Heike Wulff - US grants

Cerebellar ataxia is a lethal neurological disease, which afflicts about 150,000 people in the US. There are currently no known preventive, neuroprotective or symptomatic treatments for this devastating disease. Using a transgenic mouse model we recently identified a novel mechanism of initiation of cerebellar ataxia through hyperexcitability of the deep cerebellar neurons (DCN), the sole output pathway of the cerebellum. Small conductance Ca -activated K+ (SK) channels, key regulators of firing frequency in the DCN, were silenced in DCN neurons of Tg mice with a naturally occurring dominant-inhibitory SK isoform (SK3-1B) that suppresses the entire SK channel family. Tg mice developed severe cerebellar ataxia by the 12th day of life characterized by motor incoordination, intention tremor and gait abnormalities in the absence of neurodegeneration. This model, together with findings from other animal models for cerebellar ataxia, strongly suggests that increased DCN excitability may be an important step in the causation of this disorder. Pharmacological reduction of DCN excitability may provide a novel therapeutic approach for cerebellar ataxia. Since SK channels are critical in regulating the firing frequency of DCN neurons and their blockade causes enhanced firing, an opener of SK channels should slow down DCN firing and ameliorate the symptoms of cerebellar ataxia. Riluzole, a FDA approved drug for the therapy of amyotrophic lateral sclerosis, has been reported to be a potent SK channel opener. In a preliminary study in a Tg mouse model for human spinocerebellar ataxia type 2 (SCA2), we found that riluzole produced a dramatic improvement in motor performance after only 4 days of treatment. We plan to extend these exciting preliminary findings by pursuing three specific aims: Aim 1: Evaluation of riluzole in two animal models of ataxia; Aim 2: Design of a more potent and selective SK opener that unlike riluzole does not block slowly- inactivating sodium channels; Aim 3: Evaluation of our new SK opener in two animal models of ataxia. Taken together these important proof-of-concept studies will help to determine whether SK channel openers constitute a new therapeutic approach to improve motor performance in dominant cerebellar ataxias.

[unreadable] DESCRIPTION (provided by applicant): Specific K+ channel modulation offers an enormous potential for the development of new drugs. 1 channel that constitutes an especially promising therapeutic target is the voltage-gated Kv1.3 channel. Homomeric Kv1.3 channels are found in T and B lymphocytes and their expression is up-regulated in terminally differentiated effecter memory T (TEM) cells and class-switched memory B cells suggesting that Kv1.3 blockers should be useful for the treatment of autoimmune diseases such as multiple sclerosis, type-1 diabetes, psoriasis, contact dermatitis, rheumatoid arthritis and myasthenia gravis. This concept has been validated by the demonstration that autoreactive T cells from patients with multiple sclerosis and type-1 diabetes are predominantly Kv1.3-high TEM cells and that the Kv1.3 blocking peptide ShK can treat an animal model of multiple sclerosis. However, despite Kv1.3's obvious therapeutic importance, the pharmaceutical industry has so far been unsuccessful in developing selective and potent small molecule Kv1.3 blockers. With the alkoxypsoralen PAP-1 my laboratory recently identified the first small molecule inhibitor of Kv1.3 that blocks the channel with an EC50 of 2 nM and displays selectivity over the cardiac potassium channel Kv1.5. PAP-1 does not exhibit cytotoxic or phototoxic effects, is negative in the Ames test, potently inhibits the proliferation of human TEM cells and suppresses delayed type hypersensitivity (DTH), a TEM cell mediated reaction, in rats when administered intraperitoneally or orally. PAP-1 therefore, seems to constitute an excellent new tool to further explore Kv1.3 as a target for immunosuppression and could potentially be developed into orally available immunomodulator. With the help of this proposal we intend to thoroughly explore the therapeutic potential of PAP-1. Under Aim-1, we will determine PAP-1's pharmacokinetics and test which effect long-term suppression of memory cells with PAP-1 has on the immune system. Under Aim 2, we will test whether PAP-1 treats allergic contact dermatitis, an animal model for CD8+ T cell mediated skin reactions like psoriasis, and experimental autoimmune myasthenia gravis, a model for the T-cell dependent antibody-mediated autoimmune disease myasthenia gravis. Since TEM cells also play an important role in early and late-stage transplant rejection, we will further test whether PAP-1 can suppress acute and chronic rejection in a rat kidney transplant model (Aim 3). Lay: Potassium channels are proteins that tunnel the cell membrane and conduct potassium ions. 1 of these channels, called Kv1.3, is expressed in white blood cells and has been proposed as a potential new therapeutic target for the treatment of autoimmune diseases. The aims of our proposal are to test a Kv1.3 blocker that we designed in animal models of autoimmune diseases and transplant rejection. