Kenneth Norman Raymond - US grants

A number of polycatechol chelating agents have been prepared using the 2,3-dihydroxybenzoyl (DHB) group or derivatives of it in which the benzoic acid ring has been substituted at the five position by -SO3- or at the four position by -CO2-. In each case the DHB groups are appended to a polyamine backbone. To date, linear triamines (such as spermidine), 1,3,5,-triaminomethylbenzene, and the tripod amine triaminoethylamine have all been used as molecular skeletons. During the next year we intend to continue to explore the synthesis of 4-carboxyl-substituted derivatives of DHB in tricatechol chelating agents. Depending upon the results of animal tests, attempts will be made to optimize the geometries of these complexes through variation of the backbone structure. We also plan to explore new molecular architectures for ferric chelating agents, and examine the thermodynamic properties of those prepared to date.

Microbial iron transport and mobilization is achieved using very powerful low-molecular-weight complexing agents which solubilize ferric ion through the formation of pseudo-octahedral complexes of high-spin Fe3+. These compounds, called siderophores, are manufactured by microorganisms specifically in response to their need for iron, an essential element for growth. The availability of iron to pathogens has been directly linked to the onset of disease in a number of illnesses associated with microbial infection. Transport of the iron occurs via several different mechanisms, as found either between different microorganisms or as parallel systems within one microorganism. We continue to use the replacement of kinetically labile Fe(III) by kinetically inert ions such as Cr(III) or Rh(III) in order to produce metal siderophore complexes of known and stable geometry to be used as biological probes of the transport process. Similarly, use of Ga(III) as a substitute for Fe(III) produces complexes of approximately the same thermodynamic stability and kinetic lability, but of dramatically different redox behavior. Thus Ga(III) substituted compounds can be used as specific probes for parts of the transport and uptake process which depend upon redox behavior. The synthesis of chelating agents which at least in part mimic particular siderophores can be designed so as to test for which parts of the siderophore are critical in the recognition process associated with microbial receptor proteins. Finally, the fundamental coordination chemistry and solution thermodynamic and ligand exchange behavior of the siderophores and their analogs will continue to be characterized as fundamental aspects of the biochemistry of this important class of compounds. In particular, the ligand exchange kinetics in non-aqueous media approximating the hydrophobic environment of the cell membrane will be examined for any significant difference from the aqueous ligand exchange behavior.

This award will support a seminar, "Marine Bioinorganic Chemistry", organized jointly by Prof. Kenneth L. Raymond of the University of California, Berkeley, and Prof. Clifford Hawkins of the University of Queensland, Australia. Participants will meet on Heron Island, Australia in June 1989 to conduct joint field experiments and plan collaborative projects in this new field. Bioinorganic chemistry is the study of biological processes involving metal ions as key components. As yet, in this rapidly growing field, the bioinorganic chemistry of the sea has not been studied, although the discipline should be of great importance due to the concentration and transport of metal ions across concentration gradients, from seawater to organisms. The Heron Island Marine Research Station, the largest and best-equipped on an Australian island, is an ideal location for a seminar intended to stimulate research in this important field. This research could lead to new discoveries in the biology of terrestrial and marine inver- tebrates, and possibly eventually to the development of new therapeutic compounds for humans.

In this project supported by the Inorganic, Bioinorganic and Organometallic Chemistry Program, Kenneth N. Raymond of the University of California at Berkeley will synthesize various ligands designed to specifically bind with oxo-metal cations, and in particular, uranyl and vanadyl. This work has relevance to the nuclear power industry, mining technology and environ- mental monitoring and control. The strategy to be employed is based on the premise that oxo- metal cations do not fit the "billiard ball" model for other cations, and to effectively encapsulate the uranyl and vanadyl cations, the ligand design will incorporate nitrogen-hydrogen groups which are constrained in position to hydrogen bond to the oxo-group(s) of the cation(s). Characterization of the resultant complexes will include structure determination by x-ray diffraction, stability constant determination, electro- chemical studies in solution and various spectroscopic analyses.

