Wayne Bowen, Ph.D. - US grants

Endogenous opioid peptides play a major role in brain function, and opiate drugs are both clinically important and socially abused. Based on pharmacological and ligand binding studies, receptors for these compounds have been divided into five major classes: mu, delta, kappa, sigma, and epsilon. However, physico-chemical evidence for distinct opiate binding proteins is lacking and thus little is known of the physical and chemical characteristics which differentiate these receptor types. Derivatives of opiate alkaloids and opioid peptides will be synthesized which will irreversibly bind to mu, delta, and kappa receptor types. The derivatives will be analyzed for binding affinity, receptor selectivity, and irreversibility by competition studies with labeled reversible mu, delta, and kappa receptor probes. The tritiated or radioiodinated form of suitable ligands will then be synthesized. Brain membrane homogenates and cryostat-cut, slide-mounted thin sections of brain will be treated with these compounds. Labeled membranes will be solubilized with detergents and the extracts subjected to electrophoretic analysis. Proteins labeled by mu, delta, and kappa receptor probes will be characterized with regards to their molecular weights, isoelectric points, and peptide maps to determine the degree of similarity. Finally, a structural model for the receptor, Type 1 receptor model, will be tested. This model involves interacting mu and delta binding sites either in a protein complex or binding domains on a single polypeptide chain. This will be accomplished by ascertaining the proximity of binding sites to each other in the membrane by analyzing the effect of protein crosslinking reagents on the electrophoretic-mobilities of the labeled receptor proteins. It is anticipated that this approach will lead to chemical identification of the receptor types and will define the molecular basis of opiate receptor heterogeneity. In addition, the irreversibly binding opiate ligands produced in this project may prove useful in pharmacological and behavioral studies of opiate action.

The aim of this proposal is to establish an integrated peptide chemistry facility at Brown University. This facility will be composed of a peptide synthesizer and a peptide sequencer, along with supporting equipment. The facility will be staffed by a full-time Ph.D. peptide chemist. There is no such facility pre-existing at Brown. Such a facility will be invaluable to a number of researchers at Brown and at Brown-affiliated hospitals who have need for peptide synthesis and/or sequencing in their research programs. These investigators come from various departments and sections at the university: Biochemistry, Biochemical Pharmacology, Cell Biology, Chemistry, Medicine, Microbiology and Molecular Biology, Oncology, Physiology and Biophysics, Psychology, and Surgery. Therefore, the facility will receive university-wide use and provide a common resource upon which various disciplines can draw.

Purification of opiate receptors has lagged behind that of other receptors for neurotransmitters and hormones. Thus, little is known of opiate receptor structure and its relationship to function. We propose to combine a number of approaches in order to achieve purification of kappa opiate receptors. These include affinity chromatographic, affinity labeling, and immunologic techniques. Novel, kappa-selective affinity chromatography matrices will be synthesized by attaching amine derivatives of U50-488H to activated solid supports. These columns will then be used to purify kappa receptors which retain ability to bind opiate agonists and antagonists. These columns will also be used to resolve putative U50-488H-sensitive and U50-488H-insensitive kappa receptor subtypes. Successful separation of these activities would support existence of separate kappa receptor entities. Novel electrophilic and photolabile ligands have been developed for covalent, site-directed labeling of kappa receptor proteins. These affinity probes are also based on the structure of the kappa selective agonist, U50-488H. After covalent attachment of radiolabeled probe, affinity labeled proteins will be purified by chromatographic techniques. In an alternative approach, antibodies will be raised against the appropriate kappa affinity ligand. These antibodies will then be used to isolate the corresponding covalent ligand-receptor complex. Verification that purified affinity labeled proteins are in fact kappa receptors will be obtained using an immunologic approach. Partial N-terminal sequences of the purified complexes will be obtained. Antibodies will then be raised against synthetic peptides corresponding to the sequences. These antibodies will then be tested for ability to recognize active kappa receptors. Cross-reacting antibody would confirm kappa-relatedness of the purified affinity labeled protein. Characteristics of interest in purified kappa receptors are molecular weight, isoelectric point, amino acid composition, peptide map, and sequence. Any sequence information obtained for kappa opiate receptors will be compared to known sequences of other neurotransmitter and hormone receptors.

Sigma receptors are non-dopaminergic, non-opioid receptors which bind several neuroleptic drugs and the (+)-enantiomer of some opiate benzomorphans. The biological function of sigma binding sites has, for the most part, remained obscure and there is little data available which differentiates a true drug receptor role from a drug acceptor role. Recent biochemical studies have implicated sigma receptors in modulation of the inositol phosphate second messenger system. In addition, a variety of pharmacological, anatomical, and Physiological studies have indicated that sigma receptors are involved in neural control of movement and may be involved in drug- induced and idiopathic disorders of posture and tone. The purpose of this proposal is to further investigate the biological role of sigma receptors, with particular emphasis on biochemical systems modulated by sigma receptors and their possible involvement in motor control and the pathogenesis of dystonia. Ligands which have high affinity and selectivity for sigma receptors will be developed to use as tools in the biochemical and physiological studies. Involvement of sigma receptors in modulation of second messenger systems, protein phosphorylation, and neurotransmitter release will then be investigated. Application of sigma ligands to motor nuclei of rats will be used to investigate changes in motor behavior and electrophysiological activity. Finally, experiments will be carried out to determine the extent of sigma receptor alteration in brains of mutant dystonic rats. These studies should elucidate some of the functions of sigma receptors at both the biochemical and physiological levels. In addition, the structure-activity study should yield better ligands with which to investigate other aspects of sigma receptor biology and chemistry. The possibility that sigma receptors are involved in motor function and dysfunction suggests that sigma ligands may potentially be useful in treatment of some motor disorders.

The Graduate Program in Molecular Pharmacology & Physiology is a small and very diverse program with a strong set of courses and activities, as well as extensive mentoring mechanisms. Leadership of the program is by a group of three accomplished MPIs with complementary strengths and expertise that guarantee effective oversight of the program. The request is for 4 T32 slots per year; the total number of students in the program is 20. This training program in Interdisciplinary Training in Pharmacological Sciences will replace our current NIGMS pharmacology T32, ending in June of 2021. The training in our program focuses on fundamental principles of pharmacology, rigor and reproducibility, and cutting-edge quantitative methods. Our trainees participate in many activities that promote interaction, collaboration and professional development. Here we propose a number of new initiatives in the curriculum, professional development, program oversight and recruitment. A recent innovative initiative we are developing in the program is the establishment of summer internships at Pfizer pharmaceutical company labs; these internships will give our trainees the unique opportunity to experience first-hand the scientific environment in a pharmaceutical company and make informed decisions for their future careers. Our current admissions and recruitment practices have yielded a high percentage of students from disadvantaged and underrepresented groups (in the last 5 years, 47% underrepresented minorities plus 7% disabled), and there has been 100% retention of these students in the program during the last 15 years (retention for the program in general is 95%). The career outcomes of the students are excellent, with 52% of students staying in academia (25% faculty, 27% currently postdocs) often at top institutions, about 25% as scientists in pharmaceutical or biotechnology companies, and the remainder in a variety of science and medicine- related careers. Thus, our program has been training a highly diverse group of students who have become successful in different areas of science; the new initiatives proposed in our T32 application will further enhance our unique program and improve the quality of the training.