Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening.

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme.

Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency.

Highly active antiretroviral therapy (HAART) has improved the outlook for the HIV epidemic, but does not provide a cure. The proposed "shock-and-kill" strategy is directed at inducing latent HIV reservoirs, which may then be purged via boosted immune response or targeting infected cells. We describe five novel compounds that are capable of reversing HIV latency without affecting the general T-cell activation state.

Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs).

Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B.

An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B.

Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists.

Development of fluorescent substrates and assays for the key autophagy-related cysteine protease enzyme, ATG4B.

The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis.

Ligand electronic properties modulate tau filament binding site density.

Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimer's disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (B(max)). Previously we identified polarizability as a descriptor of protein aggregate binding affinity.

Ligand polarizability contributes to tau fibril binding affinity.

Whole brain imaging of tau-bearing neurofibrillary lesions has the potential to improve the premortem diagnosis and staging of Alzheimer's disease. Diverse compounds with high affinity for tau aggregates have been reported from high-throughput screens, but the affinity driving features common among them have not been determined. To identify these features, analogs of compounds discovered by high-throughput screening, including phenothiazine, triarylmethine, benzothiazole, and oxindole derivatives, were tested for their ability to displace fluorescent thioflavin dyes from filaments made from recombinant tau protein or authentic paired helical filaments purified from Alzheimer's disease tissue.

Modulation and detection of tau aggregation with small-molecule ligands.

Recent results from high-throughput and other screening approaches reveal that small molecules can directly interact with recombinant full-length tau monomers and fibrillar tau aggregates in three distinct modes. First, in the high concentration regime (>10 micromolar), certain anionic molecules such as Congo red efficiently promote tau filament formation through a nucleation-elongation mechanism involving a dimeric nucleus and monomer-mediated elongation. These compounds are useful for modeling tau aggregation in vitro and in biological models.

Structure-activity relationship of cyanine tau aggregation inhibitors.

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3'-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration.

Small-molecule mediated neuroprotection in an in situ model of tauopathy.

Small-molecule inhibitors of neurofibrillary lesion formation may have utility for treatment of Alzheimer's disease and certain forms of frontotemporal lobar degeneration. These lesions are composed largely of tau protein, which aggregates to form intracellular fibrils in affected neurons. Previously it was shown that chronic overexpression of human tau protein within identified neurons (anterior bulbar cells) of the sea lamprey induced a phenotype-resembling tauopathic neurodegeneration, including the formation of tau filaments, fragmentation of dendritic arbors, and eventual cell death.

Tau aggregation and toxicity in tauopathic neurodegenerative diseases.

Since its discovery as a structural component of neurofibrillary lesions of Alzheimer's disease more than twenty years ago, tau protein has been implicated in the cascade of events associated with neurodegeneration. Specifically, the "tau hypothesis" posits that misfunction of tau, which occurs in response to unknown stimuli, results in its intracellular assembly into filaments that eventually prove toxic to the cells that produce them. The tau hypothesis is supported by numerous neuropathological and genetic observations of authentic human disease cases.

Potent inhibition of tau fibrillization with a multivalent ligand.

Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy.

Differentiating Alzheimer disease-associated aggregates with small molecules.

Alzheimer disease is diagnosed postmortem by the density and spatial distribution of beta-amyloid plaques and tau-bearing neurofibrillary tangles. The major protein component of each lesion adopts cross-beta-sheet conformation capable of binding small molecules with submicromolar affinity. In many cases, however, Alzheimer pathology overlaps with Lewy body disease, characterized by the accumulation of a third cross-beta-sheet forming protein, alpha-synuclein.

Disulfide connectivity and reduction in pheromone-binding proteins of the gypsy moth, Lymantria dispar.

Males of the gypsy moth, Lymantria dispar, are attracted by a pheromone released by females. Pheromones are detected by olfactory neurons housed in specialized sensory hairs located on the antennae of the male moth. Once pheromone molecules enter the sensilla lymph, a highly abundant pheromone-binding protein (PBP) transports the molecule to the sensory neuron.

Structure-activity studies with pheromone-binding proteins of the gypsy moth, Lymantria dispar.

Pheromone olfaction in the gypsy moth, Lymantria dispar, involves accurate distinction of compounds with similar structure and polarity. The identified sex pheromone is (7R,8S)-2-methyl-7,8-epoxyoctadecane, 1a, and a known antagonist is (7Z)-2-methyloctadec-7-ene, 4a. The first step in pheromone olfaction is binding of odorants by small, soluble pheromone-binding proteins (PBPs), found in the pheromone-sensing hairs.