Scientific Report 2007

Chemistry

Strategies to Ameliorate Protein Misfolding Diseases

J.W. Kelly, J. Bieschke, S. Choi, E. Culyba, M.T.A. Dendle, W. D'Haeze, D. Du, T.R. Foss, D.M. Fowler, A. Fuller, J. Gao, M.-Y. Gao, S.M. Johnson, T. Mu, A. Murray, E.T. Powers, P. Rao, L. Segatori, S. Siegel, J.Y. Suk, K. Usui, Y. Wang, I. Yonemoto, Z. Yu

Our goal is to better understand the molecular mechanisms that lead to compromised protein homeostasis, resulting in illnesses that include Alzheimer's, Parkinson's, and Gaucher diseases and type 2 diabetes. Protein homeostasis refers to the maintenance of functional proteins both inside and outside human cells, which is essential for development, reproduction, and successful aging, consistent with the central role of proteins as the workhorses in the physiology of all organisms. Understanding the mechanisms of protein homeostasis, especially the defects in these pathways associated with aging, enables us to design new strategies to ameliorate protein misfolding diseases, a main goal of our research. We use organismal and cell biological disease models and biophysical approaches in combination with medicinal chemistry and structure-based drug design. Successful collaborations with W.E. Balch, Department of Cell Biology, J. Buxbaum, Department of Molecular and Experimental Medicine, and A. Dillin, Salk Institute for Biological Studies, La Jolla, California, are critical to achieve our goals.

Transthyretin Amyloidogenesis

Transthyretin is a 55-kD homotetrameric protein that transports holo-retinol binding protein and L-thyroxine in the blood and cerebrospinal fluid. More than 99.5% of the transthyretin binding sites for L-thyroxine remain unoccupied in blood, and only about 50% of transthyretin tetramers in plasma are bound to a single holo-retinol binding protein. As a consequence of a mutation or a protein homeostasis stress associated with aging and/or oxidative stress, dissociation of the native transthyretin tetramer and subsequent changes in the tertiary structure of the monomer make the monomeric subunits competent to misassemble into cytotoxic aggregates, including amyloid fibrils. Deposition of transthyretin amyloid is linked with a number of human diseases, including senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy, and CNS-selective amyloidosis.

A suitable strategy to slow or prevent the formation of aggregates is to inhibit the rate-limiting dissociation of the transthyretin tetramer by using small molecules, which we discovered, to stabilize the normal functional state. Two of these molecules are in placebo-controlled clinical trials for treatment of familial amyloid polyneuropathy, a disease similar to Alzheimer's that attacks the peripheral nervous system instead of the brain. This year we determined the mechanism behind the efficacy of one of these compounds. We also synthesized and identified even more potent native-state kinetic stabilizers of transthyretin for the amelioration of familial amyloid cardiomyopathy, a disease leading to congestive heart failure. We anticipate that trials of the stabilizers for treatment of cardiomyopathy will begin after the results of the peripheral neuropathy trials are known.

Aberrant Oxidative Metabolites and the Onset of Sporadic Parkinson's and Alzheimer's Diseases

Understanding the molecular or mechanistic basis for the age-associated onset of Alzheimer's and Parkinson's diseases is one of our key goals. The α-synucleinopathies, including Parkinson's disease, are characterized by cytoplasmic α-synuclein–rich aggregates within degenerating dopaminergic neurons in the substantia nigra. Clinical observations suggest a correlation between oxidative stress/inflammation and protein misfolding diseases. We found that overexpression of α-synuclein in a neuronal cell line is sufficient to increase the production of oxidative metabolites that, in turn, significantly accelerate aggregation of α-synuclein in vitro. The data suggest that the acceleration of aggregation occurs predominantly via a noncovalent mechanism.

We recently found that a prominent oxidative metabolite, 4-hydroxynonenal, long correlated with Alzheimer's disease, covalently modifies amyloid β-peptide (Aβ), whose aggregation is thought to cause Alzheimer's disease. This covalent modification makes amyloid formation much more efficient. Thus, we continue to explore the hypothesis that enhanced local oxidative stress, perhaps as a result of aging, results in a vicious cycle that forms reactive oxidative metabolites that exacerbate aggregation of α-synuclein and Aβ, resulting in even more oxidative stress and higher concentrations of oxidative metabolites that enable even more aggregation and proteotoxic effects, causing sporadic Alzheimer's and Parkinson's disease.

Diminishing Proteotoxic Effects Leading to Alzheimer's Disease

Aberrant protein aggregation is a common feature of late-onset neurodegenerative diseases, including Alzheimer's disease, which is associated with the misassembly of the Aβ_1-42 peptide. In collaboration with Dr. Dillin and coworkers, we found that the aggregation-mediated proteotoxic effects of Aβ_1-42 in a Caenorhabditis elegans model of Alzheimer's disease were reduced when aging was slowed by a decrease in insulin/insulin growth factor-1–like signaling (IIS). We discovered that the downstream transcription factors heat-shock factor-1 and DAF 16 regulate opposing disaggregation and aggregation activities to promote cellular survival and protein homeostasis in response to constitutive aggregation that can become toxic. Because the IIS pathway is central to the regulation of longevity and youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated proteotoxic effects that we strive to manipulate with small-molecule drugs as a fundamentally new approach to ameliorate Alzheimer's disease.

