2003 — 2007 |
Du Bois, Justin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Reaction Design For the Synthesis of Neuroactive Agents
DESCRIPTION (provided by applicant): This proposal is guided by an overarching interest in the chemistry of nitrogen-containing compounds that display selective activity as neurochemicals. The guanidinum toxins, tetrodotoxin and saxitoxin, represent two such targets, each having a unique molecular architecture and remarkable potency to block ion passage through voltage-gated Na+ channels. Accordingly, both compounds have been invaluable tools in neurophysiology and ion channel research. Efforts to interrogate the structure and mechanism of these large (260-280 kDa) and complex transmembrane channel proteins would be advanced greatly with access to labeled and analogue structures of the natural products. Synthesis of either toxin, or variants thereof, is made particularly challenging, however, because of the elaborate and dense positioning of functional groups within these molecules. Thus, new chemical strategies have been devised to help reduce the difficulties associated with synthesizing structures of this type. Methodology for the selective conversion of saturated C-H bonds to carbinolamine centers through metal-catalyzed C-H insertion underlies our approach. The prevalence of amine functional groups in natural products and pharmaceuticals makes this chemistry broadly applicable to problems encountered in both academic and industrial research. The amination reaction can be performed with any number of starting materials possessing secondary, tertiary, allylic, benzylic C-H bonds, and may be used to construct 1,2-amino alcohols or 1,3-difunctionalized amine derivatives from simple alcohol precursors. In addition, the process is stereospecific, thereby enabling the assembly of enantiopure tetrasubstituted amine groups from substrates possessing optically defined tertiary centers. Insertion products can be manipulated through selective nucleophilic addition reactions to myriad amine derivatives. For our purposes, these types of reactions will expedite the preparation of tetrodotoxin and saxitoxin as well as select toxin analogues. Such compounds will be used to examine the molecular workings of the Na+ channel with a focus on understanding the ion selectivity region of the channel pore. Collectively, these studies are stimulated by the essential role of ion channel proteins for electrical signaling in cells and their widely recognized importance as targets for treating central nervous system-related disorders.
|
1 |
2008 — 2011 |
Du Bois, Justin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Guanidinium Toxins as Tools For Sodium Ion Channel Study
DESCRIPTION (provided by applicant): The overarching aim of this program is to develop small molecule tools for understanding ion channel protein function associated with the highly complex ionic mechanisms of electrical transmission in neuronal cells. Naturally occurring guanidinium poisons - tetrodotoxin, saxitoxin, gonyautoxin 2/3, and zetekitoxin AB - form the bedrock of these investigations. Despite evident differences in molecular size and topology, all four molecules are exquisitely potent blockers of voltage-gated sodium ion channels (NaV) that operate by occluding the extracellular mouth of the ion conductance pore (Site I). Studies of NaV structure, of which there exist ten mammalian isoforms, and function have been advanced with the availability from natural sources of tetrodotoxin, saxitoxin, and small number of structurally related forms. In the absence of crystallographic data, molecules such as gonyautoxin 2/3, zetekitoxin AB, and designed saxitoxin mimics in combination with protein mutagenesis experiments would enable current homology models of the channel pore to be challenged and refined. Knowledge accrued from these types of studies could lead to new chemical agents patterned after the guanidinium toxins that demonstrate NaV subtype specific activity. Such tools are desirable for mapping the spatial and temporal distribution of specific channel isoforms in developing or injured neurons. As NaV channels are considered lead actors in mechanisms for inflammation and neuropathic pain response, drugs that act on specific channel subtypes could represent next-generation therapies for the treatment of such ailments. PUBLIC HEALTH RELEVANCE: We are interested in understanding at a molecular level how nerve cells conduct electricity and how the process of electrical signaling is affected when a nerve is injured. Chemical synthesis is the engine that drives our program and will make possible the preparation of selective reagents that can be used to investigate these complex biological phenomena. Results from these studies could help guide the development of new therapies for the treatment of acute and/or chronic pain.
|
1 |
2010 — 2011 |
Du Bois, Justin |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Saxitoxin-Antibody Conjugates as Tools For Na+ Ion Channel Study and Therapeutics
DESCRIPTION (provided by applicant): Opioid analgesics, such as morphine, hydromorphone and fentanyl, are broadly prescribed for the management of acute, post-operative and chronic pain. Despite this widespread clinical use, a number of side effects persist including drowsiness, confusion, nausea, hyperalgesia and respiratory depression. Opioids are also highly addictive, and considered drugs of abuse by the NIDA. We wish to develop new pharmacological tools for interrogating specific biochemical mechanisms that underlie pain sensation with the longer-term goal of revealing next-generation therapeutics for pain treatment. Voltage-gated Na+ ion channels are integral membrane proteins responsible for electrical communication between cells. Ten mammalian genes have been sequenced that encode for ten different channel isoforms (NaV1.1- 1.9 and NaX), each having unique biophysical characteristics, and cellular and tissue distribution patterns. Drugs that inhibit NaVs non-specifically (e.g., lidocaine) find application as short-lasting, local anesthetics, but are less than desirable for any type of systemic or chronic use. A compelling body of evidence, however, suggests that specific inhibition of a single NaV isoform could reduce pain sensitivity without the accompanying side effects (numbness, ataxia) associated with local anesthetic treatments (and without chance of addiction, as noted with opioids). Similarities in the macromolecular structures of the nine NaV isoforms have thwarted most efforts to develop drugs that function as antagonist against only a single channel subtype. Our approach will capitalize on the highly specific binding of a monoclonal antibody engineered to target a single NaV isoform. We envision utilizing antibodies raised against NaV1.7, a channel isoform of particular interest as a target for pain treatment. Ion conduction will be inhibited by covalently linking to this antibody a potent, small molecule channel antagonist. Saxitoxin is a low molecular weight, naturally occurring product that acts with nanomolar potency on NaV1.1-1.4, 1.6, and 1.7 by lodging in the outer mouth of the channel pore. Strategies will be developed for conjugating modified forms of (+)-saxitoxin to the antibody and for testing the efficacy of these agents as isoform-specific blockers of NaV function. The success of this program will provide: 1) a tool that can be used to validate NaV1.7 as a target for pain treatment;2) a novel therapeutic lead in the form of an antibody-small molecule conjugate;and 3) a blueprint for preparing specific inhibitors of other NaV isoforms. PUBLIC HEALTH RELEVANCE: Opioid analgesics, such as morphine, cause a range of side effects and are subject to abuse, yet remain the most frequently prescribed drugs for the treatment of pain. We wish to develop new pharmacological tools that act by intervening with specific pain-producing signals in order to gain a deeper understanding of the etiology of pain. Results from these studies could help guide the development of next-generation therapies for pain management.
