CONFIDENTIAL:  The material below should be treated as confidential for review purposes only.  Some content has been removed to protect the investigator's intellectual property.

Update (January 2, 2004).  This is an operative project, involving students Mariah McMasters, Nadia Edwin and Professors Hammer, McCarley and Russo.  Space for new students in this project (under IGERT support) is now  limited, but interested students are encouraged to enquire.   

This project will: investigate the fundamental macromolecular basis for the plaques believed by some to cause Alzheimer's disease and develop basic strategies that may prevent their formation.

Primary faculty co-advisors:

• Robert P. Hammer, Chemistry Dept. (Organic Synthesis, Bioorganic Chemistry, Peptide Mimics)
• Mark L. McLaughlin, Chemistry Dept. (Organic Synthesis, Bioorganic Chemistry, Peptide Mimics)
• Robin L. McCarley, Chemistry Dept. (Surface Science, Self-assembly)

Secondary helpers:

• William Henk, Veterinary Medicine (Electron Microscopy)
• Vince Licata and Terry Bricker, Biological Sciences Dept. (Solution Methods—e.g., ultracentrifuge)
• Paul Russo, Chemistry Dept. (Solution methods—e.g., scattering & fluorescence photobleaching)

Potential Off campus participants (to be invited as outside thesis committee members):

• Professor Peter Lansbury, Harvard Medical School
• Professor William DeGrado, University of Pennsylvania

Technical Proposal: An interdisciplinary team of 3 faculty and 2-3 students will study new ways to inhibit fibril formation of peptides such as the b-amyloid (Ab) peptide, the proposed caustive agent in Alzheimer’s disease. The students and mentors will synthesize and evaluate novel b-sheet peptide-mimic inhibitors ("blockers") of fibrillogenesis. As an integral part of the iterative molecular design process, the solution and surface polymer physics of Ab in the presence and absence of the peptide "blockers" will be studied. Each student will have a main expertise (synthesis, microscopy, solution properties, etc.) but through the integrated classroom/lab experience and close research contact with team members will also acquire hands-on knowledge of the other areas of the project. This should improve communication among the team members, expedite trouble shooting when difficulties arise, and increase throughput on the project by empowering team members with skills to independently accomplish more than one aspect of the research.

Background. Background. Several age-associated degenerative diseases are characterized by the formation of protein aggregates that assemble into very large fibrillar structures. These include Alzheimer’s, Parkinson’s and Huntington’s diseases (Kelly 1998). The brains of patients with Alzheimer’s disease (AD) exhibit extracellular amyloid plaques, consisting mostly of b-amyloid peptide (Ab). There have been two extreme hypotheses on what role these fibrils play in the actual pathology of AD. Some say the plaques are the causative agents of the disease. Others believe that the fibrils are merely linked to the disease, but not the causative agent. There is evidence to support either view. Recently, it has been suggested (Lansbury 1999) that smaller protofibrils (~1 x 50 nm) may be the toxic species in AD. This hypothesis seems to be consistent with all of the data, so great emphasis is now placed on preventing even small protofibrils from forming.

Our long-range goal is development of inhibitors of amyloidogenesis and evaluation of such potentially therapeutic molecules as suppressors of toxicity. To do this, we must develop and evaluate the tools for fundamental characterization of the range of aggregation states of amyloidogenic proteins, from the small oligomeric assembly to large fibril. Additionally, the effect of amyloid-blocker molecules on these aggregation states must be assessed. Using the well-studied Ab as a test case, we propose to develop new b-strand mimics (b-sheet "blockers") that can specifically interact with the b-sheet structure of the developing amyloid fibril. The interaction of known "blockers" and our new inhibitors with the full range of Ab aggregates will be examined by a powerful combination of macromolecular techniques including light scattering, scanning force microscopy, analytical ultracentrifugation, and fluorescence photobleaching recovery. We have these following specific aims: (1) Design and synthesize new b-strand mimics that may potentially block amyloid fibril and protofibril formation. (2) Elucidate the inhibition mechanism of protofibrillogenesis with b-strand mimics: are the fibers capped, dissolved or both? (3) Assess blocker peptide efficiency in the presence of large fibril seeds and measure Ab residence time in large fibrils. (4) Compare fibrillogenesis on hydrophobic and hydrophilic surfaces.

