Method
Briefly we assayed Chlamydomonas growth in 96 and 384 well plates over four days with and without drug. By assaying OD every 30 minutes in our automated Yeast Grower platform, we were able to construct highly detailed growth curves. Drugs that inhibited growth by 35% or more were deemed active algal inhibitors. We also assayed motility/phototaxis and photosynthesis in a short-term exposure screen.
Detailed Materials and methods
Strains and growth conditions
Screens were performed using the Chamydomonas reference strain CC-125 (nit1-, nit2-; a gift from the Dutcher Lab) grown in TAP media (Harris 1989). Cells for screening were inoculated from pre-cultures in which 4 ml of TAP was inoculated from a single colony, cells were cultured to OD600~0.1 for chronic or OD600~0.4 for acute screens.
Growth assays were performed in clear, flat-bottom 96-, and 384-well microtitre plates (Greiner) sealed with adhesive plate seals (Cat. No. AB-0580, AB- gene) using a custom developed platform incorporating microtitre plate reader Safire2 and the Freedom EVO (Tecan-US, Durham, NC). Chronic screens were carried out in 96-, and 384-well microtitre plates at an initial cell density of 1.5X105 cells/ml (OD600~0.1). A final screen concentration of 25 µM (0.5 µg/ml FDA library) with a DMSO solvent concentration at 0.5% in a total volume of 190-200 µl/well was chosen based on initial testing. We found that concentrations of DMSO greater that 0.5%, in liquid assays, resulted in aberrant growth (aggregating cells)(Supplemental Figure S1). Plates were shaken in constant light to saturation at 150 rpm, for 3-4 days, with at least 4 DMSO controls/plate. Algal growth was assayed using a Tecan Safire2 plate reader measuring optical density (OD) every 30 minutes at OD600 nm. OD readings are output to provide detailed growth curves (OD over time) that can distinguish percent variations in growth. We were also able to assess fitness in 1536 well plates as above. Plates were prepared by inoculating 50 µl of cells with drug or solvent to the appropriate concentration, aliquoting 9 µl to each well, and spinning down at 500 rpm for 10 s to remove bubbles.
Acute screens were prepared as the chronic screens except that they were performed only in 96-well microtitre plates at an initial cell density of 2.0X106 cells/ml (OD600~0.4) and incubated between 3-6 hours. Phototaxis response was assayed by placing 96-well plates in a strong directional light (LED strip light, Lumicrest) for 3 minutes. Images for each plate were captured and the assay was repeated at least 15 min later on the other side of the plate, to improve resolution of wells far from the light. Images were scored for inhibitors of motility, no movement, or movement towards light, the opposite of untreated cells. Photosynthetic efficiency was assayed by fluorescent induction in the Safire2 plate reader. Plates were kept in low light prior to the assay. We assessed photosynthetic efficiency by exciting with 470 nm actinic light and measuring the emission at 680 nm, in which a higher reading reports inhibited photosynthesis. To determine cytotoxicity (cytocidal/cytostatic) we pinned treated cells onto 2% agar in 96-well format and allowed them to grow for 10 days at 24°C before they were analyzed.
Chemicals and libraries
The chemical libraries screened were FDA BML-640 (Enzo Life Sciences, Farmingdale, NY), Yactives, a prescreened yeast actives set derived from NOVACore and DIVERSet (SciencesChemDiv and ChemBridge, San Diego, CA) and two plates from NOVACore (ChemBridge, San Diego, CA). TimTec NPL-280 (TimTec LLC, Newark, DE) was supplied at 5.0 mM in DMSO and was a gift from D. Desveaux (University of Toronto). Lugol’s stain was obtained from Sigma (62650-100ML-F, St. Louis, MO) and diluted to 10 µM in water. 10% Formaldehyde (04018, Polysciences, Warrington, PA) was diluted to 1% in water. Clozapine Fluperlapine was ordered from Enzo Life Sciences (BML-NS109, Farmingdale, NY) and diluted to a working stock of 100 mM in DMSO.