|
1 |
2012 |
Du Bois, Justin |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Guanidinium Toxins as Tools For Ion Channel Study
DESCRIPTION (provided by applicant): Proper neuronal function relies on the tightly regulated expression and discrete localization of voltage-gated sodium ion channels (NaVs), large protein complexes that control the movement of ions across cell membranes. A desire to better understand the role of NaVs in axonal plasticity and signal conduction, and the relationship between their disregulation and specific human pathologies motivates the development of high precision methods for their study in living systems. Real-time investigations of NaVs in live neuronal cells, however, are limited by the lack of available methods with which to modulate the function of individual NaV subtypes and to 'mark' their cellular distribution. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins - saxitoxin, gonyautoxin, and zetekitoxin AB. These agents function as molecular 'corks' to occlude the extracellular mouth of the ion conductance pore. De novo chemical synthesis makes available modified forms of these toxins, which we will use in combination with protein mutagenesis and electrophysiology to gain insights into the three-dimensional structure of the toxin binding site. Such information is needed to advance a NaV homology model that we have constructed, and will empower the rational design of toxin derivatives that show selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informing the development of new fluorescent imaging and affinity-based tools for investigating dynamic events associated with NaV function. We are motivated to understand how modulation of NaV expression influences the input-output responsiveness of neuronal cells (i.e., cellular plasticity) Toxin conjugates will be employed in initial experiments to measure channel synthesis and turnover rates, and ultimately to analyze quantitatively the extent to which these kinetic data vary as a function of nerve cell stimulation and nerve cell injury. The temporal control afforded by small molecule agents and the minimally invasive nature of such probes offer significant advantages over biological methods for labeling endogenous NaV channels. As such, the availability of toxin derivatives for NaV imaging studies should offer unprecedented insight into the dynamic role of these channel proteins in electrogenesis.
|
1 |
2016 — 2019 |
Du Bois, Justin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Guanidinium Toxins as Molecular Probes For Nav Study
PROJECT SUMMARY Proper neuronal function relies on the tightly regulated expression and discrete localization of voltage-gated sodium ion channels (NaVs), large protein complexes that control the movement of ions across cell membranes. A desire to better understand the role of NaVs in electrical signal conduction and the relationship between channel disregulation and specific human pathologies motivates the development of high precision reagents for their study in living systems. Real-time investigations of NaVs in live neuronal cells, however, are limited by the lack of available methods with which to modulate the function of individual NaV subtypes and to mark their cellular distributions and membrane expression levels. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins ? saxitoxin, gonyautoxins, and zetekitoxin. These agents function as molecular `corks' to occlude the extracellular mouth of the ion conductance pore. De novo chemical synthesis makes available modified forms of these toxins, which we will use in combination with protein mutagenesis and electrophysiology to gain insights into the three- dimensional structure of the toxin binding site. Such information is needed to advance a high fidelity NaV homology model, and will empower the rational design of toxin derivatives that display selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informing the development of new fluorescent imaging and affinity-based tools, which will be utilized to explore dynamic events associated with NaV function. We wish to understand how modulation of NaV membrane expression and post-translational protein modifications influence the input-output responsiveness of neuronal cells following nerve injury. Toxin-derived fluorescent probes will be prepared and used to measure the spatial distributions and concentrations of membrane-inserted NaVs in live cells. These investigations will provide a quantitative analysis of how NaV structure (i.e., post- translational modification), ion gating, membrane distribution, and protein turnover rates are altered in neuronal cell injury models. In addition, our experimental design will allow us to assess the influence of investigational drugs, protein factors, and/or other small molecules on regulating NaV trafficking and restoring proper neuronal signaling. Ultimately, this work could lead to the identification of new therapeutic targets or lead compounds for pain treatment.
|
1 |
2020 — 2021 |
Du Bois, Justin Huguenard, John R (co-PI) [⬀] Maduke, Merritt C [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Small-Molecule Probes For Study of Clc-2 Chloride-Channel Function in the Central Nervous System
The CLC chloride channel family is a class of membrane proteins that controls the flux of chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in mammalian tissue and function in diverse physiological roles, ranging from electrical excitation of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage-dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain has been known for decades, the role of this CLC homolog in neuronal signaling and proper brain function remains poorly understood, in part due to the absence of potent and selective small-molecule tools that enable studies of the molecular physiology of this channel. A recent breakthrough in our laboratories now opens the door to developing small molecule tools specific to CLC-2. Through a compound-library screen, we identified 'hit' compounds that inhibit CLC-2 activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2, we will develop novel probes, including small-molecule activators and fluorescent imaging probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to query the role of CLC-2 in excitatory synaptic transmission and network excitability in the thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy. Our team's combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function (Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions us to advance this research program.
|
1 |