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that affects approximately 0.4 million people in the US and more than 2 million worldwide. Memory T cells specific for components of the myelin sheath appear to be crucial for the pathogenesis of MS. With the voltage-gated potassium channel Kv1.3 we have recently identified an exciting new molecular target that allows selective pharmacological suppression of effector memory T (TEM) cells. Expression of this channel is increased in both CD4+ and CD8+ TEM cells and Kv1.3 blockers have been shown to potently suppress their proliferation without impairing the function of naive and central memory T cells. We have further validated Kv1.3 as a new therapeutic target for MS by demonstrating that myelin-reactive T cells from patients with MS are predominantly Kv1.3-high TEM cells and that the Kv1.3 blocking peptide ShK can treat EAE in rats. Despite Kv1.3's obvious therapeutic importance, specific and potent small molecule inhibitors of Kv1.3 have so far not been developed by the pharmaceutical industry. Following up on anecdotal reports from the late 1980s that tea prepared from Ruta graveolens, the common rue, had beneficial effects in MS, we discovered that Ruta contained a small molecule called 5-methoxypsoralen (5-MOP), which blocks Kv1.3 in the micromolar range. Using 5-MOP as a template we started a medicinal chemistry effort and have since then improved the potency of the alkoxypsoralens into the low nanomolar range and proposed an essential pharmacophore for small molecule Kv1.3 blockers. Our most potent compound so far is PAP-1, which inhibits Kv1.3 with a Kd of 2 nM and displays 22-fold selectivity over the cardiac K+ channel Kv1.5. Under Aim-1 of this proposal we intend to further explore the structure-activity relationship (SAR) around our pharmacophore model in order to further improve the potency and selectivity of our compounds. Under Aim-2 we will then test the best new compounds in adoptive-transfer EAE, an animal model of MS. The overall goal of this proposal is to obtain an ideal small molecule Kv1.3 blocker for pre-clinical development as a new drug candidate for MS. Lay: In multiple sclerosis the immune system attacks proteins in the brain of afflicted patients. The aim of this study is to identify and test new drugs that can suppress the white blood cells that are invading the brain.

DESCRIPTION (provided by applicant): Sodium-activated potassium (KNa) channels are widely expressed throughout the central nervous system. Activation of these channels is known to protect cells from hypoxic injury. The molecular correlate of KNa currents, however, was unknown until the genes underlying this new family of K+ channels were cloned relatively recently. Slack (Sequence like a calcium-activated K channel) and Slick, which are also referred to as Slo2.2 (KCa4.1) and Slo2.1 (KCa4.2), currently have no pharmacological tools that allow for modulation of their function. With the help of this grant we are therefore proposing to design potent and brain-penetrant Slack channel activators that could be used to explore the therapeutic potential of these interesting channels. In normal neurons, KNa channels contribute to the slow afterhyperpolarizations that follows repetitive firing, regulate rates of bursting and enhance the accuracy with which action potentials lock to incoming stimuli. Evidence further indicates that KNa channels play a crucial role in protecting cells from injury under ischemic conditions, when inhibition of the plasma membrane Na+-K+-ATPase by the lack of oxygen leads to an increase in intracellular sodium levels. Activation of KNa channels under these circumstances is likely to prevent calcium entry by stabilizing the membrane potential and protecting neurons from overloading with calcium. In proof of this concept, mutation of the ortholog of Slack in the nematode C. elegans renders these animals hypersensitive to hypoxia indicating that KNa channels provide endogenous protection against hypoxia in this species. Compounds that increase the activity of KNa channel therefore should be therapeutically useful for the treatment of stroke and the prevention of the effects of global cerebral ischemia as occurs, for example, in cerebral palsy. By increasing the slow afterhyperpolarizations, KNa channel activators may also be useful for reducing neuronal excitability in epilepsy and ataxia. By screening various pharmacophores known to activate the related large-conductance Ca2+-activated K+ channel BK (Slo1, Maxi-K) it was recently discovered that biphenylthioles and 4-arylquinolinones activate Slack channels in the low micromolar range. Interestingly, two compounds in the 4-arylquinolone series were found to increase Slack activity without exerting effects on BK channels demonstrating that it is possible to separate the two activities. By combining i) classical medicinal chemistry, ii) a recently developed high- throughput assay measuring mass redistribution at the plasma membrane to determine Slack activation, iii) electrophysiology and iv) pharmacokinetic experiments in rats we here propose to improve the potency, selectivity and brain-penetration of our leads. Our overall goal is to provide the scientific community with a Slack channel activator that is suitable for in vivo use. PUBLIC HEALTH RELEVANCE: Based on their abundant expression in the brain sodium-activated potassium (KNa) channels potentially constitute novel drug targets for the treatment of stroke, cerebral palsy, epilepsy and ataxia. However, these important channels currently have no pharmacological modulators. With the help of this grant we will attempt to design small molecule KNa channel activators that could be used as scientific tool compounds to test whether KNa channels indeed constitute novel targets for neurological diseases.