DESCRIPTION (provided by applicant): Nearly half a million cases of bacterial sepses are reported annually in the USA and approximately one third of the cases are fatal. Increasingly, metal regulation and transport are being found of great significance in a wide range of biological processes and disease states. It is difficult to overestimate the significance of iron as a limiting nutrient in microbial growth, as documented in this proposal. This project was the first to study the coordination chemistry aspects of siderophore-mediated iron transport. As it has evolved, increasingly the focus has shifted to include studies of the transport process itself, including receptor structure and function. The first example of a direct role of the human immune system in binding and inactivating siderophores was found in the structure of the enterobactin adduct of NGAL (now called siderocalin), a human protein secreted by neutrophils. We intend to characterize the role of siderophore-binding and inactivation by proteins of the human immune system (in collaboration with Dr. Roland Strong). The "siderophore shuttle" mechanism of microbial iron transport is a completely different kind of transport system first observed by this research group in Aeromonas hydrophila. A search will be made to characterize how widely spread this transport system is and further studies of the remarkable features of this mechanism will be made to probe the mechanism of the iron exchange, which is a key feature of this uptake system (in part in collaboration with Prof. Alain Stintzi). Little is known about the iron transport processes of Gram-positive (single cell membrane) bacteria. It is proposed that the transport systems of some Gram-positive bacteria be studied to see if they follow the same patterns of those of better-characterized Gram-negative organisms. One Gram-positive siderophore, corynebactin, is a close relative of the much better known catecholate siderophore enterobactin. The remarkable structure of corynebactin raises a number of questions about its function and what possible advantages it can confer on the producing organisms. These will be probed through a wide range of activities, from chemical synthesis of the compound through receptor binding studies and characterization of the receptor protein (in part in collaboration with Profs. Alain Stintzi and Phillip Klebba). The coordination structures of non-crystalline samples of siderophores and analogs will be proved using EXAFS (in collaboration with Dr. David Shuh) and magnetic behavior will be probed by Mossbauer studies (in collaboration with Prof. Matzanke).

This proposal seeks continuation of support for a project to develop new approaches to the chelation therapy of human iron overload that is a consequence of beta-thalassemia (Cooley's anemia). The project will continue to focus on three goals: 1) The development of new ligands for Fee+. 2) The thermodynamic evaluation of new ligands. 3) The biological evaluation of new ligands. For goal #1, the failure of 3-hydroxy-l, 2- dimethyl-4(1H) -pyridinone (L1) in clinical trials has again emphasized the need for a new chelating agent for human iron overload. The research in our laboratories and the early promise of L1 continue to point toward hydroxypyridonate (HOPO) ligands as promising. However the limited thermodynamic stability of a simple bidentate ligand such as L1 makes such ligands poor candidates relative to analogous hexadentate ligands. We now have synthetic procedures to introduce either 3,4-HOPO or 3,2-HOPO ligand groups into hexadentate ligands with a geometry optimal for octahedral coordination to Fe3+. This has not been true of other hexadentate ligands reported to date. Several new - synthetic routes to 3,2- and 3,4-HOPO ligands have been found and are being explored. We have also found that the incorporation of one catechol group into a multidentate ligand such as desferrioxamine B (the trihydroxamate ligand in current clinical use) increases the rate of iron removal from the human iron transport protein transferrin by two orders of magnitude. It is proposed that this feature be exploited by combining catechol groups into mixed function ligands. The thermodynamic stability of new ligands with Fe3+ and competing physiological metal ions will be examined. An initial biological screen will be the rate of iron removal from transferrin. The kinetics and mechanisms of iron exchange with mammalian iron storage and transport proteins will be studied. Preliminary toxicity studies will be carried out in collaboration with Dr. P. Durbin. Screening for iron removal will be carried out for the most promising compounds in collaboration with Dr. R. Bergeron.

