External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

Comment: Data analysis method and criteria for scoring active compounds:

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: HMS1419

Protocol: MELAS human cybrid cells: derived from bone, osteosarcoma; mt-tRNA mutation is m.3243A>G, MTTL1
Rieske KO cells: derived from connective tissue, fibroblasts; Rieske/UQCRFS1 knock-out

Prior to screening, cells were passaged in high glucose DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and 50 ug/ml uridine. Media was changed daily to prevent starvation and maintain cellular health.

On the day of screening, columns 1 & 3-23 of assay plates (Corning 3570) were filled with 30 uL of MELAS or Rieske KO cell suspension (50 cells/ul). Media for resuspension was DMEM (2.5 mM glucose, 22.5 mM galactose) supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and 50 ug/ml uridine. Positive control columns were 2 and 24 for most plate setups and media was DMEM (10 mM glucose, 15 mM galactose) with 10% FBS, 1% P/S, 1 mM pyruvate, and 50 ug/ml uridine. Next, 33 nL of each compound were pin-transferred to each plate in duplicate. Final assay well volume was 30 uL. Plates were stacked 2 high, covered with lids, and incubated at 37 degrees C for 2-3 days depending on cell line: MELAS 2 days, Rieske KO 3 days.

Library plates were screened in duplicate for each cell type, with all assay plates in a given set prepared on the same day.. After 2-3 days of incubation, CellTiter-Glo (10 ul/well) was added to assay plates and luminescence was read using a PerkinElmer EnVision plate reader.

Positive control: 10 mM glucose
Negative control: 2.5 mM glucose

Comment: Analysis and positive scoring method:

Crosstalk corrected luminescence values were normalized as Z-scores using the plate average and standard deviation of non-control well values (empty and compound). Each plate replicate was individually analyzed to measure reproducibility across plates. Z-scores were then averaged across replicates for both conditions (MELAS and Rieske) and an average Z-score threshold of 1.96 was selected for activity. Compound activity scores were derived by scaling replicate average MELAS Z-scores from 0 (0% activity; Z-score <= 0) to 100 (100 % activity; Z-score >= 4), with activity score of 50 having Z-score = 2. Compounds with an activity score >= 50 were considered active for MELAS; some were scored active for Rieske despite having an activity score < 50 since this value was based only on the MELAS condition Z-scores.