DESCRIPTION (provided by applicant): Small-conductance calcium activated potassium channels are encoded by the KCa2.1-2.3 (= SK1-3) genes and are best known for underlying the apamin-sensitive medium afterhyperpolarization current (mAHP) in neurons. Depending on the type of neuron, the function of KCa2 channels varies from determining instantaneous firing rates, over setting tonic firing frequencies, to regulating burst firing and potentially catecholamine release. Pharmacological modulation of KCa channels therefore offers the opportunity to significantly affect neuronal excitability. While KCa2 channel blockers like the bee venom apamin increase firing rates and induce seizures in rodents, KCa2 channel activators slow down neuronal firing and have therefore been proposed for the treatment of CNS disorders that are characterized by hyperexcitability such as epilepsy, ataxia, and neuropathic pain. However, this compelling therapeutic hypothesis currently remains largely untested because none of the existing KCa2 channel activators such as EBIO (EC50 300 μM) or NS309 are suitable for in vivo use. Using the neuroprotective drug riluzole as a synthetic template, our laboratory recently designed SKA-31 (EC50 2 uM), the first KCa2 channel activator, which is potent enough to be used in vivo, and demonstrated in collaboration with the NIH Anticonvulsant Screening Program (ASP) that the compound and several of its derivatives are effective anticonvulsants. Unfortunately, SKA-31 also activates KCa3.1 channels, which are expressed on vascular endothelium, and thus reduces blood pressure in mice. Using a combination of classical medicinal chemistry and automated and manual electrophysiology we intend to further explore the structure activity relationship around SKA 31 and EBIO in order to improve selectivity for KCa2 over KCa3.1 as well as potency and brain penetration. The best new KCa2 activators will then be evaluated for selectivity over a panel of cloned ion channels and characterized for activity on native KCa2 channels using hippocampal slices. Compounds selectively activating cloned and native KCa2 channels will further be evaluated for pharmacokinetic properties and brain penetration in rats using HPLC/MS. In parallel, we will submit selected compounds to the ASP, where the compounds will we tested in acute seizure models. Promising compounds will then be tested in amygdala kindled mice and rats with kainate-induced epilepsy, two models that are more representative of human refractory epilepsy. The design of brain penetrant and potentially subtype selective KCa2 channel activators would help to validate KCa2 channels as novel pharmacological targets for the treatment of epilepsy and would further provide the scientific community with tool compounds to study the role of KCa2 channels in ataxia, neuropathic pain and cognition. PUBLIC HEALTH RELEVANCE: Project Narrative KCa2 potassium channels play important roles in determining neuronal excitability. Activators of these channels have therefore been suggested as new therapeutics for the treatment of diseases that are characterized by neuronal hyperexcitability such as epilepsy and ataxia. With the help of this grant we will attempt to design a KCa2 channel activator that is potent and selective enough to be used as a scientific tool compound or even to be developed into a drug.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) afflicts approximately 25 million people worldwide and is the most common cause of dementia in the elderly. There is an urgent need for new therapeutic target discovery and corresponding new compound development. A protein deposited in AD brains called amyloid-¿ (A¿) has been hypothesized to play a critical role in AD pathogenesis. Ab can activate microglia to clear A¿ but at the same time releasing cytotoxic substances to cause neuronal damage. Recently it was found that the voltage-gated potassium channel Kv1.3 (KCNA3) plays an important role in microglia activation. Therefore we intend to study if Kv1.3 plays a role in microglia activation, neurotoxicity, and amyloid deposition in AD. We found in our in vitro and in situ experiments that the selective Kv1.3 blocker PAP-1 blocked the neurotoxicity induced by A¿- activated microglia, but did not block the beneficial effect of microglia to phagocytose A¿. We also found strong Kv1.3 immunoreactivities in microglia associated with amyloid plaques in two AD mouse models. Our results suggest the involvement of Kv1.3 in microglia activation and neurotoxicity in AD. With the help of this grant we now wish to obtain in vivo proof of principle that Kv1.3 could be a therapeutic target and its specific inhibitors may have a therapeutic potential for AD. We will determine: 1. Do microglia activation and associated neuronal damage in response to pro-inflammatory stimuli in vivo require microglial Kv1.3 channel activity? To answer this question we will determine if blockade of Kv1.3 activity reduces microglial activation and dendritic degeneration in mice after treatment with lipopolysscharides or Ab oligomers. We will also examine the microglial Kv1.3 activity and its influence on the microglial activation state in response to these pro-inflammatory stimuli. 