Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction.

Numerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann-Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.

Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells.

Cell-based functional assays are becoming popular in many HTS laboratories because of recent advances in detection and automation technologies. However, the supply of large amounts of live cells with consistent cellular response for day-to-day screening operations over several days/weeks is a tremendous challenge. The high cost of cell culture, labor-intensive nature of the work, and inherent variability in cellular responses from time to time tend to be prohibitive for extensive applications of cell-based assays in HTS. We therefore tested division-arrested cells that were prepared in a single batch and frozen at -80 degrees C before use in several cell-based assays and in a robotic screening campaign. Chinese hamster ovary cells expressing a Gq-coupled receptor were analyzed for the agonist-induced intracellular Ca2+ response measured on a fluorescent imaging plate reader. In this case, the division-arrested cells showed consistent agonist-induced intracellular Ca2+ concentration response as reflected by signal-to-basal ratio and EC50 even 48 h after cell plating. In comparison, the responses from untreated frozen cells and fresh cells declined significantly approximately 30 h after cell plating. In other cell-based assays tested (cyclic AMP assay, reporter gene beta-lactamase assay, and ion-channel assay), the division-arrested cells performed as well as frozen, or fresh cells. We thus conclude that the use of alternate strategies such as frozen cells or division-arrested cells may alleviate the need for several batches of cell plating each day during HTS while maintaining the desired robotic throughput and assay quality.

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader.

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.

A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.

Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.

Identification of small molecule antagonists of the human mas-related gene-X1 receptor.

The recently identified mas-related-gene (MRG) family of receptors, located primarily in sensory neurons of the dorsal root ganglion, has been implicated in the perception of pain. Thus, antagonists of this class of receptors have been postulated to be useful analgesics. Toward this end, we developed a cell-based beta-lactamase (BLA) reporter gene assay to identify small molecule antagonists of the human MRG-X1 receptor from a library of compounds. Single-cell clones expressing functional receptors were selected using the BLA reporter gene technology. The EC50 for the MRG agonist peptide, BAM15, appeared to be comparable between the BLA assay and the intracellular Ca2+ transient assays in these cells. Ultra high-throughput screening of approximately 1 million compounds in a 1.8-microl cell-based BLA reporter gene assay was conducted in a 3456-well plate format. Compounds exhibiting potential antagonist profile in the BLA assay were confirmed in the second messenger Ca2+ transient assay. A cell-based receptor trafficking assay was used to further validate the mechanism of action of these compounds. Several classes of compounds, particularly the 2,3-disubstituted azabicyclo-octanes, appear to be relatively potent antagonists at the human MRG-X1 receptors, as confirmed by the receptor trafficking assay and radioligand binding studies. Furthermore, the structure-activity relationship reveals that within this class of compounds, the diphenylmethyl moiety is constant at the 2-substituent, whereas the 3-substituent is directly correlated with the antagonist activity of the compound.