Of note, the IIS pathway plays a role in modulating other forms of toxic protein aggregation, such as in the aggregation of the huntingtin protein leading to Huntington's disease, suggesting that the aggregation/disaggregation activities we discovered in the past year may be quite general. Additionally, it is becoming clear that small perturbations in the activities responsible for protein homeostasis have a profound impact on organismal integrity, suggesting that the protective mechanisms regulated by the IIS pathway may link longevity to protein homeostasis, enabling new therapeutic strategies to be conceived.

New Therapeutic Strategies to Ameliorate Lysosomal Storage Diseases

Mutations in glucocerebrosidase lower the concentration and activity of this lysosomal glycolipid hydrolase, lead to an accumulation of the glucocerebrosidase substrate, glucosylceramide, in the lysosome, and cause Gaucher disease, the most common lysosomal storage disorder. We showed previously that administration of N-(n-nonyl)deoxynojirimycin increases the activity of the glucocerebrosidase variant N370S in a cell line derived from tissue from a patient with Gaucher disease. This 'chemical chaperoning' effect is due to the binding of N-(n-nonyl)deoxynojirimycin to the native state N370S, allowing the glucocerebrosidase to fold properly within the cell and be trafficked from the endoplasmic reticulum to lysosomes.

We are now seeking compounds that can be used in combination with these protein- and disease-specific small molecules to enhance the cellular protein folding and trafficking capacity. Compounds that influence the cellular folding and trafficking of classes of glycolipid hydrolases have the potential to be useful for more than a single lysosomal storage disease. Recently, we discovered molecules of this type, drug candidates that offer the potential to change the economics of health care by treating multiple diseases in a family of diseases with a single compound.

Publications

Bieschke, J., Zhang, Q., Bosco, D.A., Lerner, R.A., Powers, E.T., Wentworth, P., Jr., Kelly, J.W. Small molecule oxidation products trigger disease-associated protein misfolding. Acc. Chem. Res. 39:611, 2006.

Cohen, E., Bieschke, J., Perciavalle, R., Kelly, J.W., Dillin, A. Opposing activities protect against age-onset proteotoxicity. Science 313:1604, 2006.

Cordeiro, Y., Kraineva, J., Suarez, M.-C., Tempesta, A.-G., Kelly, J.W., Silva, J.L., Winter, R., Foguel, D. Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for aggregates of transthyretin. Biophys. J. 91:957, 2006.

Fu, Y., Gao, J., Bieschke, J., Dendle, M.A., Kelly, J.W. Amide-to-E-olefin versus amide-to-ester backbone H-bond perturbations: evaluating the O-O repulsion for extracting H-bond energies. J. Am. Chem. Soc. 128:15948, 2006.

Jager, M., Zhang, Y., Bieschke, J., Nguyen, H., Dendle, M., Bowman, M.E., Noel, J.P., Gruebele, M., Kelly, J.W. The structure-function-folding relationship in a WW domain. Proc. Natl. Acad. Sci. U. S. A. 103:10648, 2006.

Kelly, J.W. Structural biology: proteins downhill all the way. Nature 442:255, 2006.

Reixach, N., Adamanski-Werner, S.L., Kelly, J.W., Koziol, J., Buxbaum, J.N. Cell based screening of inhibitors of transthyretin aggregation. Biochem. Biophys. Res. Commun. 348:889, 2006.

Sawkar, A.R., Schmitz, M., Zimmer, K.-P., Reczek, D., Edmunds, T., Balch, W.E., Kelly, J.W. Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants. ACS Chem. Biol. 1:235, 2006.

Sekijima, Y., Kelly, J.W. Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 13:236, 2006.

Siegel, S.J., Bieschke, J., Powers, E.T., Kelly, J.W. The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation. Biochemistry 46:1503, 2007.

Tojo, K., Sekijima, Y., Kelly, J.W., Ikeda, S.-I. Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci. Res. 56:441, 2006.

Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan, C., Kellner, W., Matteson, J., Plutner, H., Riordan, J.R., Kelly, J.W., Yates, J.R. III, Balch, W.E. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127:803, 2006.

Yu, Z., Sawkar, A.R., Whalen, L.J., Wong, C.-H., Kelly, J.W. Isofagamine- and 2,5-anhydro-2,5-imino-D-glucitol-based glucocerebrosidase pharmacological chaperones for Gaucher disease intervention. J. Med. Chem. 50:94, 2007.

Zhang, Y., Kim, Y., Genoud, N., Gao, G., Kelly, J.W., Pfaff, S.N., Gill, G., Dixon, J.E., Noel, J.P. Determinants for dephosphorylation of the RNA polymerase II C-terminal domain by Scp1. Mol. Cell 24:759, 2006.