2. Can a selective Kv1.3 blocker called PAP-1 inhibit microglia activation in vivo and improve cognitive function of an AD mouse model called 3xTg-AD mice? To address this question, we will perform a therapeutic trial by treating 3xTg-AD mice with PAP-1 and determine the effect of PAP-1 on microglia activation state, amyloid deposition, tau pathology, and cognitive performance. Lay: Microglia, a type of white blood cells found in the brain, have been shown to contribute to the pathogenesis of Alzheimer's disease. The aims of our proposal are to test whether a potassium channel called Kv1.3 is important in microglia-caused damage. We will further test whether an inhibitor for Kv1.3 reduces microglia activity in an animal model of Alzheimer's disease.

DESCRIPTION (provided by applicant): In addition to directly causing neuronal damage ischemic stroke elicits a delayed neuroinflammatory response that is characterized by lymphocyte infiltration, hyperthermia and robust microglia activation. Reactive microglia in particular contribute to this secondary damage by producing inflammatory cytokines, reactive oxygen species, NO, and cyclooxygenase-2 reaction products. However, activated microglia might also be neuroprotective by releasing neurotrophic factors and phagocytosing cellular debris. The goal of microgliatargeted therapies therefore should be to reduce the neurotoxic effects of activated microglia while at the same time maintaining their beneficial functions. We here hypothesize, that blockers of the microglial K+ channels Kv1.3 and KCa3.1 might be able to do exactly this based on the preliminary data presented in this application. We previously designed potent and selective small molecule inhibitors for both channels, PAP-1 for Kv1.3 and TRAM-34 for KCa3.1, and demonstrated that these compounds can prevent or treat various autoimmune diseases and inflammatory conditions in rodents such as contact dermatitis, type-1 diabetes, inflammatory bowel disease, atherosclerosis and EAE. More recently we made the exciting observation that our KCa3.1 blocker TRAM-34 reduces infarct area and neurological deficit scores following ischemic stroke in rats even if treatment is commenced 12 hours after reperfusion. Another strong rationale for our study is a report that TRAM-34 does not prevent microglia from phagocytosing damaged neurons but increases the number of surviving retinal ganglion cells following optic nerve transection in rats by reducing the production and/or secretion of neurotoxic molecules in the retina. Taken together with previous work from our laboratory and other groups implicating both Kv1.3 and KCa3.1 in microglia mediated neuronal killing, these results suggest Kv1.3 and KCa3.1 as novel targets for CNS pathologies involving inflammation. With the help of this grant we therefore intend to test the hypothesis that both channels constitute novel targets for the treatment of stroke. Under Aim-1 we will more rigorously evaluate Kv1.3 and KCa3.1 as targets for stroke by testing the effect of both pharmacological blockade and genetic deletion in reperfusion MCAO and by performing parallel in vitro studies to investigate the role Kv1.3 and KCa3.1 in microglia functions. Under Aim-2 we will use our expertise in medicinal chemistry to design a less lipophilic and more brain penetrant small molecule Kv1.3 inhibitor than our existing lead compound PAP-1 (IC50 2 nM). We further will resynthesize a brain-penetrant KCa3.1 inhibitor, which was abandoned by Bayer, when the company pulled out of stroke research. Under Aim-3 we will directly compare the new Kv1.3 and KCa3.1 blockers to minocycline in a 4-week trial by assessing in vivo cytokine production, neurogenesis and functional recovery. As a first step towards translating our findings to humans, we will further obtain brain sections from stroke patients and controls and perform immunohistochemistry for KCa3.1, Kv1.3, and microglia activation markers.

