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.
This proposal focuses on the identification and development of small molecules that a) direct the differentiation and purification of dopaminergic (DA) neurons from human embryonic stem cells (hESCs) and b) stimulate their survival. The applicants propose 3 specific aims. Aim 1 consists of the optimization of two previously identified hit compounds useful for eliminating non-neuronal and potentially tumorigenic proliferating cells without affecting postmitotic neurons. Aim 2 consists of the identification of molecules that control key steps in DA differentiation from hESCs using high throughput (HTS) and high content screening (HCS). For the screens, the Principal Investigator (PI) proposes to use an assay based on a DA neuron-specific reporter hESC line and traditional drug discovery methods with chemical optimization of hits. In Aim 3, compounds will be used as probes to identify molecular pathways and targets involved in hESC DA differentiation. Finally, since compounds that promote DA neuron differentiation from hESC might also aid in the regeneration of cells in the substantia nigra, the applicants propose to develop some of the lead compounds into drug therapy candidates for stimulating endogenous regeneration in Parkinson’s disease (PD) patients.
Parkinson’s disease is a devastating neurodegenerative disorder for which disease-modifying therapies are not available to date. Reviewers agreed that this proposal addresses an unmet medical need and if successful could have significant impact on the treatment of PD. Current methods for the differentiation of DA neurons from hESCs are inefficient and the transplantation of DA neuron progenitors raises the possibility of in vivo proliferation and tumor formation This proposal addresses both of these issues. The reviewers noted that the strength of the proposal lies in Aim 2 but were less enthusiastic about Aims 1 & 3. While recognizing the importance of purifying DA neuron populations and eliminating potentially tumorigenic cells prior to transplantation, they questioned the rationale for in vivo testing of drugs toxic to proliferating, non-neuronal cells. These compounds may be toxic to endogenous stem cells and thus would seem to have limited potential in vivo. One reviewer commented that while the studies of specific pathways described in Aim 3 are of interest, the integration of these studies into the overall program is not well described. Overall, the reviewers recommended that the applicant focus on the development and testing of differentiation compounds as described in Aim 2.
Reviewers raised a number of questions about the design of the research plan and had serious concerns about its feasibility. The project is overly ambitious, has unrealistic goals and employs too many methodologies. While they appreciated the well-defined milestones and timelines they felt the proposal lacked sufficient preliminary data. One reviewer noted that while the strategy for structure-activity relationship (SAR) studies in Aim 1 is clearly articulated, the methodology for testing the non-neuronal specificity of the compounds is unclear. This reviewer also commented that in Aim 2 the applicant will need to employ a clear approach to interpreting conformational data to prioritize the assessment and SAR development of “hits”, but noted that no strategy is described. Another reviewer expressed concern about the use of the proposed DA neuron-specific reporter hESC line, citing experience that reporter assays often yield large numbers of false positives. This reviewer raised the possibility of using antibodies to detect tyrosine hydroxylase or other downstream markers as a more reliable approach. Another reviewer would have appreciated more discussion around the selection of the host site for the DA neuron transplantation in Aim 2, as the neostriatum should be considered in addition to the substantia nigra. Reviewers also registered some confusion with the research plan, finding three different numbers in different parts of the application describing how many compounds would be screened in Aim 2.
The reviewers praised the applicant’s expertise in medicinal chemistry and the strength of collaborations with experts in stem cell differentiation and HTS. However, one reviewer noted a lack of neurobiology expertise in the team and felt it was reflected in the proposal, as e.g. human DA neuron numbers were grossly overstated and cited literature did not reflect the most recent developments in the field. The resources and environment were judged to be adequate to outstanding.
Overall, while the reviewers agreed that this proposal addresses important issues and could have significant impact, they raised serious doubts about the research design and its feasibility.