Chemical Library Supplier Screened Notes FDA BML-640 Enzo Life Sciences, Farmingdale, NY 640 TimTec NPL-280 TimTec LLC, Newark, DE 280 Yactives Yactives, ChemBridge, San Diego, CA. Total size 7476. 4357 Library derived from NOVACore, SciencesChemDiv; DIVERSet, ChemBridge, San Diego, CA NOVACore NOVACore, SciencesChemDiv, San Diego, CA 168 Screening Chemical Libraries on Chlamydomonas reinhardtii
A compound was considered active on Chlamydomonas reinhardtii if the area under the growth curve after reaching saturation was less than 65% of the DMSO control (ratio [compound/control] <0.65). Automatic flagging of actives was confirmed by visual inspection of the data. Compounds were added to the culture using a 2 µl or 600 nl pin tool (V&P Scientific, San Diego, CA) for 96-well or 384-well microplates, respectively, to dilute the compounds 200 times to a final DMSO concentration of 0.5%.
Cheminformatic Analysis
For all chemicals ECFP_4 similarity was calculated using the cheminformatic package in Pipeline Pilot version 6.1 (Scitegic Inc. Accelyrs, San Diego, CA). Chemicals with a similarity of greater than 0.5 (1.0 being identical) were visualized using Cytoscape version 2.8.2, results from phenotypic screens were added as attributes and were used to alter node shape and color. Marvin version 5.4.1 (ChemAxon, http://www.chemaxon.com) was used for drawing and displaying chemical structures. Naïve Bayes model building was performed as described (Wallace et al., 2011).
Flagellar regeneration
Cells were grown for two days in a 16 hr/8 hr light/dark cycle to an OD600~0.4. Six ml of cell culture was deflagellated using the pH shock method (Witman et al., 1972) by adding 350 µl of 0.5 N acetic acid, inverted for 40 seconds, and neutralized with 125 µl Na2CO3. Deflagellation was confirmed by observation of cells at 40X using a DMIL inverted microscope (Leica). Deflagellated cells were aliquoted into 1.5 ml tubes and drug or solvent was added. Flagella were observed every 20-30 minutes by fixing cells with 6.67X10-3% formaldehyde, a concentration that preserved flagella during the 20-minute observation period. Flagella were observed with the 100X objective and acquired using AxioVision software on an Axiovert 200 M microscope (Carl Zeiss).
HPLC analysis
A 100 ml culture was grown for four days, spun down and resuspended in 20 ml fresh TAP. 1 ml aliquots were treated with drug or solvent for 3 hours. Cells were then washed three times with TAP, resuspended in 50 µl TAP media, transferred to clean tubes 1.5 ml tubes and stored frozen at -20°C. The samples were later lysed with 50 µl SDS-EB buffer (2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0) and incubated at 60°C for 1 hour. Samples were frozen at -80°C and later processed on HPLC as described (Burns et al., 2010).
Burns, A.R., I.M. Wallace, J. Wildenhain, M. Tyers, G. Giaever, G.D. Bader, C. Nislow, S.R. Cutler, and P.J. Roy. 2010. A predictive model for drug bioaccumulation and bioactivity in Caenorhabditis elegans. Nature chemical biology. 6:549-557.
Wallace, I.M., M.L. Urbanus, G.M. Luciani, A.R. Burns, M.K.L. Han, H. Wang, K. Arora, L.E. Heisler, M. Proctor, R.P. St Onge, T. Roemer, P.J. Roy, C.L. Cummins, G.D. Bader, C. Nislow, and G. Giaever. 2011. Compound prioritization methods increase rates of chemical probe discovery in model organisms. Chemistry & Biology. 18:1273-1283.
Witman, G.B., K. Carlson, J. Berliner, and J.L. Rosenbaum. 1972. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. Journal of Cell Biology. 54:507-539