Summary: The overall objective of Core A is to provide analytical support to Projects 1, 2, and 3 and the Probe and Pharmaceutical Optimization Core (Core B). Core A is an integral part of the center by providing advanced analytical support, training, and cutting edge analytical techniques for drug and biomarker detection. More specifically, Core A will develop methods for the detection of target compounds and their metabolites by LC-MS or GC-MS and provide QC analysis of standard solutions prior to their use in projects. Detailed rodent ADME studies for the anticonvulsants and neuroprotectants will be performed to assist the center projects in dose selection. Core A will work with Project 2 in the identification of biomarkers of seizure as a biochemical test of how therapeutic efficacy. Metabolomics techniques, both targeted and global will be employed. Targeted metabolomics will focus on both oxylipins and neurosteroids since levels in both pathways are altered after a seizure. Global metabolomics, as a broader approach, can identify biomarkers of seizure and therapy if needed. Current methods of detection of tetramethylenedisulfotetramine (TETs) are insensitive. TETs seems like an ideal candidate for an immunoassay, since it has several heteroatoms to provide recognition points for the antibody. An additional benefit of immunoassay is its potential to be packaged in a field deployable platform for on-site detection. When there is a clear need, immunoassays to other toxins and their metabolites will be created. Objective-1: Provide general analytical support using GC-MS or LC-MS for the detection of toxins or drugs and their metabolites in biological matrices and formulations. Objective-2: Determine the metabolomic profiles of brain tissue from Projects 1 and 2 as biomarkers of seizure damage for use in assessing neuroprotective efficacy of candidate therapeutics. Objective-3: Develop innovative immunoassay methods for detection of TETs in biological and environmental matrices.

A number of characteristics distinguish TETS and organophosphorus (OPs) cholinesterase inhibitors as credible chemical threat agents. 1) These agents can be easily manufactured on a large scale. 2) They are widely available in some countries even though banned in the US. 3) Exposure to either TETS or OPs dose dependently results in lethality or profound and sustained damage to the brain. In rodents, acute TETS or OP intoxication elicits delayed neuronal injury as evidenced by increased apoptosis and oxidative stress in the CNS that persists for up to several days following exposure. In humans, indivuduals that survive acute TETS or OP intoxication often experience significant brain damage. A limited number of therapeutic agents are available to prevent mortality induced by OP threat agents but these do not sufficiently protect against brain injury. Therapeutic approaches for TETS intoxication are less well known. To address these gaps, Core B will synthesize TETS and an inactive analog to be used as a negative control in mechanistic studies, as well as characterize and validate primary standard stocks of TETS, DFP and parathion. This will enable Projects 1-3 to advance applied therapeutic and mechanistic knowledge on these agents. Furthermore we will synthesize, characterize, test and optimize the pharmacokinetic (PK) properties and CNS penetration of two distinct classes of therapeutic agents, sEH inhibitors and KCa2 channel activators. Overall, the successful realization of the proposed aims in Core B is likely to improve therapeutic approaches for the treatment of acute OP and TETS intoxication by providing novel therapeutics for physicians and emergency first-responders to effectively intervene in cases of human intoxication with these seizurogenic chemical threat agents.

Ischemic stroke elicits a strong neuroinflammatory response characterized by massive microglia activation. However, microglia do not only cause damage by releasing pro-inflammatory cytokines and reactive oxygen species, they can also exert beneficial functions. Similar to macrophages, microglia can assume a classically activated (M1) or alternatively activated (M2) phenotype. While M2 microglia are presumably neuroprotective and anti-inflammatory and have been described to peak relatively early in rodent models of ischemic stroke, M1-polarized microglia begin to appear later in the infarct area, especially in the border zone, and expand neuronal injury. An effective anti-inflammatory treatment for stroke should therefore not be a general immunosuppressant but instead suppress microglia in a subtype specific manner by preferentially targeting pro-inflammatory M1 microglia. Our group has a long history of studying K+ channels in the immune system and previously developed small molecule inhibitors for the voltage-gated KV1.3 and the Ca2+-activated KCa3.1 channel as immunomodulators. We recently obtained exciting new data showing that M1 and M2 microglia significantly differ in their K+ channel expression profiles and here propose to test whether KV1.3 blockers can preferentially inhibit M1 microglia functions and preserve beneficial M2 functions. We propose to test this therapeutic hypothesis with three interrelated Specific Aims: Under Aim-1 we will investigate the expression profile and the functional role of K+ channels in cultured M1 and M2 microglia and macrophages. In Aim-2 we will study microglia in a more ?natural environment? and use organotypic slices exposed to hypoxia/aglycemia or acute slices from Cx3cr1GFP/+ mice subjected to reversible middle cerebral artery occlusion (MCAO) to determine K+ channel expression and function using whole-cell patch-clamp, immunohistochemistry, qPCR and flow cytometry. As part of these experiments we will characterize the time courses of K+ channel and M1 and M2 marker expression and correlate them with brain cytokine profiles and pathology. Parallel immunohistochemical experiments will be performed on brain sections from stroke patients to evaluate K+ channel expression in the context of M1 and M2 markers in humans. Finally, in Aim-3 we are proposing to test our hypothesis that selective targeting of M1 microglia with KV1.3 blockers is beneficial in ischemic stroke by evaluating the effect of KV1.3 knockout and pharmacological blockade with our KV1.3 blocker PAP-1 in MCAO. These experiments will include studies where PAP-1 administration will match the time-course of the presence of KV1.3 on microglia in the infarct. Overall, we expect that KV1.3 blockade will spare beneficial microglia functions such as phagocytosis of debris and production of neurotrophic factors and preferentially target detrimental M1 microglia functions. This strategy could be very beneficial for ischemic stroke but could also be applied to other neuroinflammatory brain disorders, where dynamic M1/M2 activation of microglia is pathologically significant.