The figure above (adapted from Lomakin 1996) describes two main pathways for Ab amyloid fibril initiation and growth and how those equilibria could be influenced by the presence of "blocker" peptides. Path A describes the initiation and growth when Ab is above a critical concentration (~10-30 µM) to form M (micelle). M then rearranges to a protofibril P that can grow and eventually aggregate to form a fibril F. At lower (more biologically relevant) concentrations (nM), M cannot form, so growth must occur from an alternative initiator I as shown in Path B. Initiation in biology is likely to involve cell surfaces, other proteins, and protein aggregates. The accumulation of Ab onto I gives rise to a protofibrillar-like species P’ which can then lead to fibrillar structures F’. The kinetics of Path B probably differ greatly from those of Path A, especially if I is on a surface like a cell membrane. Blocker or inhibitor molecules can interact with all of these species. If one wanted to prevent fibrillogenesis (i.e., formation of F or F’, but not necessarily M or P/P’) then one would design molecules to interact specifically with P or P’. If we want to dissolve already formed fibrils, then we should design molecules that interact with F/F’ and that promote dissolution of Ab from F/F’. In practice, the surfaces of M, P/P’ and F/F’ may be very similar, presenting an extended peptide hydrogen-bonding edge and side-chain groups (hydrophobic or hydrophilic depending on the part of Ab targeted) for interaction with the blocker. Initiators I are expected to be unique and thus may interact very differently with peptide inhibitors. This latter hypothesis lays the groundwork for the research proposed in Aim 4.

b-Sheets and "blocker" design. The -Sheets and "blocker" design. The -Sheets and "blocker" design. The b-sheet is one of three major secondary structures found in proteins The b-sheet can be parallel or anti-parallel with two or more individual strands, which have peptide backbone extended conformations. The dimer has two distinctly different a-carbon environments. We define the endo a-H as the one on the inside of two given strands in a b-sheet conformation, whereas the exo a-hydrogen is the one on the outside of two given strands in a b-sheet conformation. The pleating of the sheet is largely due to the avoidance of crowding by the endo a-H's. Replacing the endo a-H with a larger alkyl group would not allow sheet formation. On the other hand, replacing the exo a-H would not prevent dimerization, but would prevent another extended peptide from adding to the sheet. This is the strategy we have adopted for one of our designs, to replace alternate a-H's with alkyl groups that will prevent further addition of sheets, while still allowing hydrogen bonding to occur on the opposite edge. Substituents larger than -CH₃ in the exo pro-R position are likely to stabilize individual strand extended conformations (Di Blasio 1993) and any substituent larger than a proton in the exo pro-R position will inhibit exo hydrogen-bond-mediated oligomerization. Thus, we plan to test our hypothesis by preparing peptides with alternating a-amino acids and aaAAs, which have substituents larger than -CH₃ groups. 

An alternative way to block one face of extended peptide from forming further b-sheet structure is to replace exo amide hydrogens with alkyl groups. This strategy has been used effectively (Doig 1997) to make a monomeric, three-stranded b-sheet model peptide in which N-methyl amino acids were used on the putative exo positions of the three-stranded b-sheet model. To further lock in the extended conformation, our design involves annulating the exo amide of the N^th residue onto the exo pro-R position of the (N-1)^th residue in the chain to form a six-membered ring lactam 2. This results in a design in which the repeating unit becomes a bridged dipeptide repeat or "extendamer" dipeptide unit (Dpu).

We propose both parallel (Benzinger 1998) and anti-parallel constructs as binders to amyloid by a "like-likes-like" in-registry design. In addition to the blockers proposed here, we will study derivatives with solubulizing groups or "disruptors" (oligo-lysine, oligoglutamate, etc.) at either end of the molecule. As part of Aim 4, we will also study how these disruptors affect kinetics of fibrillogenesis on surfaces varying from hydrophobic, to polar (hydrogen bonding), to charged (anionic and cationic). In a separate study, molecules that inhibit fibril formation according to a fluorescence assay (Ghanta 1996) will be evaluated in the neuronal cell cytotoxicity test. A more advanced design of 1 will use chiral aaAAs with charged groups in the exo pro-R position of the blockers. Charged groups can also be incorporated "in plane" for the extendamer design 2 through modification of the synthesis strategy and conjugation of polar functions onto the piperidone ring by oxime formation. This will provide an orthogonal method for incorporating charged or polar functionality that can enhance solubility of Ab-blockers and/or block initiation of fibrillogenesis at interfaces and may also modulate their bioactivity and bioavailability.

