John T. Hackett, Ph.D. - US grants

An analysis of the presynaptic physiology at a vertebrate central synapse will be undertaken in this research. Synaptic potentials will be recorded with two intracellular microelectrodes, one presynaptic in the Mauthner axon and the other postsynaptic in a cranial relay neuron. The investigation is focused on a single synaptic contact. How transmembrane potential, ions, pharmacological agents and proteins associated with presynaptic vesicles act on the number of quanta available and the probability for release will be studied. The hypothesis to be examined is that quantal presynaptic events are produced by specific proteins interacting with Ca2+ to trigger neurosecretion. The experiments may elucidate the fundamental process(es) by which neurotransmitter is released from central nerve terminals and are important for understanding the mechanisms of cell communication.

Neurosecretion of transmitter substance is a cellular process whereby information transfer occurs in the nervous system. Thus, normal nervous system signaling and resultant behavior depend upon an orderly and closely regulated release mechanism for these transmitters. This neurosecretory machinery is present at every chemical synapse in the organism. Many neurological disorders and psychiatric conditions are thought to involve disturbances in neurotransmission, thus clear and detailed knowledge of this subject at a molecular level holds promise for identifying the basis for rational therapeutic strategies. This research, will clarify, in terms of individual quanta of transmitter released, the final steps in exocytosis of neurotransmitter vesicles. It is well established that Ca++ entering the terminal triggers a cascade of events leading to exocytosis, but the details of this cascade are not clear. The model synapse used in the study is the contact formed in culture between avian ciliary ganglion neurons and pectoral myotubes, a cultured neuromuscular junction. Whole cell patch clamp recording methods will be applied to both sides of the synapse, and presynaptic microinjection of specific presynaptic vesicle-associated proteins through the patch pipette will be used to investigate the molecular machinery of neurosecretion. Other useful probes available for this project include antibodies against the vesicular proteins as well as other agents which modulate Ca++-dependent and Ca++-indicating dye. Finally, the presynaptic electrical signs of vesicle fusion with the terminal will also be recorded by analysis of capacitative transients. These results will test the hypotheses that specific proteins (synapsin 1, calcium/calmodulin protein kinase II, calelectrin, synexin) are involved in neurosecretion, the phosphorylations are involved, and that the quantal postsynaptic response is directly related to vesicle fusion.

Animals initiate movements either in response to specific stimuli (such as touch, sound or light signals), or as a result of an internal change in state (such as hunger). One important aspect of such voluntary animal movements that has received relatively little attention is their episodic nature, with distinct beginnings and endings. Much is already known about the networks of nerve cells that control rhythmic movements like flying and swimming, but the nervous system components that control the initiation and termination of rhythmic behavior are largely unknown. The experiments funded by this grant are designed to significantly advance the understanding of how nervous systems process information generated by brief sensory stimuli and how they thereby produce episodic locomotion.
Experiments will be conducted on isolated central nervous systems of the medicinal leech using two different approaches. One approach is electrophysiological, with experiments to investigate connections among nerve cells that control the initiation and maintenance of swimming movements. The second approach is pharmacological, with studies on the neurohormone serotonin and on other messenger molecules to learn how these substances are involved in converting the quiescent motor system into one that is functionally active. Because there is significant functional similarity between swimming and related locomotory movements in all animals and because the transformation of a system from quiescence to activity and back to quiescence is a feature of all episodic animal movements, insights gained from this research will have a major impact on our understanding of how the nervous system controls animal movements generally.
The activities funded by this grant have a broader impact on science by increasing the opportunities for undergraduates, primarily women, to conduct scientific research at Bryn Mawr and at the University of Virginia. Also, experiments conducted in the research laboratory are subsequently incorporated into laboratory exercises for advanced neurobiology courses at both institutions. Finally, training is provided to graduate students in modern electrophysiological recording techniques, data acquisition, and analysis. Results from these experiments are widely disseminated through posters at scientific meetings, including student presentations at local science fairs and scientific meetings; publication in scientific journals; lectures open to the public; and demonstrations on animal behavior at K-12 schools.