Ischemic stroke elicits a strong neuroinflammatory response characterized by massive microglia activation. However, microglia do not only cause damage by releasing pro-inflammatory cytokines and reactive oxygen species, they can also exert beneficial functions. Similar to macrophages, microglia can assume a classically activated (M1-like) or alternatively activated (M2-like) phenotype. While M2-like microglia are presumably neuroprotective and anti-inflammatory and have been described to peak relatively early in rodent models of ischemic stroke, M1-polarized microglia begin to appear later in the infarct area, especially in the border zone, and expand neuronal injury. An effective anti-inflammatory treatment for stroke should therefore not be a general immunosuppressant but instead suppress microglia in a subtype specific manner by preferentially targeting pro-inflammatory microglia. Our group has a long history of studying K+ channels in the immune system and previously developed small molecule inhibitors for the voltage-gated KV1.3 and the Ca2+-activated KCa3.1 channel as immunomodulators. We recently obtained exciting new data showing that M1 and M2 microglia significantly differ in their K+ channel expression profiles and here propose to test whether KV1.3 blockers can preferentially inhibit M1-like microglia functions and preserve beneficial M2-like functions. We propose to test this therapeutic hypothesis with three interrelated Specific Aims: Under Aim-1 we will investigate the expression profile and the functional role of K+ channels in cultured M1 and M2 microglia and macrophages. In Aim-2 we will study microglia in a more ?natural environment? and use organotypic slices exposed to hypoxia/aglycemia or acute slices from Cx3cr1GFP/+ mice subjected to reversible middle cerebral artery occlusion (MCAO) to determine K+ channel expression and function using whole-cell patch-clamp, immunohistochemistry, qPCR and flow cytometry. As part of these experiments we will characterize the time courses of K+ channel and M1 and M2 marker expression and correlate them with brain cytokine profiles and pathology. Parallel immunohistochemical experiments will be performed on brain sections from stroke patients to evaluate K+ channel expression in the context of M1 and M2 markers in humans. Finally, in Aim-3 we are proposing to test our hypothesis that selective targeting of M1-like microglia with KV1.3 blockers is beneficial in ischemic stroke by evaluating the effect of KV1.3 knockout and pharmacological blockade with our KV1.3 blocker PAP-1 in MCAO. These experiments will include studies where PAP-1 administration will match the time-course of the presence of KV1.3 on microglia in the infarct. Overall, we expect that KV1.3 blockade will spare beneficial microglia functions such as phagocytosis of debris and production of neurotrophic factors and preferentially target detrimental pro-inflammatory microglia functions. This strategy could be very beneficial for ischemic stroke but could also be applied to other neuroinflammatory brain disorders, where neuroinflammation is pathologically significant.

