The transmissible spongiform encephalopathies (TSEs) are a class of neurodegenerative diseases that are characterized by proteinaceous deposits in the brain. The deposits consist largely of an abnormal form of prion protein which is highly aggregated and resistant to degradation by proteases. The function of prion protein (PrP) is unknown and its normal form (PrPC) is sensitive to protease digestion. Some of the TSEs include scrapie in sheep, mice, and hamsters, bovine spongiform encephalopahty, and Creutzfeldt-Jakob disease in humans. Animals with scrapie accumulate a disease-specific form of PrP designated PrPSC. The identity of the infectious agent of the TSEs is unclear. No conventional agent or disease-specific nucleic acid has been found and treatments that destroy most normal viruses have no effect. Based on that information the infectious particle has been surmized to consist solely of protein.The "protein only" theory has been strengthened by the discovery of PrP and that preparations of PrPSc are greatly enriched for infectivity. Previously, the simplest system producing protease-resistant PrP was cell culture. Here is described a system that produces protease-resistant PrP in vitro. This system was used to study the mechanism of the conversion of PrPC to the protease-resistant form. A threshold concentration of aggregates of PrP S c was required for conversion. Guanidine-HCI solubilized PrPSC had no converting activity. The need for aggregates and a threshold concentration favors a seeded polymerization mechanism of conversion. The system was also used to study species barriers and different strains of scrapie. Different combinations of mouse and hamster PrPs were used in conversion reactions. The in vitro conversion results correlated with infectivity data on transmission. Scrapie strains with different PrPSc N-terminal protease cleavage sites were used with this system. These different cleavage sites were passed to newly-resistant PrPC depending on which strain was in the reaction. This system provided evidence that a "protein only" theory is compatible with the properties of TSEs.Thesis Supervisor: Dr. Peter T. Lansbury, Jr. Title:Associate Professor of Chemistry Chapter 1 CJD is characterized by dementia, ataxia (a loss of muscle coordination), and the formation of small holes in the brain known as spongiform degeneration.This spongiform degeneration is the result of a loss of neurons. CJD is often accompanied by abnormal proteinaceous deposits known as amyloid plaques.The onset of CJD can be months to years after infection, but once the disease presents itself a gradual deterioration of mental capabilities ensues. The deterioration ends in death usually within a year of onset. Heparin sulfates have been shown to offer some protection to mice inoculated with scrapie,7 but as of now there is no way to stop the deterioration and the disease is always fatal.GSS is a familial version of CJD with different pathology. All GSS cases are accompanied by the formation of amyloid plaques 2 and it i...

The transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by amylold formation in the brain. The major amylold protein is the prion protein (PrP). PrP and the (-amyloid protein of Mzhelmer disease share a similr sequence that, in both cases, may be respondble for the initiation of protein aggregation in Wivo. We report here that a peptide based on this sequence in PrP (PrP96-111M) forms amyloid fibrils. The existence of a kinetic barrier to amyloid formation by this peptide was demonstrated, suggesng that formation of an ordered nudeus Is the rate-determining step for aggregation. Seeding was demonstrated to occur with PrP96-111M amyloid fibrfls but not with amylold fibrils of a related peptide. This effect is consistent with the p al that the agegation of PrP, which characterizes TSE, involves a nucleation event analogous to the seeding of a crytlition.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.

A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

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