Lopac1280

The Library of Pharmacologically Active Compounds (LOPAC¹²⁸⁰, Sigma-Aldrich) was plated in 1536-well format with an Evolution P3 liquid dispenser (PerkinElmer) in columns 5–48 of 1536-well clear polypropylene compound plates (Greiner Bio-One). Five stock concentrations were tested: 10 mM, 2 mM, 400 µM, 80 µM, and 3.2 µM. General compound library preparation in 1536-well format, compound storage, and further details of compound management have been described previously [41] (link).
1536-well control compound plates contained either DMSO or varying concentrations of GKA-EMD in DMSO in columns 1–4 added using a Cybi-Well (CyBio, Jena, Germany). Column 4 contained 26 mM GKA-EMD (n = 16 wells) while column 2 contained a 1.5-fold 11-point serial dilution of GKA-EMD (n = 2 for each concentration, starting concentration 26 mM); all other wells contained DMSO. For all screening experiments, 23 nl from control plates or compound plates were pin-transferred into each well of the 1536-well plate using a Kalypsys PinTool equipped with a 1536-pin head (Wako Chemicals USA, Richmond, VA, USA) [42] (link). Immediately following compound addition, a pre-read was taken utilizing the appropriate detection wavelengths to detect compound autofluorescence using a ViewLux Microplate Imager (PerkinElmer).

Cell culture reagents were from Invitrogen. Lipofectamine 2000 was from ThermoFisher. Libraries were sourced as follows: LOPAC₁₂₈₀ (Sigma), the Pathogen Box (Medicines for Malaria), Scripps Drug Discovery Library (Scripps Institute). FLIPR Calcium 5 assay kits were from Molecular Devices. Additional supply of AG1 (3-(3,4-Dimethoxyphenyl)-6-(3-propan-2-ylphenyl)-[1,2,4]triazolo[4,3-a]pyridine; PubChem CID:122196572, C₂₃H₂₃N₃O₂) was sourced from Evotec and ANT1 (1-(9H-fluoren-9-yl)-4-(5-methyl-3-phenyl-1,2-oxazole-4-carbonyl)piperazine; PubChem CID:2813918, C₂₈H₂₅N₃O₂) was sourced from Maybridge. All other routinely used chemical reagents were purchased from Sigma.

The LOPAC 1280 (LOPAC stands for library of pharmacologically active compounds) collection of 1,280 pharmacologically active compounds was purchased from Sigma-Aldrich. A diverse collection of ∼26,000 compounds was selected from various commercial suppliers by applying “lead-like” filters in silico (e.g., logarithm of a compound's partition coefficient between n-octanol and water [cLogP] of 0 to 4, polar surface area [PSA] of <120, H-bond donors of <3, H-bond acceptors of <6, freely rotatable bonds of <6). A kinase-focused set of 8,100 compounds was purchased from Biofocus and ChemDiv.

A small‐molecule compound library (LOPAC^®1280; Sigma‐Aldrich LO4200) was used for screening. All the compounds were used at 100 µM for primary screening. Compounds were mixed with 5 pmol DIG‐labeled ε WT for the reaction in each well and incubated at R.T. for 3 hr. After washing three times with the wash buffer, 100 µl of anti‐DIG‐POD antibodies (final concentration: 60 mU/ml) were added and the wells were incubated at 37°C for 1 hr. The luminescence was finally measured as above. All the compounds which showed over 80% inhibition of binding between Strep‐TP‐spacer‐RT‐His8 and ε RNA were used for the second time screening. For the second time screening, each of the compounds was diluted threefold at the indicated concentration (see Figure 3). 6‐hydroxy‐DL‐DOPA (DL‐DOPA, Sigma H2380) and N‐oleoyldopamine (OLDA, Sigma O2139) were purchased from Sigma‐Aldrich. The assay was performed at least three times, and the data are shown as the mean value with the standard deviation.

The Library of Pharmacologically Active Compounds library (LOPAC¹²⁸⁰, Sigma-Aldrich) contains 1,280 compounds with well-known pharmacological properties that target a wide range of cellular activities. Many compounds in this library have been approved for use in the clinic, which greatly increases the possibility of re-purposing relatively non-toxic and cheap drugs for new treatment of cancers. Canine kidney epithelial MDCK cells were transduced to stably express a GFP-tagged N-Ras (GFP-N-Ras). These cells were seeded in 96-well plates to which 10 µM of each compound was added. The cells were incubated with the drug for 2 days before being analyzed by microscopy, for which we have developed an algorithm to identify individual GFP⁺ cells in order to calculate GFP levels in the cell in an automated fashion^{25 (link)}.