DESCRIPTION (provided by applicant): The cytochrome P450 enzymes (CYPs) are essential for the biosynthesis of numerous natural products, steroid hormones, and eicosanoids, as well as the clearance of most drugs. Due to their central role in xenobiotic disposition, CYPs mediate many adverse drug interactions of therapeutic significance. The mechanisms of CYP catalyzed O2 activation and substrate oxidation have been challenging to unravel, in large part because of the reactivity of intermediates. The CYPs that cleave C-C bonds are among the most mechanistically flexible of such enzymes; however, it is not usually realized that the pathways and reactive intermediates of this group of CYPs have not yet been investigated extensively. Most studies on CYP have been primarily focused on the hydroxylating CYPs. Thus relatively little attention has been paid to the CYP enzymes which use multiple oxidants and catalyse the more complicated transformations. Thus the presently available experimental data cannot be generally extrapolated to the C-C bond cleaving CYPs. Mycobacterium tuberculosis CYP51 constitutes a valuable and prototypical example for the study of O2 activation and C-C bond cleavage mechanisms. Moreover, many mycobacterial, trypanosomal, and fungal pathogens utilize bond cleaving CYPs in their own biosynthetic pathways, each of which is a drug target. Given that these pathogens are responsible for millions of deaths annually, there is a profound need for a clearer mechanistic understanding of these particular enzymes in support of the development of therapeutics of broad public health importance. The long-term goal of this project is to understand the catalytic mechanisms of C-C bond cleaving CYPs, and to answer questions surrounding how these enzymes tune the reactivity of their putative oxygen intermediates. Applying molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics techniques (QM/MM), the objective of the first Specific Aim is to explore the several possible reaction mechanisms utilized by M. tuberculosis CYP51 to activate O2 and perform substrate oxidation. The objective of the second Specific Aim is to validate the computationally-derived structure-function relationships governing the lifetimes of reactive oxygen intermediates. To meet these objectives, organic chemical syntheses of catalytic intermediates, site-directed mutagenesis, stopped-flow UV-vis, and resonance Raman techniques will be utilized. Guided by computational results, the objective of the third Specific Aim is to characterize relevant CYP intermediates using cryoradiolysis and resonance Raman spectroscopy to shed light on the C-C bond cleavage mechanism. Taken together, the interplay between these three Specific Aims will provide a feedback loop between theory and experiment, allowing incremental refinement of mechanistic hypotheses to provide a more complete understanding of reactive oxygen intermediate chemistry in CYP enzymes with important implications for human health.

? DESCRIPTION (provided by applicant): There is little doubt that cytochrome P450 CYP3A4 is the single most important protein in human xenobiotic metabolism. The prominence of CYP3A4 in drug metabolism results in routine investigation of the activity of thousands of molecules annually as substrates for this enzyme. Numerous computational methods have been developed to predict CYP3A4 metabolism. Ligand-based methods have considered structure-activity relationships for a baffling array of potential substrates, with a complex set of rather unreliable predictions. The key problem is that the conformational flexibility, or plasticity, of CYP3A4 has not been available and hence, has not been factored into these predictions. The long-term goal of this research program is to develop and apply novel approaches that dramatically expand our understanding of the P450 enzyme mechanisms. The objective of this application is to combine state-of-the-art tools of computational chemistry, chemical biology, and molecular spectroscopy to gain insight into the coupling of heme reactivity and protein dynamics, and the influence of interactions with redox partners with the adaptation of CYP3A4 to ligands. To meet this objective, there are three Specific Aims, we will: 1) Delineate the range of CYP3A4 conformational states in solution and the transition pathways between these conformers. Molecular dynamics methods capable of sampling low frequency protein motions will afford a description of CYP3A4 solution conformations, that to date, have been elusive by other means. 2) Define the functional consequences of ligand and redox partner interactions on CYP3A4 heme dynamics. Resonance Raman spectroscopy will be used to probe the conformational shifts and electronic structure changes resulting from interactions between CYP3A4, cytochrome P450 reductase, and cytochrome b5 that pre-organize that active site to facilitate electron transfer. 3) Map changes in CYP3A4 electrostatics by selective incorporation of Raman-active vibrational probes. The selective incorporation of amino acid analogs with vibrational probes will permit direct observation of local electrostatic changes through solvatochromic shifts induced by ligand binding, protein-protein interactions with redox partners, and resultant conformational interchanges. To afford accurate metabolic predictions for CYP3A4 metabolism, the conformational plasticity and the interactions between conformer and heme dynamics must be understood. We propose to approach these holes in our current understanding using a suite of interactive experimental designs. The significance of this set of studies is the promise of a clearer insight into ligand- and redox-partner induced changes in P450 dynamics and heme reactivity and the impact of these heretofore understudied factors in the adaptation of CYP3A4 to new substrate structures.