Project Summary ? Probe and Pharmaceutical Optimization Core (PPOC) ? Core B The overall goal of the UC Davis CounterACT Center of Excellence is to identify and advance improved medical countermeasures for stopping seizures and preventing long-term consequences resulting from acute intoxication with chemical threat agents, specifically organophosphate cholinesterase inhibitors like diisopropylfluorophosphate (DFP), paraoxon and soman, or GABAA receptor blockers like tetramethylene- disulfotetramine (TETS), picrotoxin and bicuculline. The role of Core B, the Probe and Pharmaceutical Optimization Core, is to promote the overall Center goal by providing general medicinal chemistry, formulation and pharmacology support. Core B will function as an integral component of the Center by supporting Projects 1, 2 and 3, and by closely collaborating with Core A, the Analytical Chemistry Core. Core B will synthesize the chemical threat agent tetramethylenedisulfotetramine (TETS) and specific mechanistic probes for the individual projects, including TETS-haptens and analogs for the development of a TETS detection assay in Core A. Core B will further use its medicinal chemistry expertise to synthesize and characterize potential novel therapeutics, including soluble epoxide hydrolase (sEH) inhibitors, dual sEH/cyclooxygenase-2 (COX-2) or sEH/phosphodiesterase inhibitors, activators of small-conductance calcium- activated potassium channels (KCa2), blockers of the microglial voltage-gated potassium channel Kv1.3, as well as AMPA, ryanodine or GABA receptor antagonists, as required by the projects. Having a core dedicated to probe and reagent design, synthesis and/or verification will help ensure consistency, efficacy and reproducibility across the projects of the UC Davis Center and the CounterACT program. In contrast to the first project period, where the emphasis was on delivering libraries of diverse compounds for in vitro screening, the emphasis now will be on optimizing previously identified leads and candidate therapeutics for bioavailability, half-life and central nervous system (CNS) penetration. Core B will further devote significant effort to performing rapid efficacy and safety screens of candidate therapeutics and therapeutic combinations to help inform compound-combinations and dose- selection for Projects 2 and 3.

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage- independent and share a common Ca2+/calmodulin mediated gating mechanism. Their lack of voltage- dependence enables KCa2/3 channels to remain open at negative membrane potentials and the channels therefore play important roles in physiological processes that require hyperpolarization. While the three KCa2 channels, KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3 (SK3) are best known for their role in neuronal afterhyperpolarization, KCa3.1 (IK) has mostly been studied in the immune system, vascular endothelium and in secretory epithelia, where the channel is involved in activation, proliferation and secretion processes through modulation of Ca2+ influx events. Small molecule KCa2/3 channel modulators constitute both useful chemical biology probes as well as potential novel drugs for the treatment of autoimmune diseases, hypertension, and various neurological disorders such as ataxia, epilepsy, and alcohol dependence. Our laboratory has been working on the pharmacology of KCa2/3 channels for many years. After we initially developed KCa3.1 blockers such as TRAM-34, we later discovered the mixed KCa2/3 activator SKA-31 and the KCa3.1 selective activators SKA-121 and SKA-111, which display 40- or 100-fold selectivity for KCa3.1 over KCa2 channels. All these compounds, which have been widely used to probe the physiological and pathophysiological functions of KCa channels, were designed using classical medicinal chemistry approaches without any structural input. However, using the recently solved crystal structures of the KCa2.2 calmodulin binding domain (CaM-BD) in complex with CaM from our consultant Miao Zhang, we generated Rosetta homology models of the KCa2.3 and KCa3.1 CaM-BD/CaM complexes and discovered that an extensive hydrogen bond network stabilizing SKA-121 in KCa3.1 is key to its KCa3.1 selectivity. Using this atomistic scale structural insight into KCa channel subtype selectivity we are now proposing to switch selectivity under Aim-1 and perform hypothesis-driven structure-assisted drug design of novel napthothiazole/oxazole-type KCa activators that make unique contacts with KCa2-specific residues using the Rosetta Ligand and the new RosettaDrug Design approach. After synthesizing and experimentally testing KCa channel potency and selectivity by electrophysiology, we intend to first confirm the binding mode by mutagenesis and then turn the new KCa2 activators into a useful pharmacological probe for the scientific community under Aim-2, where we will determine selectivity over other ion channels and evaluate pharmacokinetic properties and brain penetration. The innovation in our proposal is twofold: 1) This work will be one of the first attempts at hypothesis-driven structure based drug design for a small molecule ion channel modulator; 2) This work will provide the scientific community with KCa2 channel selective gating modulators which will be useful tools to explore the pathophysiological role of KCa2 channels and their suitability as therapeutic targets for epilepsy, ataxia, substance dependence and post-traumatic stress disorder.