The University of California Berkeley will receive ARI Facilities support to create modern facilities for researchers working in a unique, integrated Program in Biological Chemistry and Engineering within the College of Chemistry. The renovated facilities will promote a strong interaction among bioinorganic chemists, biochemical engineers, and bioorganic chemists working in the areas of biotechnology and environmental research. The renovations activity funded by this award will be directed at the improvement of 1,305 square meters in 30-year old Latimer Hall and will consolidate researchers. Program activity will be enhanced as researchers are currently located in geographically distributed campus laboratories that are between 32 and 77 years old. A 7 month- long space assessment precedes the project which will involve gutting much of the interior space, installation of modular laboratory units and new fume hoods and upgrades to the mechanical, electrical and plumbing services for increased safety and improved efficiency. Provision of adequate facilities will enable the basic discovery processes to be linked more closely with the development process. Biochemical engineers will study separation techniques for the recovery of biological products, biomimetic adsorbents for metal removal and recovery and new approaches to bioremediation. Bioinorganic chemists will study metal ion transport that is essential to life processes, the use of chelating agents in the sequestration of heavy metals, and lanthanide complexes for possible use in enhancing MRI. A total of 8 professors and 117 graduate students and postdoctoral fellows will benefit from these improvements.

This award from the Chemistry Research Instrumentation and Facilities Program will assist the Department of Chemistry at University of California at Berkeley in the purchase of a new X-ray diffractometer equipped with a high-resolution, high-sensitivity area detector with associated computers. This new instrumentation will enhance greatly research in a number of areas including the following: 1) Stereognostic Coordination Chemistry 2) Lanthanide and Actinide Coordination Chemistry 3) Coordination Chemistry of Biological Iron Transport 4) Metal Complexes with Te and Se-Containing Ligands 5) Transition Metal-Silicon Chemistry 6) Synthesis and Study of New Electroactive Polymers 7) Single-Source Precursors to Elaborate Solid-State Structures 8) Structures of the Oxide Superconductors and Related Materials and 9) Conformationally Constrained and Templated Peptides. The X-ray diffractometer is used to make accurate and precise measurements of the full three- dimensional structure of a molecule. The information obtained gives the precise values of all the bond distances and bond angles of a given molecule and it gives accurate information about the spatial arrangement of the molecule relative to the neighboring molecules.

DESCRIPTION: Iron is an essential growth element, but the availability of iron in an oxidizing environment is severely limited by the insolubility of Fe(OH)3. Microbes respond to this limitation by synthesizing low-molecular-weight chelating agents, siderophores, for which there are membrane-bound receptors. Often bacteria synthesize several siderophores and/or express multiple receptors. There is a clear link between iron and microbial virulence. Pathogenicity increases of up to 107 have been observed when the bacteriostatic effects of human iron complexing proteins are overcome. Previous studies have focussed on the structures of siderophores and the relationship of stereochemistry to siderophore mediated iron transport. Future work will address the dynamics of siderophore complexation in addition to the identification of new siderophores and their structure/function relationships. Fluorescent probes will be attached to siderophores to monitor both the spatial and temporal uptake of iron mediated by siderophores. The kinetics of ligand exchange and complex isomerization in catechol siderophores will be studied by NMR, and competition experiments will be used to obtain full thermodynamic characterization of siderophores. The overall focus of this research continues to be the coordination chemistry of siderophores, the connection of this chemistry to their biological activity, and the consequent medical significance of the results.

This award from Inorganic, Bioinorganic, and Organometallic Chemistry Program of the Chemistry Division supports research of Professor Kenneth N. Raymond, Department of Chemistry, University of California at Berkeley into superamolecular clusters assembled by mean of metal-ligand coordinate bonds. The project will focus on the synthesis of expanded host-volume clusters, the formation and rearrangement of supramolecular clusters, the control of guest molecule reactivity by supramolecular hosts. It is the goal of the research to precisely define what can be programmed into a ligand in terms of predetermined symmetry based linkages and to exploit this to develop new chemistry based on supramolecular self-assembly.

Large, supramolecular structures of tetrahedra, cubes and dodecahedra will be synthesized with metal ions at the vertices and organic linkers along the edges. Symmetry-based principles developed in this work will be generally applicable to the preparation of other nanostructures. The ability to systematically prepare very large molecules with controlled shapes and pore cavities will be exploited to catalyze chemical reactions.

