Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic (DA) neurons present in the midbrain. Current treatments try to overcome the loss of DA neurons by replacing lost dopamine, attempting to halt or delay loss of neurons, or modulating other parts of the circuit. Cell replacement transplantation of DA neurons to restore function via local DA secretion has shown some success but has been limited by the availability of pure DA cells for transplantation. Embryonic stem cells (ESCs) are an ideal source for cell replacement therapy. In advance of an investigational new drug application for cell-based treatment of PD we will study the following Aims: 1. Optimize “hits” useful for eliminating tumorigenic proliferating cells. Analogs of hits will be synthesized and tested in assays with quantitative results guiding design and synthesis of more potent drug-like agents, and also to develop affinity probes used in Aim 3 to identify cellular targets. 2. Identify molecules that control key steps in DA differentiation. Automated screens are proposed for identifying small molecule activators of pathways that will stimulate DA differentiation in hESCs. Recognizing additional pathways are likely important; unbiased phenotypic screens will be carried out to identify novel small molecules that stimulate progression through discrete stages in DA differentiation. Screens are also proposed for compounds that stimulate DA neuron survival. Hits will be confirmed and analyzed by secondary assays that assess independent function. 'Smart' libraries of analogs of the most promising hits will be synthesized and tested for solubility, chemical and metabolic stability, permeability, and toxicology. Cell-based differentiation assays will provide feedback for a medicinal chemistry “synthesis engine”, an iterative process of analog design, synthesis and biotesting. The tools are in place for efficiently developing hits into more effective PD drug candidates. Leads will be optimized with a battery of pharmaceutical property tests, and leads will go into safety tests of transplanted cells in suitable animal models. 3. Identify cellular targets of the active compounds. Affinity versions of the optimized leads (Aim 2) will be used to directly label and characterize cellular proteins. Signaling pathways targeted by optimized leads will be profiled for phosphoproteins after cell treatment. Data will yield hypotheses about the target pathways, verified using selective inhibitors to block the leads. Our goal is to use the leads to yield new insight into basic mechanisms of DA differentiation, and to exploit the cellular proteins themselves as druggable targets. Developmental candidates will be optimized for ADMET properties, and scaled up for in vivo testing. Our proposal of developing ESC-based therapy for a non-curable disease (PD) meets CIRM's primary goal for this award by developing PD drug candidates and cells for unmet medical needs.
Statement of Benefit to California:
Several million adult Californians suffer from Parkinson’s Disease (PD) or other neurodegenerative conditions. PD is a devastating movement disorder caused by death of dopaminergic (DA) neurons in the midbrain. The economic burden to the State of California for individuals suffering from PD or their families is in excess of one billion dollars per year. Treatments try to overcome loss of DA neurons by replacing lost dopamine, halt or delay loss of neurons, or modulate other parts of the circuit. DA neuron transplantation to restore function via local DA secretion has shown some success but is limited by availability of DA cells. Adult DA cells retain little if any ability to replicate, and substantia nigral (SN) failure is principally a disease of DA cell loss. The ameliorating effects on SN reported have been achieved by improving other processes that are impaired in the failing SN. Hypothetical replacement therapies work via transplantation or stimulation of endogenous regeneration. Whether endogenous SN stem cells exist and can be mobilized remains controversial. Embryonic stem cells (ESCs) are an ideal source for cell replacement therapy. However, currently, DA cell yield is inefficient for large-scale production. With current survival of transplanted cells at < 5%, improving replication of committed precursors either pre- or post-implantation would be desirable. Another issue is co-transplantation of small numbers of proliferating cells that can produce tumor cells. Addressing both of these issues would close a major gap in our knowledge about enrichment of large numbers of pure, postmitotic differentiated DA cells. Needed are new approaches and new agents that enrich highly purified DA cells. Advances in dynamic medicinal chemistry utilizing high throughput screening (HTS) has provided promising and highly potent candidates for further drug development. Additionally, high content screening (HCS) using automated image acquisition and analysis will be used to identify molecules that stimulate differentiation and cell cycle entry detectable by specific biomarkers for differentiated DA-producing midbrain neurons. Our proposal addresses both issues of DA cell purification and differentiation required for a developmental candidate through chemical screening and biological target identification. Lead compounds will mimic action of known regulators and activators of novel, unanticipated pathways that control cell purity and differentiation. Using a validated, iterative approach of synthetic chemistry and biotesting we will optimize lead candidates for improved potency and other pharmaceutical properties. The candidates will then be tested in vivo for safety and efficacy in animal models of PD. Applying proven approaches, we expect to deliver safe and effective PD drug candidates as small molecules and/or transplantable cells for clinical trial tests, thus fulfilling the CIRM mission of ESC-based therapies to address a major unmet medical need.