Inhibition of ?-aminobutyric acid type A (GABAA) receptors (GABAAR) is the presumed mechanism of the seizure-inducing activity of the natural product picrotoxin (PTX), the rodenticide tetramethylenedisulfotetramine (TETS) and the high-energy explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). TETS, which has been banned from production worldwide, is still readily available on the black market, and is associated with thousands of human poisonings per year. It is incredibly stable in the environment. RDX is an environmental contaminant found in both groundwater and soil due to its worldwide military and civilian use, and it is an illicit abuse substance. PTX is of toxicological concern, because like TETS and RDX, it is listed as a credible threat agent by the United States Department of Homeland Security. All three compounds can cause seizures that rapidly progress to status epilepticus and death; however, TETS is by far the most potent with a lethal dose of 7 to 10 mg in humans and an LD50 of 0.1 mg/kg in rodents, which makes it roughly ~40x more potent than PTX and ~1000x more potent than RDX. There currently is no approved medical countermeasure for individuals acutely intoxicated with these convulsant chemicals. While some information is available regarding the molecular site of action and the GABAAR subtype selectivity of PTX, practically nothing is known about the molecular mechanism of action of TETS and RDX other than that they are most probably GABAAR inhibitors. We intend to use molecular modeling, whole-cell patch-clamp electrophysiology and gene knockdown techniques in zebrafish to identify the subunit specificity of TETS and RDX interactions with GABAA receptors and to determine whether it differs from that of picrotoxin. Treatment with subtype selective compounds/drugs should allow us to confirm which GABAA receptor subunit combinations are important for the seizure activity of TETS and RDX, and whether they differ from the receptor subunit profile that mediate PTX action. Detailed understanding of the molecular mechanism(s) of action of TETS and RDX will not only provide novel insight as to the biological reasons for the toxicologic differences between these agents, but will also be important for evaluating the validity of ?read across? risk assessment approaches for GABAA receptor antagonists, and for developing effective medical countermeasures for terminating SE in intoxicated individuals.

Abstract Recent studies have begun to uncover the central role of microglia-mediated neuroinflammation in Parkinson?s disease (PD) pathogenesis. Increasing evidence suggests that microglia-driven innate immunity could further potentiate deleterious ?-synuclein (?Syn) aggregation and progressive neurodegeneration. However, we lack an in-depth understanding of the cellular mechanisms regulating ?Syn-induced innate immunity. Therefore, identifying signaling mechanisms that regulate microglial function in response to Parkinsonian pathology may lead to the development of novel immunomodulatory therapies for PD. We recently discovered that the transcript and protein expression levels of the calcium-activated potassium channel KCa3.1, best known for its role in immune cell calcium signaling, are elevated in activated microglia in both postmortem PD brains and in preclinical models of PD. We further identified that disruption of either FYN or STAT1 dampens reactive microglia activation responses via modulation of inflammatory mediators in aggregated ?Syn (?Synagg)-stimulated primary microglia. Importantly, the highly selective and orally active KCa3.1 inhibitor Senicapoc reduced neuroinflammation and nigral dopamin(DA)ergic neurotoxicity in a preclinical mouse model of PD, suggesting that KCa3.1 plays a multifaceted role by governing disease pathology. Despite these encouraging findings, the exact cellular mechanisms by which KCa3.1 regulates microglial function in the context of synucleinopathy remain poorly characterized. Herein, we propose three integrated aims to test the central hypothesis that KCa3.1 promotes ?Synagg-mediated progressive nigral DAergic neurodegenerative processes via activation of the microglial Fyn- STAT1 signaling axis and that the in vivo inhibition of KCa3.1 restores microglial homeostasis and affords DAergic neuroprotection in the context of synucleinopathy. In Aim-1, we will test the hypothesis that upregulation of KCa3.1 induces the proinflammatory microglial activation phenotype and nigral DAergic neuronal loss in the context of synucleinopathy. In Aim-2, we will test the hypothesis that the Fyn-STAT1 signaling axis drives microglial responses to PD-like pathology in a KCa3.1-dependent manner. In Aim-3, we will test the hypothesis that inhibiting KCa3.1 activation is efficacious in reducing reactive microglial activation and progressive PD-like disease pathology. The proposed studies are innovative, utilizing a combination of transcriptomic profiling, RNA in situ hybridization (ISH), imaging analysis, the RT QuIC assay for ?Synagg seeding, CRISPR/Cas9 KCNN4 knockout (KO) mice, transgenic conditional KO mouse models, and electrophysiological recordings to test how microglial KCa3.1 influences progressive neurodegenerative processes in PD. These studies address key mechanistic aspects regarding the functional roles of KCa3.1 in PD pathogenesis and may aid in the identification of new molecular determinants that can be targeted for slowing or halting PD progression and/or repurposing Senicapoc for PD therapy. 1