Progress Report for Neurotherapeutics Unit

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Within the NTDU group, we continue to utilize many automated, medium-throughput cell-based assays to screen small compound collections against human neural stem cells (NSC) and human NSC-derived neurons and astrocytes. We've utilized live cell imaging-based assays with human neurons, as well as developed fluorescence-based assays to probe high content imaging endpoints. These cell-based screening assays, in 384 well plate format, have been successfully employed in multiple screens with multiple investigators. We continue to produce and purify a number of recombinant proteins, such as HERV-K Envelope proteins and HIV Tat protein for our own studies and provide that Tat protein to extramural researchers. SINS (PI: Nath) With the Section of Infections of the Nervous System (SINS), we have continued to work on multiple research projects. We continue to characterize the neurotoxicity resulting from the expression of the HERV-K env protein in NSC and human neurons, and have already identified some compounds and proteins which attenuate HERVK env mediated neurotoxicity. Exogenously added recombinant HERV-K Env protein also elicits significant neurotoxicity, which is reduced by select small molecules and anti-HERV-K antibodies. Mechanism of action studies in HERV-K activation are continuing. We have initiated screening assays to determine how the expression of the human endogenous retrovirus is activated and which agents may drive that activation. Additional will continue to elucidate these pathways of activation, in order to prevent activation and expression of these toxic HERV-K proteins. The HIV Tat antagonist program has incorporated direct label-free binding assays, including protein thermal shift and Octet biolayer interferometry assays, as well as Tat-dependent selective LTR activation assays to identify new lead Tat inhibitors. The resultant active compounds continue to be characterized for potency and biopharmaceutical properties, as well as activity against HIV viral replication. Additional Tat antagonist identification and characterization has been completed by collaboration with NCATS scientists. Subsequent studies of these compounds will focus on their antiviral effects. The NTDU has continued multiple studies in collaboration with other intramural investigators: Sibley Laboratory: We continue to work with the Sibley laboratory to characterize the dopamine D3 receptor modulators that they have identified, using our live cell imaging platforms for quantitative analysis. This work includes working with C elegans models as well as iPSC derived neuronal cultures. Chittiboina Laboratory: We worked toward purification of protein targets that display elevated expression and activation in Cushings Disease. We have characterized the binding interaction of these two proteins. Yesh Banasavadi Siddegowda laboratory: We characterized a potential chemosensitizer for GBM and have screened a chemical library for compounds that may work synergistically to eliminate the GBM cells. Characterization of these active compounds is underway. Grunseich laboratory: We are setting up neurotoxicity assays with his motor neuron cultures from patient derived and normal control patients. The first step is to knock in the TdTomato reporter into the iPS cells to facilitate their quantitation. Laboratories outside NINDS: Simeonov NCATS We have been working with their group to screen compounds for inhibition of Tat mediated HIV LTR activation, evaluating and characterizing the active molecules in confirmatory assays. This work has continued and potential Tat antagonists are being characterized. Additional binding studies are being conducted to complete their biological characterization. Dr. Peng Loh laboratory NICHD: We utilized the Epic BT to determine the binding affinities of a series of compounds to the serotonin receptor 5HTR1E. The binding of serotonin, the prototypic ligand carboxypeptidase E (CPE), Z124, R9 and R10 were completed, with some of these compound interacting with 5HTR1E with Kd in the 1-25 nM range. Safavi Laboratory NIAID: Their laboratory generated iPSC lines from patient derived samples and characterized the derived neuronal populations with MEA and fluorescence microscopy. The current work includes characterization of modified patient derived lines to reverse the neurodegenerative effects. Extramural collaborations: Jonathan Geiger Laboratory (Univ North Dakota School of Med) We are collaborating with the Geiger laboratory to study the role of endolysosomal function in the neurotoxic activities of the HERV-K Env protein. Asish Shah laboratory (Univ Miami School of Med) Glioblastoma cells with stem cell like properties contain elevated levels of HERV-K Env and the HERV-K elements contribute to the stem cell niche of the glioblastoma, suggesting that HERV-K may be a therapeutic target for these tumor cells. Cell line generation NeuroTherapeutics Development Unit (NTDU): As part of its role in facilitating translational research, the NTDU develops cell lines in human neural stem cells (NSCs) for both internal and collaborative use. Lines have been developed to ubiquitously express the tdTomato fluorescent protein to aid in conducting screening assays. Collaborations between the NTDU and intramural and extramural groups has led to the development of more than ten such NSC lines that will make it possible to conduct medium throughput screens aimed at targeting several neurological diseases including bipolar disorder and Alzheimer diseases. Additionally, the NTDU has also generated additional cell specific lines for internal use that are also available to collaborators. These lines use cell specific promoters to express tdTomato in neurons and astrocytes and could have also have broad utility for conducting screens in distinct neural cells.