Iron is essential - yet toxic both in deficiency and excess. Increasingly the medical literature show that iron plays a key role in areas of human health. The focus of this research project is the essential role iron plays in bacterial growth, and hence the role of iron in human disease caused by bacteria or fungi. The goals are to characterize the mechanisms of siderophore-mediated iron uptake and to understand the role of siderophores in bacterial disease. Previous progress in this on-going project have involved the characterization of several siderophores from human pathogens, the quantitative determination and explanation of the stability or ferric siderophore complexes, the preparation and in vivo use of siderophore analogs, the characterization of the role stereochemistry at the metal center plays in the siderophore recognition and transport process and other aspects of siderophore coordination chemistry. The focus of this proposal now turns in a more microbiological direction. We will study the role of amonabactin in the virulence of Aeromonas hydrophyla, a human pathogen. The ability of this sideophore to remove iron from transferrin will probed and the gene it encodes for an enzyme in the synthesis of amonabactin, amoA, will be isolated to determined the importance of amonabactin in Aeromonas pathogenicity. Recognition and transport of ferric amonabactin will be probed using both four natural siderophores and synthetic analogs. The amonabactin receptor protein will be characterized. The siderophore for the organism that causes whooping cough in humans will be investigated with regard to its kinetic ability to remove iron, and its role in iron uptake will be probed. The latter stages of iron uptake into E.coli. mediated by enterobactin will be probed and the enterobactin esterase will be isolated and characterized. Collaborative studies on siderophores will include their biogeochemistry, marine biochemistry and the characterization of new siderophores.

[unreadable] DESCRIPTION (provided by applicant): Human iron overload as a consequence of beta-thalassemia, hemochromatosis, or sickle cell anemia is a serious clinical problem. Iron chelation therapy is effective, but the goal of developing readily available, orally-active, effective and non-toxic sequestering agents have proven to be remarkably difficult to achieve. Desferrioxamine (Desferal(r)), a trihydroxamate ligand, remains the iron chelator of clinical use in the United States. Desferal(r) is expensive, has a short half-life in vivo, does not efficiently remove iron from transferrin, must be given on a regular, frequent basis by a subcutaneous or intravenous route, and its use can result in significant, irreversible toxicity. While there are other agents being investigated and developed as potential successors to Desferal(r), there is no established chelator that has yet met this target. The goal of this project is to develop new sequestering agents that would meet that need. In the previous period of support, in collaboration with the Children's Hospital of Oakland Research Institute (CHORI) and Dr. Patricia Durbin- Heavey at the Lawrence Berkeley National Laboratory (LBNL), both new ligand systems and new screening protocols have been developed. The radioactive tracer mouse model at LBNL has been found to be accurate and reliable and has been selected as this project's routine screening protocol. Several promising new multidentate ligands have been developed based on the continuing hypothesis that multidentate, rather than bidentate or tridentate, hydroxypyridonate ligands will be effective as oral chelating agents effective at concentrations below toxic levels. A kinetic animal model has been developed for the metabolism of these agents that accurately fits the observed behavior. It is now proposed to extend these animal studies and to develop the chelators to the point where they can be accepted for large-scale testing, with the ultimate goal being the production of a safe, orally-active iron chelating agent that will prevent the toxicity of iron accumulation in patients who require chronic red cell transfusions. Several new ligand types are proposed with specific hypotheses about structural function relationships. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In the past two decades, magnetic resonance imaging (MRT) has revolutionized diagnostic medicine. MRI enables the acquisition of high resolution, three-dimensional images in the detection of a wide variety of physical abnormalities and recent advances in dynamic MRJ show the prospect of real-time imaging. About 30 percent of MRI scans are now acquired using a paramagnetic contrast agent, which enhances the image quality. Gadolinium complexes are most widely used and these complexes currently are all based on a poly(aminoearboxylate) ligand scaffold . 'While effective, the water proton relaxivity of these agents are only a few percent of that theoretically possible. Attachment of the agent to macromolecules, either synthetic or in vivo biomolecules, can increase the relaxivity by lowering the rotational correlation time but the rate of water exchange from the gadolinium center then becomes the limiting factor. We have a new family of gadolinium complexes (based on a hexadentate hydroxypyridonate ligand scaffold) that are stable and have substantially higher relaxivity than most poly(amino-carboxylate) complexes due to a water exchange rate about two orders of magnitude higher. The main objective of this project is to synthesize a novel series of Gd(III) MRI contrast agents for different diagnostic needs through selective structural modifications of the hexadentate ligand scaffold. The solution properties, including stability, will be assessed and the fundamental physical chemistry of their relaxivity explored in collaborative studies. Other collaborative studies will investigate initial biodistribution and toxicity screening (with a cell assay) and further studies will be done in a mouse or rat model. Our ultimate goal is to develop second generation MRI contrast agents with greater accuracy and specificity than those currently available or theoretically possible based on amino carboxylate ligands. These second generation agents will be made by introducing a new Gd complex unit that enables relaxivity of one or two orders of magnitude greater than what is routine today.

