David M. Wilson - US grants

The functions of and changes in proteins occuring in regenerating nerves will be examined. Proteins in regenerating axons and sprouts will be labeled in cell bodies and, after slow and fast axonal transport, will be analyzed upon arrival at terminals. The lifetimes, modifications and processing of such proteins will be examined in both synaptic terminals and regenerating axons by two dimensional gel electrophoresis. Neurons from fish and frogs will be studied. The modifications in membrane proteins and states of polymerization of cytoskeletal proteins during regeneration will be described. Changes in protein synthesis in glial cells during nerve degeneration and regeneration also will be analyzed. We will be testing hypothesis for analyzing the failure of most central nervous system neurons to regenerate. We hope to identify the sequence of biochemical events underlying the reorganization of damaged nerves for regeneration.

Polymerization of the microtubule-associated protein tau is responsible for the neurofibrillary pathology observed in Alzheimer's disease. This is a prevalent illness which causes dementia and death in the elderly. Attempts to understand the etiology of tau polymerization , and the subsequent impact of tau polymers on neuronal function have been limited by a paucity of suitable model systems. The goal of this research is to produce a cell culture system in which tau pathology can be selectively induced. Evidence indicates that tau polymers possess an ordered substructure which would necessarily result from the ordered structure of their constituent proteins. Amino acid sequences predicted to be involved in folding events associated with tau polymerization will be systematically altered by site directed mutagenesis, and he resultant proteins will be assayed for their ability to assemble. Tau sequences coding for proteins that demonstrate an increased potential for polymerization will be inserted into a.

Genetic material is subject to spontaneous and mutagen-induced modifications that contribute to the process of carcinogenesis. The most frequently formed damages are likely those produced by free radical attack of DNA. Free radicals, which include superoxide and hydroxyl radicals, are generated during normal oxygen metabolism and from exposure to anti-cancer agents such as bleomycin and ionizing radiation. Mutagenic or lethal oxidative DNA damages include sites of base loss (abasic or AP sites) and 3'-obstructive termini (3'-phosphate or 3'- phosphoglycolate). Enzymes that initiate the repair of AP sites and 3'- damages have been identified in all organisms studied to date, from bacteria to humans, and are a central component of base excision DNA repair. The predominant AP endonuclease in mammals is Ape/Hap1/Ref-1, but unlike other AP endonucleases, Ape possesses only a minor 3'-repair activity. Although many of the general features of Ape have been elucidated, the specifics of its recognition and binding to abasic site-containing DNA are not well understood. Using biochemical (e.g. footprinting, protein-DNA crosslinking, and site-directed mutagenesis) and structural (Nuclear Magnetic Resonance and Circular Dichroism) approaches, we intend to unravel the details of the Ape-DNA interaction and to identify the critical points of contact. In addition, we will employ computer modeling to define the structural features of DNA substrates utilized in these and other studies to uncover aspects of DNA that are important for recognition and incision. Elucidating the molecular detail of the Ape-DNA interaction may permit the design of effective inhibitor substrates, which may aid cancer treatment regimes and Ape-DNA structural studies, or of a dominant-negative Ape protein, which would help to clarify the biological importance of this enzyme. Since Ape displays a relatively poor 3'-diesterase activity in vitro, particularly at double-strand break ends, the biological role of this activity is unclear. A recent study reporting an interaction between Ape and Ku could suggest that these proteins act in cooperation to efficiently process 3'-damages, and may indicate a role for Ape in non- homologous recombination (i.e. DNA end-joining). We intend to characterize this interaction biochemically and determine whether Ku influences the 3'-repair capacity of Ape. We intend to identify and characterize other nucleases or accessory factors that are involved in the processing of 3'-oxidative damages. The studies described within will go a long way towards defining the repair activities of Ape and will help understand the intracacies of two critical DNA repair pathways.