Project 1. Design and synthesis of new b-strand mimics that may potentially block amyloid fibril and protofibril formation. -strand mimics that may potentially block amyloid fibril and protofibril formation. This project can occupy 1 or 2 students, who would ideally take an intense interest in helping with some aspects of Project 2 research as well (see below). We hypothesize that oligomerization of b-sheet structures, including those that lead to amyloid plaques, can be inhibited or reversed by extended peptide structures that have only one edge available for hydrogen bonding. We will concentrate on two different designs for our b-strand mimics: (a) Alternating natural L-amino acids and a,a-disubstituted amino acids (aaAAs) having side-chains larger than methyl (propyl, isopropyl, isobutyl, benzyl, etc.), which are known to favor extended conformations. A key aspect of these structures is that they can only form hydrogen bonds from one edge of the b-strand, as one face is blocked by the pro-R alkyl group of the aaAAs. (b) Dipeptide analogs in which the pro-R position of the N-terminal amino acid is cyclized to the connecting (amide) nitrogen of the C-terminal amino acid to give a piperidone ring. Repetitive structures of these 3-aminopiperid-2-on-1yl) alkanecarboxylate dipeptide analogs, which we refer to as "extendamer" dipeptide units (Dpu), as they have the dipeptide locked into an extended conformation, will again have one face of potential hydrogen bonding sites blocked by the pro-R alkyl group of the piperidone ring. Initial screening for inhibition of large fibril formation will use fluorometric quantitation with thioflavine T.

Project 2. Inhibition mechanism of protofibrillogenesis with b-strand mimics: capping vs. dissolution or both.-strand mimics: capping vs. dissolution or both. This project can occupy 1 student, who would work closely with Project 1 teammates to understand their synthetic objectives. Assays for large fibril formation (e.g. turbidity, Congo Red staining, thioflavine-T fluorescence) are usually chosen to judge the effectiveness of fibrillogenesis inhibitors. To test for inhibition of the much smaller protofibrils it will be necessary to combine scanning probe microscopy and solution-based measurements. To test the hypothesis that potentially toxic protofibril formation can also be inhibited or reversed by "blocker" molecules, we will apply scanning force microscopy (SFM) to study the formation and dissolution of b-amyloid protofibrils in the presence and absence of molecules known to inhibit fibril formation. SFM will be augmented by several solution methods. Standard techniques taught in the MS-I and MS-II courses, such as analytical ultracentrifugation (AU), dynamic light scattering (DLS) and static light scattering (SLS), will be joined by fluorescence photobleaching recovery (FPR). This marks the first application of the highly selective FPR method to amyloid research; it should prove a valuable complement to fluorescence correlation spectroscopy.(Eigen & Rigler, 1994) The inhibiting molecules will include short peptides developed by the Soto (Soto 1996) and Murphy groups (Pallitto 1999), as well as molecules developed in Project 1. Such work may lead to convenient in vitro assays for development of anti-amyloid molecules. We also hypothesize that small, mainly hydrophobic peptides which block fibrillogenesis may do so simply by capping the growing amyloid, whereas peptides with the key hydrophobic core and other hydrophilic groups may be able to block as well as dissolve amyloid fibrils or protofibrils. Alternately, such blocker molecules may actually increase the rates of amyloid aggregation, but with a change in aggregate structure, detectable by scattering (esp. SAXS), that renders them non-toxic.(Pallitto 1999)

Number of IGERT apprentices to be recruited and probable home departments: 2-3 from Chemistry or Biological Sciences (biophysics). At least one student must be synthetic organic (Project 1). Ideally, at least one student would be a "macromolecular generalist"—willing to tackle some synthesis and some macromolecular solution characterization (Project 2).

Consistency with the Macromolecular Education, Research & Training theme: The Ab fragment is itself a macromolecule. It assembles into huge structures, posing a serious characterization challenge that will require students to master a wide range of techniques. The synthetic chemist(s) from Project 1 will need the language and methods of macromolecular science to judge the claims made by the physical/analytical team concerning the effectiveness of their inhibitors. The physical/analytical scientist of Project 2 can hardly ignore the delicate peptide structural details that underly the assembly phenomena and its inhibition, and must have faith bred of experience in the synthetic process that produces the inhibitors.

How does the project form a vector cross-product of existing research themes by the participants?

Existing research directions. Hammer designs and synthesizes peptide-nucleic acid mimics for fundamental study of enzyme inhibition and applications in molecular biology. McLaughlin synthesizes novel constrained amino acid derivatives designed to stabilize known protein secondary structures when incorporated into short peptide sequences; he also studies the antimicrobial and antineurodegenerative activity of these peptides. McCarley is an electrochemist by training, with extensive experience in atomic force microscopy, one of the more illuminating techniques used to study fibril formation.