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Dr. Kenneth N Raymond of the University of California at Berkeley to design, synthesize and evaluate high-symmetry, nanometer scale molecular clusters. The clusters are designed to be self-assembled from a number of small and simple, identical subunits. Modeling studies will focus on making clusters with cavities much larger than a cubic nanometer. The work will address the trade-off between loss of stability in host/guest formation due to the hydrophobic cavity of the cluster as compared to expanding the ease of access to the interior to allow more rapid guest exchange. New designs will include icosahedra and clusters with positive as well as negative charges (changing the host/guest preference characteristics). The broad areas of supramolecular properties under investigation include calorimetric quantification of thermodynamic quantities, kinetic studies of self-assembly, supramolecular chirality, guest-host interactions and electron and molecular transport. Among questions to be addressed is whether guest molecules enter the supramolecular cavity through an opening made by a the dissociation of a ligand forming the cavity or whether the guest molecule squeezes in through a seam in the host structure. Modeling studies suggest that exit of the guest probably occurs through a cooperative distortion, rather than partial dissociation, of the cluster. Because of trigonal propeller chirality at the metal vertices and mechanical linkage between the metal vertices, the clusters are homochiral, resolvable and retain their chirality even when components of the cluster are replaced. Clusters will be prepared with guest interiors that are much more enantiomerically selective. Other goals are the attachment of clusters to solid supports and the preparation of switchable clusters whose host/guest properties can be altered either electrochemically or photochemically. Clusters with varying oxidation state metal ion vertices can act as electron carriers through as many as nine different redox states. Non-labile higher oxidation state clusters will be prepared from the more labile lower oxidation state complexes by oxidation to a less labile state.


The ability to do chemistry inside designed, chiral, nanometer sized flasks opens promising applications that may improve chemical separations and selective chemical reactivity. Students will be trained in supramolecular design, synthesis, characterization, and supramolecular reaction mechanisms.

Magnetic resonance imaging (MRI) has provided dramatic new capabilities for diagnostic medicine. MRI enables the acquisition of high resolution, three-dimensional images in the detection of a wide variety of physical abnormalities, and recent advances in dynamic MRI are providing real-time imaging. Over 30% of MRI scans are now acquired using a paramagnetic contrast agent, which enhances the proton relaxation and hence image quality. Gadolinium complexes are most widely used, and these complexes currently are all based on a poly(amino-carboxylate) ligand scaffold . While effective, the relaxivity values (3 - 5mM"V1) of these agents are only a few percent of that theoretically possible, requiring gram amounts of Gd per administration. Attachment of the agent to macromolecules increases the relaxivity by lowering the rotational correlation time, but the rate of water exchange from the gadolinium center then becomes the limiting factor. This project has developed gadolinium complexes (based on a hexadentate hydroxypyridonate ligand scaffold) that are stable and have substantially higher relaxivity due to a water exchange rate at least two orders of magnitude higher than commercial agents. Having developed the agents and demonstrated their stability and fast rates of water exchange, it is now proposed to continue their development for enabling new kinds of imaging. Relaxivities of more than 100mM~1s"1 are the target. This would require much less Gd for images and, more significantly, enable new types of imaging with MRI. Aims include the development of stable agents with 3 (as opposed to the current 1) coordinated water molecules, thus tripling the relaxivity; novel designs of supramolecular Gd clusters;agents that dock to specific targeted biomolecules;and full characterization of those thermodynamic and kinetic properties of these complexes in aqueous solution that are related to their MRI enhancement. The goal is a second generation of MRI agents that utilize relaxivities of up to two orders of magnitude greater than the clinical agents in use today.