DESCRIPTION (provided by applicant): Remarkable progress has been made over the last decade in the characterization of in vivo metabolism using hyperpolarized (HP) 13C spectroscopy, with profound implications for the diagnosis and treatment of human cancers. The majority of studies have focused on metabolism of [1-13C] pyruvate by lactate dehydrogenase (LDH), which occurs to a larger extent in cancer and has been correlated with pathologic grade in a murine prostate tumor model. Although the molecular requirements of dynamic nuclear polarization (DNP) are rather strict, several additional hyperpolarized 13C agents have been developed that are potential markers for pH (13C bicarbonate), hexose metabolism ([2-13C] fructose), and necrosis ([1,4-13C] fumarate). More recently, we have developed the redox sensor [1-13C] dehydroascorbate (DHA), an oxidized version of Vitamin C that shares an uptake mechanism with glucose. This new probe demonstrates rapid in vivo reduction to [1-13C] Vitamin C, and is the principal agent used for the Specific Aims of this R01 proposal. In the proposed project, this new probe is employed to address the redox adaptation of tumors, which accumulate large quantities of GSH and other antioxidants, conferring resistance to therapies that are ROS- dependent, including radiation. Prostate cancer was chosen since (1) radiation therapy is a mainstay of treatment (2) several in vitro studies have implicated GSH and other redox components in resistance and (3) non-invasive biomarkers for disease aggressiveness are lacking. Our preliminary 1H studies on primary prostate cancer cells using high resolution magic-angle spinning (HR-MAS) NMR indicate high levels of GSH, and in vivo murine studies using HP [1-13C] DHA demonstrate reduction in organs that are known to be rich in GSH, including the liver, kidneys, and brain. Furthermore, numerous reports in the literature have established that reduction of DHA to VitC is GSH-mediated. However, other redox mechanisms are certainly possible for the reduction of HP [1-13C] DHA observed in vivo, and our first studies will determine which cellular redox (or transport) components are involved (Specific Aim 1). We will then turn to studies validating the use of HP [1- 13C] DHA to determine cancer aggressiveness, and predict response to radiation therapy in a murine prostate cancer (TRAMP) model (Specific Aim 2). Finally, we will compare this new 13C probe to related 18F ascorbates as well as to [2-deoxy-2-18F] fluoro-D-glucose (FDG), the radioisotope used in the vast majority of clinical positron emission tomography (PET) studies (Specific Aim 3). We anticipate HP 13C MRI emerging as an important technology to complement existing molecular imaging methods, including PET, and comparative studies (employing structurally or mechanistically related probes) will be important to validate both modalities. Our justification for pursuing 18F ascorbate probes is twofold: (1) to determine whether ascorbate-based PET probes demonstrate similar in vivo characteristics to HP [1-13C] DHA and (2) to compare the ability of PET and HP methods to predict tumor aggressiveness/ treatment response in prostate cancer.

PROJECT SUMMARY: This proposal addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Existing clinical techniques target the host immune response, for example 111In SPECT white blood cell scanning or 18F-FDG PET. Although these modalities can sometimes be useful, they lack the specificity required to distinguish living bacteria from sterile inflammation, cancer, and other highly metabolic tissues. Therefore we are proposing a PET imaging technique that exploits metabolic pathways specific to bacteria, targeting both gram-positive and gram- negative organisms. We believe a technique that detects ALL or at least a majority of pathogenic bacteria will be most useful in clinical practice. Once an imaging abnormality has been identified as infection, tissue sampling, staining and culture may still be required. An imaging method that could distinguish active infection from frequent mimics, would instantly become the standard of care in a variety of inpatient and outpatient settings. In this proposal, we develop 18F and 11C tracers for positron emission tomography (PET) using D-Met derived tracers, and study them for the first time in human patients with spinal infections. We have identified 11C D-met as a radiotracer with (1) a simple, high-yield radiosynthesis (2) good in vivo stability (3) appropriate mimicry of the endogenous substrate (4) high rate of incorporation into bacterial peptidoglycan and (5) low uptake in background tissues. We will start by refining the synthesis of enantiopure 11C D-met, and investigating close structural relatives of 11C D-met for enhanced bacterial uptake (Specific Aim 1). We will then study our lead D-met tracer in compelling preclinical infection models, including models of vertebral osteomyelitis-discitis (Specific Aim 2). In Specific Aim 3, we will take all steps needed for an investigational new drug (IND) approval for our lead tracer, and study its performance in patients suffering from spinal infection.

This proposal addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Existing clinical techniques generally target the host immune response, for example 111In SPECT white blood cell scanning or 18F-FDG PET. Although these modalities can sometimes be useful, they lack the specificity required to distinguish living bacteria from sterile inflammation, cancer, and other highly metabolic tissues. The central hypothesis of this proposal is that imaging bacteria-specific metabolic pathways will afford a highly accurate method to detect bacterial infections in vivo. We have identified 11C-PABA as a radiotracer with (1) a simple, high- yield radio-synthesis (2) good in vivo stability (3) appropriate mimicry of the endogenous substrate (4) rapid (minutes) rate of incorporation into both Gram-negative and Gram-positive bacteria, including clinical strains and multi-drug resistant organisms and (5) low uptake in background tissues. We propose a multi-PI and interdisciplinary collaboration across two institutions (University of California, San Francisco ? UCSF and Johns Hopkins) to validate 11C-PABA PET as a bacteria-specific imaging tool for spinal infections and study it for the first time in human patients. We will investigate the sensitivity of 11C-PABA for several relevant microorganisms, and compare its specificity to that of 18F-FDG and 68Ga-citrate in mouse models (Specific Aim 1). We will then study 11C-PABA in compelling preclinical infection models, assessing in vivo Gram-staining via a dual-tracer approach, rapid imaging readouts for antibiotic efficacy and evaluating tracer performance in rat models of vertebral osteomyelitis-discitis (Specific Aim 2). In Specific Aim 3, we will translate 11C-PABA under the Radioactive Drug Research Committee (RDRC) program and study its performance in patients suffering from spinal infection. Any imaging method that could confirm presence of infection as well as guide antibiotic therapy would be profoundly helpful in clinical practice, sparing patients exposure to inappropriate broad spectrum antibiotics, invasive tissue sampling and reducing the risks of antibiotic resistance. Finally, while the focus of this application is on spinal infections, a clinically translatable bacteria-specific PET tracer would revolutionize the workup and management of a large variety of clinically relevant bacterial infections.