New research direction. The combination of synthesis at the nanometer scale and physical characterization from nanometer to micrometer scales spans just about the entire macromolecular size range, resulting in a challenging project that no single research group (at LSU or elsewhere) would undertake.

How do students benefit from the team-oriented research, beyond what would be available to them from either advisor separately? Students will enjoy the scientific and social benefits of team-based research on a truly challenging biopolymer project that mixes a goal (inhibition of fibrillogenesis) with fundamental knowledge (fiber characteristics) and discovery (advancement of characterization methods and synthetic routes). This will prepare the academically-bound student for similarly difficult, broad-based biopolymer projects. Students headed for industry (pharmaceutics or polymer characterization are the most likely areas) will be have a facile and marketable knowledge of modern synthesis and characterization tools.

Briefly describe the support level available to each individual faculty or off-campus participant (i.e., without IGERT) The faculty involved are all independently supported. The sum of their individual grants and their share of multi-investigator grants exceeds $1,000,000/year. IGERT students can expect a stable environment with good access to postdocs and other graduate students. Additional grants are already being written (NIH, pending) to pursue in vivo assays of the inhibitors. Here we seek the opportunity to study at a very fundamental level, with a significant discovery component as regards characterization methods, physical parameters of the fibrils and synthetic principles.

Interdisciplinary strengths of the team project: McCarley is interested in polymer behavior at surfaces, as revealed by scanning probe microscopy, but can also direct research in solution methods, since students will be taught these in MS-I and MS-II and since help is available from the secondary advisors (Bricker, Licata and Russo). The daily physical characterizations in the McCarley lab (lower floor, vibration-free for delicate equipment) bear little resemblance to the wet chemistry found in either the Hammer or McLaughlin labs. These are bio-organic, synthetic chemists (top floor, well-ventilated to exhaust fumes). Including the secondary advisors, this group provides a rare combination of biology, synthetic organic chemistry, solution properties and surface structure.

Commitment of faculty & off-campus participants to work side-by-side with apprentices:

Hammer and McLaughlin will work with their student(s) during winter break to prepare a known peptide inhibitor such as those described by Soto and coworkers (Soto 1996, 1998; Poduslo 1999), Nordstedt and coworkers (Tjernberg 1996, 1997, 1999), and/or Murphy and coworkers (Ghanta 1996, Pallitto 1999), using commercially available materials. The project will provide the students with the basic skills to prepare and purify peptides (solid-phase techniques, protecting group chemistry, HPLC analysis and purification, mass spectrometric analysis) without excessive delay in preparing starting materials. This reinforces physical organic course material taught to first-year synthetic chemists (concurrent with MS-I). It will also provide important material for control experiments in the physical characterization projects. Thus the students will learn the value of control experiments and use them to set baselines in subsequent experiments. McCarley will work closely with his new apprentice during winter break, attempting to reproduce the results of Lansbury and Lieber for Ab on mica (Harper 1997) and more recent results on highly oriented pyrolytic graphite (HOPG) (Goldsbury 1999, Kowalewski 1999). This will quickly lead to visual results—a tangible picture of the fibrils—for all team members. The student can begin to master DLS to prepare for measurements of Ab in the absence and presence of an inhibitor molecule described by Murphy and coworkers (the inhibitor can be prepared ahead of time by the LSU Protein Facility, thus no need to wait for Project 1 molecules). This work can be compared to the results of Snyder et. al. (1994) and Pallitto et al. (1999).

Consistent with IGERT guidelines, the proposal has received external funding (from NIH). As IGERT requires students to go into new directions, the following changes are made.

Students attracted to this program may be partly paid by the NIH to do that portion of the work already described. Their IGERT responsibilities are to develop additional means of characterizing the inhibition of A-beta formation, especially: very low angle light scattering, including zero angle depolarized scattering; small angle

References:

Benzinger, T. L., Gregory, D. M., Burkoth, T. S., Miller-Auer, H., Lynn, D. G., Botto, R. E., and Meredith, S. C. (1998) Propagating structure of Alzheimer's .beta.-amyloid(10-35) is parallel . beta .- sheet with residues in exact register Proc. Natl. Acad. Sci. U. S. A. 95, 13407-13412.

Di Blasio, B., Pavone, V., Lombardi, A., Pedone, C., and Benedetti, E. (1993) Noncoded residues as building blocks in the design of specific secondary structures: symmetrically disubstituted glycines and beta-alanine Biopolymers 33, 1037-1049.