[unreadable] DESCRIPTION (provided by applicant): Therapy for radioisotope contamination of a large population by a dirty bomb or other event will require a cocktail of decorporation agents because of the wide variety of possible radionuclides and their chemical/biological properties. Decorporation is the only way to reduce exposure of certain incorporated radioisotopes. Fission product lanthanides and the actinides are among the most intractable of these elements to decorporate. While diethylenetriaminepentaacetic acid (DTPA) has been the standard therapy for actinide/lanthanide decorporation since its development and use by the U.S. Atomic Energy Commission in the 1950's, it is limited in efficacy. A new family of sequestering agents has been developed using a biomimetic design based on the similar biochemical transport properties of plutonium(IV) and iron(IIl) and siderophores, the natural iron chelators of bacteria. These chelators are more selective and have higher affinity for plutonium(IV) and a number of other actinide metal ions. Extensive toxicity and efficacy studies using a mouse model have been published and limited tests have been done in dogs and baboons. The results established that several of the new agents are much more effective than DTPA and, unlike DTPA, can be orally active. This project proposes to take two lead compounds 3,4,3-LI-i ,2-HOPO (an octadentate ligand) and 5-LIO-Me-3,2-HQPO (a tetradentate ligand) toward clinical use by scaling up the synthesis, establishing preparation methods suitable for good manufacturing practice (GMP), carrying out limited efficacy and toxicity studies for combinations of the two chelators in a mouse model, completing toxicity studies in human cell lines, and establishing preclinical safety of the candidate ligands under good laboratory practice (GLP) guidelines. The objective of this research is to bring forth two new decorporation agents in tandem and successfully accelerate their development to a pre-IND stage where only primate studies remain prior to a full IND application. This will be accomplished by an effective partnering of Lawrence Berkeley National Laboratory (LBNL) that has expertise in Iigand design, synthesis, and laboratory testing, with SRI International which possesses expertise in GLP testing and bringing pharmaceutical products to market. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nearly half a million cases of bacterial sepses are reported annually in the USA and approximately one third of the cases are fatal. Iron is a limiting nutrient in microbial growth;bacteria primarily obtain iron through production of siderophores, low molecular weight chelating agents with high ferric affinity and selectivity. The availability of iron is essential in determining the virulence of an invading pathogen. The most successful human pathogens, such as Bacillus anthracis, devise elaborate, multifaceted strategies to ensure their iron supply. This project seeks to understand siderophore transport systems: 1) from a structural level, studying the thermodynamics and kinetics of iron binding, 2) to a systemic level, following the recognition and transport of these siderophores into the bacteria, 3) to an environmental level, exploring how the surroundings, such as temperature, host immune system, presence of other bacteria and even exposure to light, affect the growth of the bacteria. We are uniquely equipped in our laboratory to carry out this range of studies and to pursue the following specific aims: 1. To understand the relationship between structure and function of siderophores. 2. To characterize siderophore-mediated iron transport in Gram-positive bacteria. 3. To explore the scope and functioning of the siderophore shuttle mechanism of microbial iron transport. 4. To further describe the mechanism of recognition of siderophores by proteins of the human immune system (siderocalin) and how the selectivity of this immune response is exploited by the most dangerous bacterial pathogens. To meet these Aims siderophore features such as thermodynamic stability and reduction potential of siderophore ferric complex, their kinetics of iron binding, and lipophilicity, will be determined for targeted siderophores and through the construction of synthetic siderophore analogs and coordination analogs we will explore siderophore function. Almost everything that is known about bacterial siderophore-mediated iron transport is in Gram-negative bacteria;Gram-positive bacteria are now our target, since this group of organisms includes many important human pathogens. We have in place collaborations to determine the crystallographic structures of membrane-associated protein receptors of Bacillus species that will complement our studies in this family. Our first report of the siderophore shuttle mechanism showed that metal exchange between two siderophores was essential for iron transport in the Gram-negative bacteria studied. We plan to see how widely distributed is this mechanism is among genera of bacteria;we now propose an extended experimental approach that incorporates the use of isotopically labeled natural siderophores. We have recently begun to develop an understanding of what we call "siderophore stealth": the evasion of siderocalin binding by structural modification of the siderophore. Through the use of synthetic analogs and bacterial siderophore isolates, as well as labeled substrates and mutant proteins, we intend to describe the selectivity and physiological course of siderocalin. PUBLIC HEALTH RELEVANCE: Nearly half a million cases of bacterial sepses are reported annually in the USA and approximately one third of the cases are fatal. Iron is a limiting nutrient in microbial growth;bacteria primarily obtain iron through production of siderophores, low molecular weight chelating agents with high ferric affinity and selectivity. This project determines how this process occurs and how the human immune system counters it.