This proposal focuses on the development and validation of glutamine-derived 11C isotopomers for positron emission tomography. Specifically, the 1- and 5-positions of exogenously administered glutamine have different metabolic fates, depending on the dominant mechanisms of conversion present. We will therefore synthesize both L-[1-11C]glutamine ([1-11C]gln) and L-[5-11C]glutamine ([5-11C]gln), and investigate their use in detecting the isocitrate dehydrogenase mutation (IDHm), seen in several benign and malignant clinical diseases including low-grade glioma (LGG). Improved diagnosis of LGG would address a major challenge that neuro-radiologists, neuro-oncologists, radiation oncologists and neuro-surgeons encounter frequently, namely differentiating glial tumor from other brain entities. Patients with IDH-mutant (IDHm) glioma harbor the oncometabolite 2-hydroxyglutarate (2HG), which represents a promising way to detect LGG. We are motivated by a recent report establishing rapid conversion of exogenous glutamine to 2HG in IDHm lesions, representing a way to sequester the 11C radionuclide. The central hypothesis of this proposal is that a positron- labelled metabolic precursor of 2HG, [1-11C]gln, can be used to detect IDHm. We further predict significant advantages in using [1-11C]gln versus the reported [5-11C]gln isotopomer, based on the suppression of background signals related to tricarboxylic acid (TCA) cycle metabolism. When validated, this approach would set the stage for clinical use of [1-11C]gln PET in evaluating LGG and other IDHm lesions. We propose a multi-PI and interdisciplinary collaboration to validate [1-11C]gln PET as an IDHm-specific tool and perform the first patient studies using [1-11C]gln to further support the methodology. We will first develop the first radiosynthesis of [1-11C]gln, via adaptation of reported methods using [11C]CO2 and [11C]CN-. We will then compare the accumulation of [1-11C]gln to that of [5-11C]gln in IDHm versus IDH-wildtype (IDHwt) cells, both in vitro and in vivo (Specific Aim 1). In Specific Aim 2, all regulatory work and site approvals needed to study [1-11C]gln at UCSF will be accomplished. In Specific Aim 3, we will translate [1-11C]gln under the Radioactive Drug Research Committee (RDRC) program and study its performance in patients suffering from LGG. While this application focuses on LGG, a clinically translatable IDHm-specific tracer would revolutionize the workup and management of a large variety of clinically relevant lesions. Furthermore, robust methods to synthesize and evaluate amino-acid derived 11C isotopomers will dramatically improve the ability of PET to detect metabolic reprogramming in human disease.

PROJECT SUMMARY: This application addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. In response to this challenge, our laboratory has developed both hyperpolarized 13C MRI and positron emission tomography (PET) tools targeting bacteria-specific metabolism. In this proposal, we employ another nuclear magnetic resonance (NMR)-observable stable isotope namely deuterium (2H) to detect living microorganisms both in vitro and in vivo. In doing so we address two major challenges of 2H magnetic resonance imaging (MRI) by (1) identifying 2H MRI-observable metabolites that have the needed chemical shift separation from HDO (water) and (2) using bacteria-specific sugar alcohols whose incorporation into microorganisms is not easily saturated. Our approach also takes advantage of two different strategies to incorporate 2H in a microorganism-specific way, and newly developed 2H MRI methods to maximize sensitivity. The two detection strategies pursued are either (1) a 2H substrate is converted to a dominant, 2H-MRI observable downstream metabolite or (2) a 2H substrate is converted to a dead-end metabolite that is accumulated as a 2H-MRI detectable species. These two concepts are highlighted by enriched variants of sorbitol, either D-[6,6'-2H2]sorbitol whose metabolism may be detected by its conversion to lactate/ethanol, or 2-deoxy-D-[2,2'-2H2]sorbitol anticipated to be ?trapped? as its 6-phosphorylated adduct in bacteria. In both cases, the 2H nuclei have the needed chemical shift for in vivo 2H MRI imaging. In addition, the technologies pursued are amenable to clinical translation; our long-term goal is application of nontoxic 2H sugar alcohols to vulnerable populations including children, without the need for ionizing radiation. We will first adapt an NMR- compatible bioreactor for use in investigating 2H substrate metabolism in bacteria in vitro (Specific Aim 1). We will show then show bacteria-specific incorporation of 2H using enriched substrates, that are either metabolized to dominant 2H-MRI detectable products, or retained as dead-end molecules by pathogens (Specific Aim 2). In Specific Aim 3, we will show that 2H MRI at 14T using these tools can detect living bacteria in vivo.