Doig, A. J. (1997) A three stranded beta-sheet peptide in aqueous solution containing N-methyl amino acids to prevent aggregation Chem Comm, 2153-2154.

Eigen, M. & Rigler, R. (1994). Sorting single molecules: Application to Diagnostics and Evolutionary Biotechnology. Proc.Natl.Acad.Sci.U.S.A. 91, 5740-5747.

Ghanta, J., Shen, C.-L., Kiessling, L. L., and Murphy, R. M. (1996) A strategy for designing inhibitors of beta-amyloid toxicity J Biol Chem 271, 29525-29528.

Goldsbury, C., Kistler, J., Aebi, U., Arvinte, T., and Cooper, G. J. S. (1999) Watching Amyloid Fibrils Grow by Time-Lapse Atomic Force Microscopy J. Mol. Biol 285, 33-39.

Harding, S.E.; Rowe, A.J.; Horton, J.C. (1992) Analytical Ultracentrifugation Biochemistry and Polymer Science. The Royal Society of Chemistry, Cambridge.

Harper, J. D., Lieber, C. M., and Peter T. Lansbury, Jr. (1997) Atomic Force Microscope Imaging of Seeded Fibril Formation and Fobril Branching by the Alzheimer's Disease Amyloid-b Protein Chemistry and Biology 4, 951-959.

Kelly, J. W. (1998) The environmental dependency of protein folding best explains prion and amyloid diseases Proc Nat. Acad. Sci. USA 95, 930-932.

Kowalewski, T., and Holtzman, D. M. (1999) In Situ Atomic Force Microscopy Study of Alzheimer's b-amyloid Peptide on Different Substrates: New Insights into Mechanism of b-Sheet Formation Proc. Natl. Acad. Sci. U. S. A. 96, 3688-3693.

Lansbury, P. T. (1999) Evolution of Amyoid: What Normal Protein Folding May Tell Us About Fibrillogenesis and Disease Proc. Natl. Acad. Sci. U. S. A. 96, 3342-3344.

Lomakin, A., Chung, D.-S., Benedek, G.B., Kirschner, D.A. & Teplow, D.B. (1996). On the Nucleation and Growth of Amyloid b-protein Fibrils: Detection of Nuclei and Quantitation of Rate Constants. Proc.Natl.Acad.Sci.U.S.A. 93, 1125-1129.

Pallitto, M. M., Ghanta, J., Heinzelman, P., Kiessling, L. L., and Murphy, R. M. (1999) Recognition sequence design for peptidyl modulators of b-amyloid aggregation and toxicity Biochemistry 38, 3570-3578.

Poduslo, J.F., Curran, G.L., Kumor, A., Frangione, B. & Soto, C. (1999). b-sheet Breaker Peptide Inhibitor of Alzheimer's Amyloidogenesis with Increased Blood-Brain Barrier Permeability and Resistance to Proteolytic Degradation in Plasma. J.Neurobiology 39, 371-382.

Snyder, S.W., Ladror, U.S., Wade, W.S., Wang, G.T., Barrett, L.W., Matayoshi, E.D., Huffaker, H.J., Krafft, G.A. & Holzman, T.F. (1994). Amyloid-b Aggregation: Selective Inhibition of Aggregation in Mixtures of Amyloid with Different Chain Lengths. Biophys.J. 67, 1216-1228.

Soto, C., Kindy, M. S., Baumann, M., and Frangione, B. (1996) Inhibition of Alzheimer 's amyloidosis by peptides that prevent b-sheet conformation Biochem. Biophys. Res. Commun. 226, 672-680.

Soto, C., Sigurdsson, E. M., Morelli, L., Kumar, R. A., Castano, E. M., and Frangione, B. (1998) .beta.-Sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer 's therapy Nat. Med. 4, 822-826.

Tjernberg, L. O., Naslund, J., Lindqvist, F., Johansson, J., Karlstrom, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) Arrest of b-amyloid fibril formation by a pentapeptide ligand J Biol Chem 271, 8545-8.

Tjernberg, L. O., Lilliehook, C., Callawya, D. J. E., Naslund, J., Hahne, S., Thyberg, J., Terenius, L., and Nordstedt, C. (1997) Controlling amyloid b-peptide fibril formation with protease-stable ligands J. Biol. Chem. 272, 12601-12605.

Tjernberg, L. O., Callaway, D. J., Tjernberg, A., Hahne, S., Lilliehook, C., Terenius, L., Thyberg, J., and Nordstedt, C. (1999) A molecular model of Alzheimer amyloid b-peptide fibril formation J Biol Chem 274, 12619-25.