DESCRIPTION (provided by applicant): In the last several years, a sense of urgency and a renewed interest in the study of radionuclide chemistry and biology have emerged, as threats of nuclear terrorism have become more plausible, and the risk of environmental contamination and human exposure to radioisotopes consequently increased. The only practical therapy to reduce the dramatic health consequences of internal actinide/lanthanide contamination is treatment with chelating agents that form excretable complexes, although fission product lanthanides and the actinides are among the most intractable radionuclides to decorporate. While diethylenetriaminepentaacetic acid (DTPA) has been the standard therapy for actinide/lanthanide decorporation for several decades, it has limited efficacy and is not yet orally available. Hydroxypyridonate sequestering agents developed in our laboratory are selective and have a high affinity for plutonium(IV), americium(III), a number of other actinide ions, and lanthanide ions. Extensive efficacy studies in mice have been published and a limited number of tests have been performed in dogs and baboons, establishing that two of the designed agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2- HOPO), are up to 30 times more effective than DTPA and, unlike DTPA, are orally active. In addition, recent methods for large-scale synthesis, preclinical toxicity studies in rats and in vitro cytotoxicity studies using human cells have demonstrated that the selected chelators exhibit low toxicity and hold promise as non-toxic orally available actinide/lanthanide decorporation agents. The objective of this two-year project application is to sustain a large-scale interdisciplinary research program that will carry forward the pre-clinical development of both selected therapeutic actinide/lanthanide decorporation agents for emergency use and to enable the establishment of a viable infrastructure dedicated to the study and understanding of actinide and lanthanide chelation in biological systems. Compound characterization, actinide removal efficacy studies in mice and dogs, pre-clinical safety studies in rats and dogs and permeability assessment using rat intestinal tissues will be performed in collaboration with scientists from the Lawrence Berkeley National Laboratory, SRI International, and the Lovelace Respiratory Research Institute (LRRI). Finally, early input from the FDA will be obtained for our ligand development plan to enable the timely and successful filing of an IND application. As underlined by the National Institute of Allergy and Infectious Diseases (NIAID) Radiation Countermeasures Program, the development of radionuclide decorporation agents responds to the urgent need to protect the general population from the consequences of a large-scale exposure to radionuclides. In the event of a radiological/nuclear terrorist event, prospective decorporation treatments must be suitable to treat a large population that could be exposed to a variety of agents with a potentially high number of casualties. This project focuses on meeting the criteria listed by NIAID for the pre-clinical development of actinide and lanthanide decorporation agents, which include (i) chelation and elimination of a range of actinides/lanthanides, (ii) oral administration, (iii) effectiveness when administration is delayed, and (iv) safety for all potential populations.

DESCRIPTION (provided by applicant): The College of Chemistry X-ray Crystallography Facility at the University of California, Berkeley is requesting a total of $500,000 in funding from the National Institutes of Health for the purchase of a new single crystal X-ray diffractometer. The facility is oversubscribed as it is and current instrumentation in the laboratory is beginning to reach the end of its useful life and will need to be replaced shortly. In addition, new advances in technology allow newer instrumentation to be much more sensitive and versatile than first generation CCD diffractometers. Considering the amount of NIH-supported structures solved in this facility (over 250 per year), a new x-ray diffractometer for the facility will instantly improve the quality and range of data we are able to collect for NIH researchers. HEALTH RELEVANCE: The determination of the chemical and absolute structure of newly synthesized compounds is of utmost importance for potential drug targets used to treat diseases and illnesses. Single Crystal X-ray Crystallography is the one technique that can unambiguously give the chemical and absolute structure information necessary in one experiment.