This work is directly relevant to human embryonic stem cell (hESC) research because it brings new ideas about novel compounds to affect cardiomyogenesis. The work addresses an urgent need to develop new agents to treat cardiovascular disease. We will develop potent and selective drug-like molecules as cardiomyocyte differentiation agents.
Heart disease is the leading cause of mortality and decline in the quality of life in the developed world. The ability of hESCs to form cardiomyocytes has spawned hope that these cells may be used to replace damaged myocardium. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in ESC cultures has not been achieved due to the unavailability of differentiation agents and an incomplete understanding of the pathways that regulate cardiac development.
Success has been achieved in developing a robust and dependable high-throughput assay to study the effects of small molecules on cardiomyocyte differentiation. Powerful cell-based assays were developed and provided readouts that led to high-content results because multiple signals were probed. The assay is capable of capturing fast or long-acting biology because of the time-course readouts. Cell-based assays are superior to molecular screens because the cell-based assay delivers active compounds or “hits” that are permeable and non-cytotoxic. Moreover, refined “hits” can be used as probes to reveal novel signaling pathways and proteins that control differentiation, in a process termed chemical biology. By taking advantage of knowledge of the current “hits” we will rapidly synthesize novel drug-like compounds in a low-risk approach to. The “hits” will be refined and improved through an efficient synthetic process we use in our lab called “Dynamic Medicinal Chemistry”.
Even after miniaturization and automation, screening is still expensive. A key to improve the screening process is to use pharmacologically active, drug-like compounds to provide rich target-relevant information. Intelligently designing libraries for screening by incorporating drug-like features into “lead” library design will improve the attrition rate and lead to more pharmacologically relevant compounds for future studies.
This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyoenesis of hESCs. Importantly, it brings new investigators and a collaborative approach to the stem cell field. The agents discovered and developed may hold great promise as the groundwork for future medications development for a new class of damaged myocardium replacement agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will also be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis.
In 2002, in the State of California, approximately 697,000 adult Californians died from heart disease. The cost as measured by loss of lifelong earnings was more than $79 billion. Setting aside the pain and suffering, the economic impact of cardiovascular disease to the State of California is staggering. Despite recent advances in cardiovascular medications development, new approaches and novel drug-like compounds are urgently needed to treat cardiovascular disease in California and elsewhere. The poor prognosis for heart disease for Californians underscores the critical need to develop alternative therapeutic strategies. The demonstrated ability of human embryonic stem cells (hESCs) to form cardiomyocytes has spawned widespread hope that these cells may be used as a source to replace damaged myocardium in humans. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in hESC culture has not been achieved due to the unavailability of differentiation agents and also because of an incomplete understanding of the pathways that regulate cardiac cell development.
Using a high-throughput whole cell assay with image analysis, we have identified four small molecules that promote cardiomyogenesis in human ESCs. This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyogenesis of hESCs. It also brings new investigators and new collaborative approaches to the field. The promising agents discovered already constitute an excellent starting point and further refinement and development of these compounds may hold great promise as the groundwork for future medications development for a new class of damaged myocardium differentiation agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis.
The compounds discovered in our whole hESC-based assays thus far are not potent enough to be developed as drug candidates. But these compounds hold great promise as agents that could be refined further into drug leads. If the leads become drugs, promise of a new class of medication to treat cardiovascular disease may become a reality. Such drugs would decrease cardiovascular disease and decrease health care costs in California. This will likely have a significant economic impact to the State of California. The proposed work represents essential translational research required for new drug development.
SYNOPSIS: In this proposal the investigators plan to develop a library of small molecules that can induce hESCs to differentiate into cardiomyocytes. Studies will be done wherein optimized compounds will be generated based on the structure of established hits. This library will then be screened on hESCs already engineered to express fluorochromes from the Nkx5.2 and MHC promoters. Hits will then be further optimized taking into account absorption, metabolization, etc.
SIGNIFICANCE AND INNOVATION: The use of hESCs to understand human cardiomyogensis and generate drugs that can induce such differentiation are of great importance in developing therapies for human cardiac diseases. Therefore, this work would be very significant if new molecules can be found to direct hESCs into cardiomyocyte lineages.
STRENGTHS: The chemical genetics approach with high-throughput screening is a major strength, as is the PI's expertise in both of these areas. The collaboration with Dr. Mercola for hES cell expertise, who has developed lineage marked hESCs, is also a plus. The PI has some preliminary hits from screening the chemical libraries.
WEAKNESSES: There is some weakness in the biological characterization of lead compounds. There are limited plans to identify the cellular targets of the lead compounds, a limited description of gene array studies to be conducted with compounds, and an inadequate analysis of what fraction of cells give the cardiomyocyte signal, or express non-cardiomyocyte cell fates in the assays. The ambitiousness of the proposal is another weakness, and it is not clear what the funding exactly will be used for as this is an ongoing project.
DISCUSSION: This proposal represents a collaborative program involving two strong investigators who will expand a library screen to find new molecules that promote the differentiation of cardiomyocytes. The facilities seem appropriate, but the robotics and the experiments are not well described. The experiments are presented in a cursory manner with respect to how the data will be interpreted. Also, this looks like an ongoing program. The PI has already screened one library and found four hits, and this proposal is for screening a second library. This makes the proposal somewhat less innovative, but finding these small molecules is very important. How new is this work? Is this truly a SEED grant? A massive amount of work is proposed and it is unclear if it can be completed in 2 years.
The proposal is weak in the biological characterization of lead compounds. One concern is that a positive readout in the cell based assay for a desired cell type does not necessarily speak to the efficiency of generating that desired cell type due to heterogeneity. What percentage of cells would become cardiomyocytes? This heterogeneity of the indicator cell population was not considered a major problem given the investigators involved, but this complication diminished enthusiasm for this project. More positively, the work is well underway, and chemical genetics approaches with hESCs should be supported as they are unlikely to be supported by NIH.