Kathleen Seyb

Identification of Translational Activators of Glial Glutamate Transporter EAAT2 through Cell-Based High-Throughput Screening: An Approach to Prevent Excitotoxicity

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

This study identifies an unusual sulfur-based chemical as a novel and specific inhibitor of the tyrosine phosphatase STEP and shows that it can improve the cognitive function of a mouse model of Alzheimers disease.

Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.

Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2—Discovery of LRRK2 inhibitors

LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.

Structure–activity relationship study of EphB3 receptor tyrosine kinase inhibitors

A structure-activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure-activity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also profiled for inhibitory activity against a panel of 288 kinases and found to be quite selective for tyrosine kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and potentially in vivo kinase-dependent function of EphB3 receptor.

Identification of Small Molecule Inhibitors of β-Amyloid Cytotoxicity through a Cell-Based High-Throughput Screening Platform

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of β-amyloid (Aβ) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimers disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Aβ. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Aβ with IC50 values of <10 µM and showed little or no toxicity at concentrations up to 30 µM, none inhibit the calpain enzyme directly. (Journal of Biomolecular Screening 2008:870-878)

Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors.

PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the Km of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer–based assay that yielded a robust Z′ factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs.

INTRODUCTION This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. METHODS Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood-brain barrier permeability. RESULTS Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ⩽ 15 μM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 μM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 μM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 μM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. CONCLUSION LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis.

Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.