Parkinson's disease (PD), a chronic and progressive neurological condition and one of aging and age- related diseases, affects approximately 1.5 million people in the US alone. PD not only places severe burden on the patients but also on their family and society. In 2003, approximately US $2.3 billion was spent on drug therapy worldwide to treat Parkinson's disease. Although great strides have been made in the development of agents to treat PD, the existing PD drugs such as dopamine (DA) agonists, levodopa and catechol-O-methyl transferase inhibitors (COMT) only treat the symptoms of the disease, and are also fraught with adverse effects and long-term complications. Consequently, there is a great need for developing disease-modifying and neuroprotective as well as neurorestorative drugs which can slow down or stop the disease from progressing. Recent research suggests that inhibition of the glycogen synthase kinase-3? (GSK-3?) by small molecules may offer an important strategy in the treatment of a number of neurodegenerative diseases including Alzheimer's disease (AD), but its usefulness in PD has not been described. We have recently found in the cellular and animal MPTP model of parkinsonism, an induction in the hyperphosphorylated form of Tau, p-Tau, with hyperphosphorylation seen at many sites, including those found in neurofibrillary tangles of AD. The increase in p-Tau levels was strictly dependent upon the presence of a- Syn. This requirement for a-Syn was mandatory, since in a-Syn-/- mice, and in transfected cells not expressing any a-Syn, the toxin failed to induce p-Tau. MPTP also caused an increase in a-Syn protein levels. Hyperphosphorylation of Tau lead to its dissociation from the cytoskeleton, and aggregates of p-Tau were seen in sarkosyl-insoluble and Triton X-100-insoluble fractions. a-Syn was able to form stable heteromeric protein complexes with p-Tau, and p-Tau aggregates were seen in mature inclusion bodies of a-Syn. MPP+ caused the activation of several p-Tau-specific kinases, such as GSK-3? and p-ERK. Blockade of GSK-3? not only prevented, but also reduced, MPP+-induced p-Tau formation, a-Syn accumulation and cytotoxicity. Very new data obtained in human postmortem PD brains, in collaboration with Dr. Jeffrey Joyce, show a similar pattern of pathology: increased a-Syn accumulation, hyperphosphorylation of Tau at sites similar to the MPTP models [pSer262 and pSer396/404], lack of phosphorylation at sites not seen with MPTP [pSer202], and large increases in GSK-3?. Interestingly, these pathological changes were further augmented in PD patients with dementia [PD + DEM]. Together, these findings in PD brains confirm the validity of our findings with the MPTP models. Importantly, prior to our findings, a possible role for GSK-3? in PD has not been previously described, although its role in AD is well studied. In addition to our findings, another study found strong linkage of two single nucleotide polymorphisms in the GSK-3? gene to sporadic PD. Thus, GSK-3? presents a novel target site in the development of novel therapies for PD. To date, we have identified some nM potency GSK-3? inhibitors that emerged from our SAR studies of staurosporine. A number of these designed staurosporine analogs have been screened against a family of 30 kinases. Among the compounds tested we found one, an indolyl-indazolylmaleimide, that was able to inhibit 98% of the kinase activity of GSK-3? when tested at a concentration of 10 ?M. After further structural modifications described below, novel 3-(indol-3-yl)-4-(benzofuran-3-yl)maleimides having a Ki value as low as 2 nM selectively against GSK-3? relative to 30 additional kinases were identified. Moreover, we have been able to show that some of these ligands are able to exert a neuroprotective and neurorestorative action in vitro. The ultimate goal would be to identify one or two GSK-3? inhibitors that could be further developed for slowing or halting the progression of PD. Through funding from this STTR grant, we intend to follow-up on the exciting preliminary findings we have made in pursuit of novel GSK-3? inhibitors as potential therapeutics for the treatment of PD. To achieve this goal, the Specific Aims of this research proposal are as follows: 1. Compound selection and synthesis: Based upon the compound library in hand, 10 potent GSK- 3? inhibitors will be resynthesized for further in vitro pharmacological studies. Then 2 or 3 of the most promising ligands based upon the in vitro profile will be scaled up (about 5 grams each) for in vivo animal studies. 2. Neuroprotective In vitro studies in transfected cells and neurons: Investigation of time course and dose response of the compounds for inhibition of GSK-3?, analyses of other kinases and phosphatases that are modulated by these compounds, and comparison of our findings with the effects using lithium. 3. Neurorestorative in vitro studies in transfected cells and neurons: After the initiation of cytotoxicity, we will examine the time and dose of GSK-3? inhibitors necessary for neurorestoration. 4. In vivo animal studies: Injection with MPTP for 5 days will be followed by simultaneous or delayed injections with increasing doses of the GSK-3? inhibitors for different time periods. GSK-3?, a-Syn, Tau, kinases and phosphatases will be examined, as specified in Specific Aims 2 and 3. Key words: GSK-3? inhibitors, Kinases, Selectivity, Parkinson's disease, Therapeutics, Staurosporine analogs, Neuroprotective agents, Neurorestorative agents, Neuroprotection, a-Synuclein, p-Tau, Taupathies, Synucleopathies, MPTP model, Aging, Neurodegenerative, Age-related diseases, Alzheimer's disease. Project Des inson's disease (PD), the second most common neurodegenerative disease next to Alzheimer's disease (AD), is a progressive neurological condition associated with aging. Our proposal entails the chemical synthesis of novel staurosporine analogues as potent glycogen synthase kinase-3? (GSK-3?) inhibitors, and in vitro studies in transfected cells and neurons as well as in vivo studies in animals of these compounds, which have the great potential to be developed as neuroprotective and neurorestorative therapies to slow down or stop Parkinson's disease from progressing. Public Health ? ? ?
