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The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999


Bioorganic Chemistry, Neurochemistry, and Composite Materials


J.W. Kelly, H. Bekele, R. Kaul, E. Koepf, J. Kowalski, H. Lashuel, V. Oza, M. Petrassi, E. Powers, H. Purkey, P. Raman, H. Razavi, G. Ratnaswamy, T. Walkup, J. White, L. Woo

The central themes of our research are to understand the chemistry and biology of proteins and to develop new approaches for manipulating these properties with purposefully designed small molecules. Under the auspices of The Skaggs Institute for Chemical Biology, we are focusing our research on neurodegenerative diseases. Our goal is to learn enough about the mechanism of these diseases to facilitate development of novel therapeutic strategies. We are also creating composite materials based on biomineralization mechanisms. In addition, we have begun preliminary studies centered on the use of peptides as starting materials for products that lack amide bonds.

Development of a Genetic Complementation System to Discover Inhibitors of Protein Aggregation

An invariant feature of the 16 human amyloid diseases (e.g., Alzheimer's disease) is the presence of amyloid fibrils close to dead or dying neurons. Amyloid fibrils are protein quaternary structures assembled through intermolecular ß-sheet formation. We are studying several amyloidogenic proteins, including transthyretin and gelsolin. Misfolding and proteolysis appear to initiate the self-assembly cascade that leads to pathologic changes. In an effort to discover small-molecule inhibitors that block self-assembly, we developed a genetic complementation system in which an amyloidogenic protein is covalently linked to an enzyme required for cell growth. Assembly of the amyloidogenic fragment of the fusion protein removes the enzyme from solution, attenuating or halting cell growth. This propensity to aggregate can be complemented by using small molecules that prevent assembly of the amyloidogenic proteins. This system is being used to screen for small-molecule inhibitors that prevent the aggregation of several amyloidogenic proteins.

A Cell Biology Approach to Understanding Fibril Formation In Vivo

How and where formation of amyloid fibrils takes place in vivo are not known. The general assumption is that the phenomenon is extracellular. However, evidence in several amyloid diseases indicates that formation of fibrils could be occurring within a cell. In collaboration with S. Schmid, The Scripps Research Institute, we discovered cells of monocyte origin that take up and convert amyloidogenic proteins into amyloid protofilaments and, occasionally, amyloid fibrils. After we identify the cells responsible for this conversion and the organelles involved, we will use this approach to evaluate inhibitors of fibril formation.

Toward Composite Organic-Inorganic Materials

To control the size, orientation, and morphology of inorganic crystals, we used an organic surface as a nucleus for growth of the crystals. We introduced a novel peptidomimetic incorporating the 4-(2´-aminoethyl)-6-dibenzofuran propanoic acid residue flanked by natural and unnatural α-amino acid residues that folds and assembles into a 2-dimensional ß-sheet monolayer at an air-water interface. A Langmuir-Blodgett film balance, circular dichroism, and infrared spectroscopic methods were used to characterize the monolayer. An acidic peptidomimetic with glutamic acid residues projecting into aqueous subphase was used as a nucleus for the formation of cadmium sulfide nanocrystallites via the {01.0} face of cadmium sulfide. Transmission electron microscopy was used to measure the width and length (each ~25 Å) of the cadmium sulfide particles, and atomic force microscopy was used to measure the height (80 Å). Epitaxial growth was supported by the face nucleated and the size of the crystal, which appear to be controlled by the lattice mismatch (Fig. 1). High-resolution transmission electron microscopy studies showed that the nanocrystals were all oriented in the same direction, implying long-range order in the monolayer as well. This approach should be useful for the preparation of composite organic-inorganic materials with applications as catalytic, magnetic, optical, and electronic materials.

