO2. This will effectively produce an active photocatalytic therapeutic agent that can be easily sprayed on to a surface. We believe that this task can be accomplished within the first three to four months of this project. These coatings can be tested in a variety of ways: Firstly, coatings can be tested using electrophoretic mobility methods. Magnetite particles display a given charge vs pH signature. TiO2 particles have a different signature. If completely coated with magnetite with TiO2, the composite particle will show a signature that is representative of the outer coat. This essentially insures that the nanoparticle magnetic core is completely covered with our photocatalytic, antimicrobial coating. Secondly, a magnet will be use to determine if these coated particles can be removed from a suspension. The effectiveness of the removal can be tested by analyzing the TiO2 remaining in the suspension after the magnetic field is applied. In yet another test, we can measure the photocatalytic activity of these materials by spraying films of these magnetic-core photocatalysts on glass slides and performing gas-phase tests to determine the ability of these coated slides to destroy organics (ref). PHASE I : 1. Making magnetite (Fe3O4) core particles: Produce a colloidal form of magnetite having particle diameters of ca. 10nm or less. One possible approach is that of Rosenwieg. 2. Characterizing magnetite particles: Electrophoretic mobility to characterize the zeta potential of the particles as a function of pH. This measurement will provide a fingerprint of the surface charge of these particles. SEM and X-ray methods can be used to characterize size, morphology and crystalline habits of these materials. 3. Coating magnetite particles with TiO2: The method of TIO2 coating of the magnetite particles must be able to form a complete cover. A method such as electrophoresis may be used to demonstrate magnetite saturation with TIO2. PHASE II: 4. Measuring photocatalytic activity of the coated particles: Simple gas –phase testing methods should be used to determine if in fact these materials are photocatalytically active. Coat glass slides should be coated with the suspensions produced in Task 3. These coated slides will then be placed in a gas-sampling system connected to an infrared spectrometer. Upon UV-irradiation of the coated slide, a volatile organic contaminant such as ethanol should degrade to form carbon dioxide if the coated particles have photocatalytic activity. 5. Testing removal using magnetic fields: In this study, particles will be removed from suspensions containing known particle concentrations using a bar magnet. Test the removal efficiency by using inductively coupled plasma atomic absorption spectroscopy to measure the amount of Ti remaining in the suspension. This approach will determine if all of the particles have been removed in a given time. PHASE III: Studying the potential as a surface and liquid sanitizer activated by sunlight for commercial use. The suspensions should be placed in pump-spray or aerosol vessels for delivery. It is envisioned to test various topical concentrations as well as selected light intensities by moving the UV source nearer to or farther from the area of application. These materials can then be sprayed onto real wounds. Direct (application self or buddy) to open surface wound that will have antimicrobial activity when exposed to sunlight or visible light and can be combined with wound dressings to keep exposed dressing surfaces from being a source of environmental contamination penetrating to the wound. Also, the magnetic properties can be used to concentrate the nano-particles and also remove them when needed. Commercially, the potential exists for specifically localizing injected magnetic particles in hospitalized patients with magnetic fields such as MRI or other magnetic scanners and then using a fiber of laser to excite therapeutic activity. REFERENCES: 1) Mills, A., Lehunte, S. L. 1997, J. Photochem. Photobiolog. A. – Chem, 108, 1-35. 2) Maness, P. C., Smolinski, S., Blake, D. M., Huang Z., Wolfrum, E., and Jacoby, W. A. 1999, Appl. Environ. Microbiol. 65, 4094-4098. 3) Ashikaga, T., Wada, M., Kobayshi H., Mori, M., Katsumura, Y., Fukui, H., Karto, S., Yamguchi, M. 2000. Mutat. Res. – Gen. Toxicol. and Environ. Mutag. 466, 1-7. 4) (Rosensweig, R. E. Scientific American 1992, 247 (4), 136.
A03-169 TITLE: Programmable Wrist-Worn Prediction Model and Environmental Stress Monitor TECHNOLOGY AREAS: Biomedical ACQUISITION PROGRAM: DSA, MRMC OBJECTIVE: Design and build a low cost, programmable wrist-worn environmental monitoring system with in built high quality, high performance, inexpensive Analog and Mixed Signal Application Specific Integrated Circuits (ASICs) for physiologic/thermoregulatory model output to assess individual warfighter physiologic responses to a variety of environmental stresses. The technical challenge is integration of key predictive mathematical algorithms to deployable environmental sensors and that displays predictive real-time outputs in a low power drain, rugged weather resistant package that does not exceed the size of a wristwatch and is non-intrusive to the warfighter. DESCRIPTION: A wristwatch size environmental heat/cold stress activity monitoring system with key ASICs to assess individual warfighter readiness across a wide range of military operational settings. Existing technology incorporates predictive heat stress algorithms and environmental sensors in a pocket-sized device for use at the small unit level, but outputs, which consider heat stress only, are based exclusively on detached, bulky environmental measurements, and depend on user estimates of average activity and other input parameters. The proposed wrist-worn device will provide the capability to estimate activity monitoring coupled with a physiologic servo-loop thermoregulatory model to quantify metabolic expenditures. In built ASICs will enable personalized predictive estimates of the physiologic responses to activity, clothing system and environmental stressors in real-time operational settings. Contractor will have wide discretion in selection of methods and materials as long as key functional requirements are met. A water resistant, rugged enclosure with optimized sensor placement will have a liquid crystal display and control buttons for selecting functions and data display modes. Environmental sensor suites can be deployed from the watch head to provide ambient temperature, humidity, wind speed, and solar irradiance for use as input to a physiological model output source. The device will have sufficient memory to store 72 hours of measured data and/or computed indices for subsequent display, download, or network transmission. Total weight will not exceed 2 ounces (56 grams) and battery life will be sufficient for at least 7 days of sustained operations by a person. Target environmental sensor accuracies should be within these tolerances: Air temperature + 0.5 C, humidity +5%, and solar irradiance + 100 Watts/m2. The activity sensor should accommodate both sleep scoring (zero crossing) and daytime activity (proportional) modes of operation. Additional micro-sensors to measure skin temperature and/or heart rate would significantly enhance operational potential of the device. The device will have primary functionality as a "smart" environmental sensor suite capable of integration and/or fusion in a network of lightweight, wearable body sensor systems. The device will have I/O ports to allow communication with a PC or network hub. The developed system will include supporting personal computer software to enable download of data and upload or direct programming of specialized performance or environmental stress index algorithms such as Cold Stress Index (CSI) and Physiological Strain Index (PSI) for near real-time determination of thermal performance in various Objective Force Warrior scenarios. Directory: osbp -> sbir -> solicitations solicitations -> Army sbir 09. 1 Proposal submission instructions dod small Business Innovation (sbir) Program solicitations -> Navy sbir fy09. 1 Proposal submission instructions solicitations -> Army 16. 3 Small Business Innovation Research (sbir) Proposal Submission Instructions solicitations -> Air force 12. 1 Small Business Innovation Research (sbir) Proposal Submission Instructions solicitations -> Army 14. 1 Small Business Innovation Research (sbir) Proposal Submission Instructions solicitations -> Navy small business innovation research program submitting Proposals on Navy Topics solicitations -> Navy small business innovation research program solicitations -> Armament research, development and engineering center solicitations -> Army 17. 1 Small Business Innovation Research (sbir) Proposal Submission Instructions solicitations -> Navy 11. 3 Small Business Innovation Research (sbir) Proposal Submission Instructions Download 1.85 Mb. Share with your friends:
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