The goal of this conference is to bring an interdisciplinary group of scientists from around the world whose research is focused on cytochromes P450 to provide networking opportunities, updates on cutting-edge technologies, opportunities for new collaborations, and to engage young scientists. These enzymes are responsible for a dazzling array of transformations in the disposition of xenobiotics and biosyntheses of hormones. Over 70% of drugs are metabolized by CYPs; hence tremendous resources are devoted to identification of substrates and inhibitors in drug development to avoid later attrition, or adverse drug interactions following introduction into the clinic. Accordingly, CYP research has positively impacted the fields of xenobiotic metabolism, endocrinology, toxicology, nutrition, and biotechnology. The ICCP450 series is the premiere meeting in the field, is small enough (~200-250 delegates) to facilitate interactions between delegates, but large enough to attract prominent speakers. The conference was last held in the United States in 2013 on the campus of the University of Washington in Seattle. ICCP450 2021 will be held at the Hyatt Regency Crystal City Hotel in Arlington, Virginia near Washington, D. C. from June 13-17, 2021. Nearby airports offer convenient access for both international and U. S. delegates. ICCP450 2021 will continue to focus on its traditional areas of strength, including advancements structural biology, novel biophysical methods, xenobiotic/endobiotic metabolism, and genetic control of P450 expression. However, additional goals of this meeting are to strengthen contributions from expert investigators that have adopted the powerful tools of metabolomics, synthetic biology, and high-performance computation. The meeting format facilitates interactions between trainees, junior and established investigators with representation from women and underrepresented minorities. The meeting will consist of a keynote lecture on Sunday (6/13) followed by twelve plenary lectures and more than 80 invited talks (6/14-17). There will be two posters sessions for scientists to present their work and participate in informal discussions. Some of the poster abstracts will be selected for short oral presentations. Here we request funds to support student travel awards (registration and lodging), plenary speaker registration, and production of electronic/printed materials.

The cytochromes P450 (CYPs) are responsible for a dazzling array of transformations in the disposition of xenobiotics and biosyntheses of hormones. Over 70% of drugs are metabolized by CYPs; hence tremendous resources are devoted to identification of substrates and inhibitors in drug development to avoid later attrition, or adverse drug interactions following introduction into the clinic. Among these, CYPs involved in steroid hormone biosyntheses are among the key targets for the pharmacotherapy of endocrine disorders and cancers. The range of catalytic selectivity and the breadth of substrates transformed by CYPs are immense; however the interplay of protein dynamics, ligand binding, and catalysis used to achieve this flexibility remains poorly understood. Within this void, nothing is known about how these are impacted by their physiological context. We have adopted human aromatase (CYP19A1), responsible for estrogen biosynthesis from androgens, as a model to address these longstanding and important questions. Phylogenetic analysis supports that CYP19A1 is among the most primordial human CYPs and it is functionally representative of those steroidogenic enzymes catalyzing sequential transformations. The translational relevance of CYP19A1 cannot be underestimated since it has vital role in maintaining numerous tissues and has demonstrated immense value as a pharmacotherapy target for gynecological disorders, cancers and infertility. There are three Specific Aims to test our hypotheses, we will: 1) Map pathways for 19A1 ligand entry, product egress, and delineate the structural underpinnings of selectivity. The working hypotheses are that i) substrates, intermediate products, and inhibitors give rise to unique equilibrium dynamics and ii) that selectivity is conferred by transient bound states as ligands enter and exit the enzyme through distinct channels. 2) Delineate the impact of membrane complexity on catalysis, ligand binding kinetics and global dynamics in 19A1. The working hypotheses are i) Selectivity is dominated by kinetics involving multiple protein states and conformational transitions, ii) catalytic and inhibitory 19A1 complexes display distinct dynamics, and iii) both protein dynamics and binding kinetics are impacted by membrane properties. 3) Determine the architecture of 19A1-NDs in variable environments by small-angle x-ray and neutron scattering. The working hypothesis is that the insertion depth and orientation of 19A1 are determined by the surface and bulk properties of the bilayer. These Aims are technically- independent, but convergent approaches that are highly synergistic in combination, and will not only allow novel insight into CYP19A1, but also unveil paradigms that will transform our understanding of CYP function.