Summary of Research Interests

General: Medicinal and bioorganic chemistry, synthesis and investigation of novel probes of enzyme structure and function, protein mass spectrometry & proteomics, enzyme inhibition and binding kinetics, pharmacophore development and computational modeling of proteins, immunoorganic chemistry.

1. Glutamate Neurotransmitter System: l-Glutamate is involved in neuropathologic events including cell death that results from excessive stimulation of post-synaptic neurons. Excitotoxic damage is believed to contribute to numerous neurologic maladies including ischemia, anoxia, stroke, epilepsy, ALS and others. The glutamate neurotransmitter system can be subdivided into classes of neuronal receptors, metabolizing enzymes and transporter proteins. Much of the characterization and differentiation of glutamate receptor and transporter subtypes has been advanced through study of the pharmacology of conformationally-restricted analogs and/or functional group isosteres of glutamate.

a. Glutamate transporter pharmacology/pharmacophores: We design and synthesize custom-tailored glutamate analogs to study and differentiate the pharmacology of the glutamate neurotransmitter system proteins. In particular, we are focusing our current attention on developing novel inhibitors and substrates of the excitatory amino acid transporters (EAAT) and glutamate vesicular transporter (VGLUT1). Because these transporters are membrane bound protein architectures, they are not likely to have their three-dimensional structure solved by x-ray analysis in the near future and therefore offer a wide opportunity for study. The immediate goal of this project is to define the structural requirements for selective and potent binding to VGLUT1 and to visualize and model these requirements (pharmacophore). The long-range goal of this study is to develop a pharmacophore model of VGLUT1 and to utilize this information to regulate vesicular storage, uptake and release of glutamate. We are also in development of EAAT2 pharmacophore.

b. Solving the [glutamate] vesicle proteome: Advances in mass spectrometry now allow for the analysis of macromolecules including protein [sub]structures. Our research has focused on solving the proteome (the protein complement of the gene) complement of the neuronal vesicle. The vesicle is responsible for storage of neurotransmitters and is trafficked from the endosome and via a ‘reserve pool’ through a series of complex, protein- and ion-regulated steps to fuse with the neuron membrane and release quanta of neurotransmitter that regulates an array neuronal activities. Although several key proteins contained in the vesicle have been identified, no systematic study has been undertaken to identify the vesicle proteins as integral, associated or anchored. Using 2D electrophoresis and mass spectrometric fingerprinting techniques (trypsin digest), proteins in the vesicle system are identified via public bioinformatics databases. We are currently completing phase I, of a three-phase research approach that involves: (I) identification of the functional and structural vesicle proteins, (II) identification of glutamatergic vesicle proteins as distinct from the other vesicles (via pull-down using VGLUT antibodies), and (III) cross-linking protein clusters and networks present on the vesicle membrane surface.

2. Mechanism of Toxicity of Organophosphates: Organophosphates (OPs) are a semi-reactive class of organic molecules that are well known for their use to control insect pests and as nerve gas agents. Following a large exposure, humans undergo typical symptoms of cholinergic poisoning, which correlates with the inhibition (covalent modification) of the enzyme, acetylcholinesterase (AChE). Our research seeks to understand the molecular level of toxicity, namely, how does AChE and non-target proteins undergo reaction with OPs to form OP-protein conjugates and how do these conjugates result in novel pathways of intoxication. Our research portfolio in OP chemistry, biochemistry and toxicology involves the following:

a. OP Covalent Modification - Identification of Non-Target Proteins: Exposure to OPs results in three toxicity profiles – acute, delayed and chronic. It is generally believed that a single dose exposure to an OP that leads to cholinergic crisis is consistent with acute toxicity. Occasionally, single or multiple dose exposures do not afford a toxic cholinergic event but result in alternate symptoms including nausea, headache, vision loss, peripheral neuropathy, paralysis and other complications. However, there has been no systematic approach that seeks to correlate the molecular event with non-cholinergic OP poisoning. Using a neuroblastoma cell line (SHSY5Y) as a model, the cell line has been treated with reactive and non-reactive OPs and will isolate and identify OP-modified proteins using mass spectrometry. In brief, a reactive fluoro-OP is tethered to a biotin molecule (FP-biotin) and reacted with the cell line. Following an incubation, proteins that have been modified by the FP-biotin are separated from non-reacting proteins by passage through an avidin column. The biotinylated proteins are separated from each other by 2D-PAGE, digested with trypsin, the peptides separated by CapLC and directly injected onto the mass spectrometer for analysis and identification. This project is termed a “directed proteomics study” because the proteome complement is pre-selected based on a specific reaction type – organophosphorylation.

b. OP Molecular Fingerprints – An Antibody Approach to Enzyme Mechanism: When OP compounds inhibit AChE they form a covalent bond at an essential serine hydroxyl group principally forming a new phosphoester bond. But unlike biological phosphorylation where an inorganic phosphate (PO₃⁼) group is added reversibly to a protein, organophosphorylation forms a neutral phosphorus diester that is relatively stable. The addition of the OP group to AChE affords a novel protein conjugate that we propose can be distinguished from native AChE using antibodies. Important to note however, is that the formation of the OP phosphoserine bond occurs deep in the enzyme nearly 20 D below the surface of the protein and it not readily accessible so whole protein cannot be used. We therefore selected an AChE active site sequence and chemically phosphorylated it at the catalytically important serine hydroxyl group. The native peptide sequence and phosphorylated sequence were used as haptens for antibody production. Our goal is to show that exposure to OP compounds can be not only be determined by antibody analysis of AChE-OP conjugates but that the type of OP compound can be determined using a panel of antibodies custom-tailored to the OP compound type.

3. Other Areas of Interest:

a. Proteomics and protein chemistry.

b. Synthesis of phosphorus-containing analogs of alpha amino acids and their utility as inhibitors of matrix metalloproteinases.

c. Synthesis and utility of novel protein probes (fluorescent, UV-Vis, spin label, etc.).