Jeffrey W. Kelly
The central theme of our research is to
understand the chemistry and biology of peptides and proteins and
to develop new approaches for manipulating these properties with
purposefully-designed small molecules. We employ spectroscopic
and biophysical methods in combination with chemical synthesis
and recombinant DNA technology to accomplish these aims. Specific
research projects include:
(1) SPECTROSCOPIC AND BIOPHYSICAL STUDIES ON THE CONVERSION OF A NORMALLY SOLUBLE AND FUNCTIONAL HUMAN PROTEIN INTO AMYLOID FIBRILS. An invariant feature of the sixteen human amyloid diseases (e.g., Alzheimer's Disease) is the presence of amyloid fibrils proximal to dead or dying neurons. We are currently studying three (3) amyloidogenic proteins, transthyretin, gelsolin and A-beta, whose conversion from a well-defined tertiary structure into a b-sheet rich quaternary structure appears to be the causative agent in their associated diseases. Analytical ultracentrifugation and mass spectrometry are employed to study the quaternary structural changes while circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopic methods are utilized to study the tertiary structural changes required for amyloid fibril formation. Electron microscopy and atomic force microscopy are also employed to study the mechanism of assembly of the amyloidogenic intermediate. The dynamic characteristics of the amyloidogenic intermediate are studied by H/D exchange employing electrospray and MALDI MS. We now know that significant tertiary structural changes are required for the conversion of these proteins into amyloid. The challenge remaining is to understand the details of these changes, the dynamic properties of the intermediates, and the self-assembly mechanism that yields amyloid.
(2) A CELL BIOLOGY APPROACH TO UNDERSTAND THE NEUROTOXICITY EXHIBITED BY AMYLOID OR THE SOLUBLE AMYLOID PRECURSORS. Neurotoxicity, which is a common feature of all amyloid diseases, is poorly understood. It is likely that amyloid exhibits toxicity through multiple mechanisms. Using our expertise in the preparation of amyloid and its soluble precusors in combination with the cell and neurobiology expertise at Scripps, we have embarked on a project to understand why amyloid fibrils kill nerve cells.
(3) STRUCTURE-BASED SMALL MOLECULE APPROACH FOR PREVENTING THE CONFORMATIONAL CHANGES ASSOCIATED WITH HUMAN AMYLOID DISEASE. We have introduced a new therapeutic strategy for preventing amyloid fibril formation whereby a high affinity ligand is used to prevent the quaternary and tertiary or tertiary structural changes required for fibril formation. In this project we employ X-ray crystallography or NMR in combination with synthetic chemistry to identify high affinity inhibitors. Recent cocrystal structures of the normally folded form of transthyretin with second generation inhibitors suggest new molecules to synthesize and test which should be much improved. Our philosphy on synthesis is to keep it simple, such that parallel syntheses can be used to prepare libraries to quickly sort out structure activity relationships.
(4) THE USE OF PEPTIDES, PEPTIDOMIMETICS, AND STRUCTURALLY DEFINED PROTEINS TO BETTER UNDERSTAND b-SHEET STRUCTURE IN AQUEOUS SOLUTION. Peptides < 40 amino acids in length typically do not adopt well-defined structures in aqueous solution, making it difficult to study beta-sheet tertiary structure with small peptides. In an effort to better understand b-sheet structure in aqueous solution, we have designed and synthesized unnatural amino acids which nucleate b-sheet formation when incorporated into a suprising range of sequences composed of six (6) or more natural amino acids. The challenge is to convert these dynamic beta-sheet structures into well-defined b-sheets that are protein like. In parallel we are studying the WW domainn (57 residues), which is an important and recently discovered signal transduction domain that adopts an isolated three standed b-sheet structure in aqueous solution as discerned from NMR and X-ray crystallography. This is an excellent protein to understand the contributions of H-bonding and hydrophobic interactions to beta-sheet structure. This will be accomplished by incorporating unnatural amino acids into the WW sequence by solid phase peptide synthesis to test the physical and spectroscopic properties of the resulting analogs.
(5) THE DEVELOPMENT OF COMPOSITE ORGANIC-INORGANIC SURFACES EMPLOYING SELF-ASSEMBLING PEPTIDOMIMETICS THAT INDUCE INORGANIC CRYSTAL GROWTH ON THEIR SURFACE. The use of small peptidomimetic beta-sheet structures described above in combination with electrostatic intermolecular interactions leads to b-sheet building blocks which self-assemble to form a monolayer coating at an air-water interface or on a hydrophobic surface. Controlled self-assembly allows a periodic presentation of functional groups on a surface which can nucleate mineral crystallization if the lattice match is good. Taking clues from biology, we have accomplished calcite mineralization. The future challenge and potential of this approach is the formation of unatural inorganic surfaces which can be used for a variety of applications such as catalysis.
(6) DEVELOPING THERAPEUTIC STRATEGIES FOR
TREATING HUNTINGTON'S DISEASE. The main focus of this research is
to develop a therapeutic strategy for Huntington's Disease. We
aim to discover inhibitors that interfere with poly-Gln
beta-sheet mediated aggregation in the nucleus. Aggregation
inhibitors will be discovered through a selection scheme and a
screening approach. The selection utilizes an oligo-Gln sequence
fused to a protein that is critical for cell viability. When the oligo-(Gln)>39 portion of the fusion mediates aggregation, the
cells are unable to grow. Cell growth can be rescued by small
molecules that interfere with (Gln)>39 assembly. The
feasibility of a second approach, using small molecules to target
the mRNA of the expanded huntingtin protein to prevent its
translation, will also be evaluated.
(7) SYNTHESIS OF NATURAL AND UNNATURAL PRODUCTS LACKING AMIDE BONDS FROM OLIGOPEPTIDES. Peptide synthesis has evolved to a point where nearly any sequence can be prepared in very high yield. We will take advantage of the stereochemical purity of peptides and their capability to undergo intramolecular reactions to prepare novel heterocycles that can bind to proteins for the purpose of inhibiting protein-protein interactions. This work will commence with oligo-Gln sequences.
Analogously, we will prepare the following sulfur-based heterocycles.:
Knowing the products we desire, we can utilize
mass spectrometry to identify productive reactions and in the
process we will likely
discover new types of chemical reactivity. Peptides have been very much underutilized as starting materials in organic synthesis and we
hope to change this.
(8) ATROPISOMERICALLY PURE BIARYLS AS LIGANDS FOR INHIBITING PROTEIN-PROTEIN INTERACTIONS. Atropisomerization refers to isomerization about the aryl-aryl bond of biphenyls substituted with
X and Y substitutents where X does not equal Y. Depending on the size of X and Y it may be possible to resolve the R and S enantiomers from one another. We envision controlling the atropisomerization to make chiral ligands designed to target proteins by making X large and chiral, such that the reaction can only yield one atropisomer. One approach is to utilize:
The main goal of this project is to decipher the appropriate methodology to do hindered aryl couplings such that the products are
atropisomerically pure or nearly pure. These ligands should prove to be excellent protein binders.