Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2.

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

Novel cell-penetrating-amyloid peptide conjugates preferentially kill cancer cells.

The goal of this study was to develop a peptide which could use the toxic effects of amyloid, a substance which is the hallmark of over 25 known human diseases, to selectively kill cancer cells. Here we demonstrate that two separate amyloid-forming hexapeptides, one from the microtubule associated protein Tau involved in formation of paired helical filaments of Alzheimer's disease, and the other an amyloid forming sequence from apolipoprotein A1, when conjugated to a cell penetrating peptide (CPP) sequence, form toxic oligomers which are stable for up to 14 h and able to enter cells by a combination of endocytosis and transduction. The amyloid peptide conjugates showed selective cytotoxicity to breast cancer, neuroblastoma and cervical cancer cells in culture compared to normal cells. Fluorescence imaging experiments showed the CPP-amyloid peptide oligomers formed intracellular fibrous amyloid, visible in the endosomes/lysosomes, cytosol and nucleus with thioflavin S (ThS) staining. Further experiments with rhodamine-conjugated Dextran, propidium iodide (PI), and acridine orange (AO) suggested the mechanism of cell death was the permeability of the lysosomal membrane brought about by the formation of amyloid pores. Cytotoxicity could be abrogated by inhibitors of lysosomal hydrolases, consistent with a model where lysosomal hydrolases leak into the cytosol and induce cytotoxicity in subsequent downstream steps. Taken together, our data suggest that CPP-amyloid peptide conjugates show potential as a new class of anti-cancer peptides (ACPs).

Development of a cost effective and robust AlphaScreen® platform for application.

The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2.5 µg/ml beads, without compromising assay integrity. We demonstrate that reducing bead concentration reduces detection costs substantially while yielding robust statistics. We believe this simple new protocol will enhance the future utilization of AlphaScreen technology in high-throughput screening.

Novel Cell Model for Tauopathy Induced by a Cell-Permeable Tau-Related Peptide.

In the present study, a cell penetrating peptide (CPP)-amyloid conjugate was prepared (T-peptide), where the amyloid-forming sequence was homologous to a nucleating sequence from human Tau protein (306VQIVYK311). Kinetic and biophysical studies showed the peptide formed long-lived oligomers which were taken up by endocytosis and localized in perinuclear vesicles and in the cytoplasm of murine hippocampal neuroblastoma cells and human HeLa cells. Thioflavin S (ThS) staining of amyloid colocalized with pathological phosphorylated Tau, suggesting that the peptide was able to seed endogenous wild-type Tau. Subsequent experiments showed that aggregates present in the lysosomes mediated lysosome membrane permeability (LMP). We observed a decrease in total Tau, irrespective of phosphorylation state, consistent with Tau fragmentation by lysosomal proteases. We found cytotoxicity of T-peptide could be abrogated by inhibitors of lysosomal hydrolases and caspases, consistent with a model where Tau fragments processed by the lysosome leak into the cytoplasm and induce toxicity in subsequent downstream steps. It is our hope that the T-peptide system may prove amenable to the evaluation of small molecule inhibitors of cytotoxicity, especially those which target either Tau aggregation or the lysosomal/autophagy system.

Optimization of a Luminescence-Based High-Throughput Screening Assay for Detecting Apyrase Activity.

Apyrase is a calcium-activated enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and Pi. It is currently used in studies involving cancer and platelet aggregation in humans, as well as herbicide resistance in plants. Inhibitors of apyrase are being investigated for their use to suppress tumors and combat herbicide resistance. Only a few inhibitors of apyrase have been reported, many of which were identified through automated screening using a 96-well plate format and colorimetric phosphate detection. However, these screens have had limitations, including large volumes, inconsistent reproducibility, high incidence of false hits, and lack of higher-throughput compatibility. A luciferin/luciferase-based detection system has been reported to examine potential inhibitors of apyrase; however, these reactions were performed in tubes with the assay completion in seconds, which necessitate the development of a high-throughput screening (HTS)-compatible format for screening. Therefore, a more cost-effective biochemical assay that improved the limitations of the previous assay formats using a commercially available luminescence-based detection system was developed. This new robust mix-and-read platform incorporates a low-volume luminescence-based protocol, formatted for use in 384-well microplates. This new format provides a simple and cost-effective method to screen for apyrase inhibitors and will facilitate larger HTS efforts to identify potent inhibitors of apyrase.