Peptides As Starting Materials For Natural and Unnatural Products That Lack Amide Bonds

We aim to develop both the chemistry and the technology to efficiently discover reactions that transform peptides into products of interest that lack amide bonds. The availability of a wide variety of functionalized natural and unnatural α-, ß-, and γ-amino acids allows synthesis of peptides tailored for the reactivity and product desired. Along these same lines, technology is being developed to efficiently follow numerous parallel reactions in which a certain type of reactivity is being sought. Thus far, we have shown that thiazoline heterocycles can be efficiently synthesized by using a variety of transition metal--based reactions and hypervalent phosphorus-based reagents. The dehydration reactions leading to thiazoline and oxazoline heterocycles shown in Figure 2 have been accomplished by the mechanism shown. This approach also can be used to accomplish tandem cyclizations, resulting in the formation of oligomeric thiazoline products.

Publications

Angeletti, R.H., Bibbs, L., Bonewald, L.F., Fields, G.B., Kelly, J.W., McMurray, J.S., Moore, W.T., Weintraub, S.T. A multicenter study of racemization during "standard" solid phase peptide synthesis. In: Peptides 1996: Proceedings of the 24th European Peptide Symposium. Ramage, R., Ephron, R. (Eds.). Mayflower Scientific, Kingswinford, England, 1998, p. 207.

Baures, P.W., Oza, V.B., Peterson, S.A., Kelly, J.W. Synthesis and evaluation of inhibitors of transthyretin amyloid fibril formation based on the non-steroidal anti-inflammatory drug, flufenamic acid. Bioorg. Med. Chem. Lett. 9:1, 1999.

Baures, P.W., Peterson, S.A., Kelly, J.W. Discovering transthyretin amyloid fibril inhibitors by limited screening. Bioorg. Med. Chem. 6:1389, 1998.

Chitnumsub, P., Fiori, W.R., Lashuel, H.A., Diaz, H., Kelly, J.W. The nucleation of monomeric parallel ß-sheet-like structures and their self-assembly in aqueous solution. Bioorg. Med. Chem. 7:39, 1999.

Kelly, J.W. Overview of the biosynthesis of amino acids, peptides, porphyrins and alkaloids with a focus on the biosynthesis of aromatic amino acids. In: Comprehensive Natural Products Chemistry: Amino Acids, Peptides, Porphyrins and Alkaloids. Kelly, J.W. (Ed.). Elsevier, New York, 1999, p. 1.

Koepf, E.K., Petrassi, H.M., Sudol, M., Kelly, J.W. WW, an isolated three-stranded antiparallel ß-sheet domain that unfolds and refolds reversibly: Evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state. Protein Sci. 8:841, 1999.

Labrenz, S.R., Bekele, H., Kelly, J.W. Enhancement of the water solubility of aromatic molecules via the Heck reaction: A comparison of ethano-ammonium, -carboxylate and -phosphonate functional groups. Tetrahedron 54:8671, 1998.

Lashuel, H., Lai, Z., Kelly, J.W. Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: Implications for wild type, V30M and L55P amyloid fibril formation. Biochemistry 37:17851, 1998.

Nettleton, E.J., Sunde, M., Lai, Z., Kelly, J.W., Dobson, C.M., Robinson, C.V. Protein subunit interactions and structural integrity of amyloidogenic transthyretins: Evidence from electrospray mass spectrometry. J. Mol. Biol. 281:553, 1998.

Oza, V.B., Petrassi, H.M., Purkey, H.E., Kelly, J.W. Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors. Bioorg. Med. Chem. Lett. 9:1, 1999.

Patricelli, M.P., Lashuel, H.A., Giang, D.K., Kelly, J.W., Cravatt, B.F. Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: Identification of the transmembrane domain as a site for oligomerization. Biochemistry 37:15177, 1998.

Peterson, S.A., Klabunde, T., Lashuel, H.A., Purkey, H., Sacchettini, J.C., Kelly, J.W. Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc. Natl. Acad. Sci. U. S. A. 95:12956, 1998.

Ratnaswamy, G., Koepf, E., Bekele, H., Yin, H., Kelly, J.W. The amyloidogenicity of gelsolin is controlled by proteolysis and pH. Chem. Biol. 6:293, 1999.

Xie, Y., Lashuel, H., Miroy, G.J., Dikler, S., Kelly, J.W. Recombinant human retinol-binding protein refolding, native disulfide formation and characterization. Protein Expr. Purif. 14:31, 1998.

 

 







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