Project Abstract: The human granzymes are a somewhat mystical class of five serine proteases (A, B, H, K, M) that are expressed and conditionally secreted by select lymphocytes like natural killer (NK) and cytotoxic T cells (CTL). Based largely on data for A and B, granzymes have been historically regarded as pro-apoptotic effectors whereby they are presented transiently by NK and CTLs at the immunological synapse with a problematic target cell (e.g. cancer cell, pathogen infected cell), and immediately shuttled into the cytosol via perforin to initiate cell death. However, emerging data has begun to challenge this canon and present a more complex model in which secreted granzymes perform essential signaling functions in extracellular space, including activation of macrophages as part of host defense. Interestingly, dysregulation of granzyme presentation and/or proteolytic activity may also underlie the pathobiology of debilitating diseases like neurodegenerative or systemic autoimmune diseases. Thus, more fully elaborating the biology of granzymes is an important unmet need, and requires technologies to study granzymes in the most clinically relevant animal models and humans themselves. To this end, we developed a novel imaging approach we term ?restricted interaction peptides? (RIP) to detect granzyme proteolytic activity in vivo with PET. Mechanistically, RIPs are administered as an inactive pro-form, whereupon internal cleavage of the RIP by the target endoprotease releases a radiolabeled peptide that immediately associates with nearby phospholipid membranes (i.e. the plasma membrane of the target cell). Thus, accumulation of the radiolabeled cleavage product adjacent to the endoprotease provides a readout of the relative units of enzyme activity within a region of interest. As preliminary data, we developed 64Cu-GB1, a RIP that measures granzyme B activity on PET. Our proof of concept data show that 64Cu-GB1 detects the cytotoxic pool of granzyme B activity trafficking to pathogenic target cells, but also an unexpected non-cytotoxic pool elicited as part of an inflammatory response to an endotoxin. During this project, we will expand upon these data in several important directions. First, we will test in Aim 1 if 64Cu-GB1 can be applied to study granzyme B proteolytic activity in the spontaneous immune responses arising due to pathogen stimulation. These data will be crucial to understanding the utility of this methodology beyond simply detecting pharmacologically augmented anticancer immune responses. During Aim 2, we will expand the RIP toolkit to develop and study probes targeting the tryptase proteolytic activities of granzymes A and K. During the final Aim, we will carry out the late stage preclinical experiments required to judge the feasibility of human imaging with RIP probes. As a model system, we will study our lead candidate 64Cu-GB1. If successful, this project will confer new translational technologies at a crucial inflection point away from the classical view as simply pro-apoptotic effectors toward multifaceted regulators of human immunology and host defense against pathogens.

PROJECT SUMMARY: This application addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. Specifically, the proposed work is motivated by the difficulty in diagnosing and treating pneumonias in cystic fibrosis patients, especially those caused by P. aeruginosa. Clinically available imaging tools either (1) are limited by their background accumulation in the lungs or (2) image the host response to infection rather than the living bacteria themselves. To address this challenge, we have developed several PET radiotracers that exploit metabolic pathways specific to bacteria, including D-amino acid derived probes most recently D-[3-11C]alanine. When D-[3-11C]alanine was applied to several compelling preclinical models of infection, we found that it was exquisitely sensitive to P. aeruginosa, which is not the case for the vast majority of reported radiotracers. We further showed that D-[3-11C]alanine is a radiotracer with (1) a simple, high-yield radiosynthesis (2) good in vivo stability (3) appropriate mimicry of the endogenous substrate (4) high rate of incorporation into both gram- negative and gram-positive bacteria and (5) low uptake in background tissues. These studies demonstrate the outstanding potential of D-[3-11C]alanine, with the proposed work necessary to further validate and understand this tracer. We will first expand our radiochemical methods, synthesizing the D-[1-11C]alanine isotopomer and structurally related 18F probes (Specific Aim 1). We will then further investigate the lead 11C isotopomer in vitro, analyzing its performance in clinical P. aeruginosa strains and validated biofilm models (Specific Aim 2). In Specific Aim 3, we will extend these concepts in vivo employing both acute and chronic models of P. aeruginosa infection.