C. albicans biofilm killing---Mixture HTS
This screen is for compounds that are potentiators of the antifungal drug clotrimazole that are active against multidrug tolerant persister cells of Candida albicans biofilms. Biofilms are notoriously resistant to antimicrobial therapy, but the mechanism of resistance remains largely unknown. The recently characterized multidrug tolerant subpopulation of cells within biofilms, known as more ..
BioActive Compounds: 19
Molecular Library Screening Centre Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Kim Lewis, Northeastern University, Boston, MA
MLSCN Grant: X01 MH080686-01
This screen is for compounds that are potentiators of the antifungal drug clotrimazole that are active against multidrug tolerant persister cells of Candida albicans biofilms. Biofilms are notoriously resistant to antimicrobial therapy, but the mechanism of resistance remains largely unknown. The recently characterized multidrug tolerant subpopulation of cells within biofilms, known as persisters, have been found to be similar to specialized bacterial survivor cells. Even the most effective antifungals, such as amphotericin B, fluconazole, and caspofungin are completely inactive against C. albicans persisters. However, a molecule that disables the mechanism of persister formation or maintenance could synergize with a conventional antimicrobial and lead to biofilm eradication.
To identify potentiators of clotrimazole we have employed a whole cell screen of wild type C. albicans biofilms for primary HTS of the MLSCN compound library. Active compounds inhibit the metabolic activity of the biofilm as measured by Alamar Blue reduction. Screening of 84,889 compounds in 384-well plates was completed earlier and reported in AID 834. Here we report the results of screening an additional 121,267 compounds as mixtures of 4 compounds per well in 384-well plates. Based on the low hit rate of 0.14% in the earlier HTS we reasoned that mixture screening would accelerate the primary HTS four-fold while requiring the retest of no more than 800-1200 individual compounds (200-300 mixtures).
Mixtures were tested in the primary HTS at a concentration of 30.4 uM, corresponding to a concentration of 7.6 uM for each individual compound. The four compounds contained in each mixture that showed >30.3% inhibition were reordered from BioFocus DPI and retested individually at a concentration of 61 uM. Compounds that gave >30.3% inhibition on retest will be tested in dose-response both in the presence and absence of clotrimazole. Those compounds that inhibit only in the presence of clotrimazole will be selected for further studies of biofilm killing and persister elimination.
A portion of the MLSCN library was screened as mixtures of 4 compounds per well in 384-well plates. Compounds were supplied by BioFocus DPI as 2.5-mM solutions in DMSO stored in 1536-well V-bottom plates. Mixtures were generated using a pintool (384-pin, V&P Scientific) to transfer 114 uL of DMSO solution from all four quadrants of each 1536-well plate into a single 384-well compound dilution plate containing 25 uL per well of media:
Wells A7, A9, C7, C9, etc. (1536-well) transferred into A5, A6, B5, B6, etc. (384-well)
Wells A8, A10, C8, C10, etc. (1536-well) transferred into A5, A6, B5, B6, etc. (384-well)
Wells B7, B9, D7, D9, etc. (1536-well) transferred into A5, A6, B5, B6, etc. (384-well)
Wells B8, B10, D8, D10, etc. (1536-well) transferred into A5, A6, B5, B6, etc. (384-well)
Thus the 2.5-mM solutions in each 1536-well plate are mixed 4-per-well in the 384-well compound dilution plate as follows :
Wells A7, A8, B7, B8 are all added to well A5
Wells A9, A10, B9, B10 are all added to well A6
Wells C7, C8, D7, D8 are all added to well B5
Wells C9, C10, D9, D10 are all added to well B6
(Columns 1-4 and 45-48 in each 1536-well plate were empty; therefore columns 1-2 and 23-24 in the corresponding 384-well compound dilution plate did not contain compounds.)
Final mixture composition:
Compound dilution plate: 11.4 uM per compound, 45.6 uM total mixture, in 1.8% DMSO
Assay plate: 7.6 uM per compound, 30.4 uM total mixture, in 1.2% DMSO.
C. albicans---Stocks were provided by Michael Lafleur, Northeastern University. Wild type Candida albicans strain CAF4-2 was streaked onto YPD media (10 g/L yeast extract; 20 g/L bacto peptone) and incubated at 30 C for 48 hr. A single colony from the YPD plate was used to inoculate 100 mL liquid YPD in a 1 L baffled Erlenmeyer flask and culture was incubated at 30 C for 24 hr. Cells were concentrated by centrifugation and resuspended in 15% glycerol in phosphate buffered saline. Stocks of the suspension in 15% glycerol were divided into single-use aliquots and frozen in liquid nitrogen and stored at -80 C.
Growth media---RPMI 1640 with L-glutamine, Phenol Red, glucose, 165 mM MOPS, pH 7.0, prepared as follows:
HyClone HyQRPMI-1640 (Cat. No. SH3001104, from Fisher)
MOPS (Fisher BP308-500)
Dissolve 10.4 g of RPMI in 950 mL water, add 34.53 g MOPS. Adjust pH to 7.0 with NaOH, bring volume up to 1 L, filter sterilize.
Phosphate buffered saline (Sigma P44714-100TAB)
Alamar Blue (Serotec BUF012B available from Fisher)
Clotrimazole (Sigma C-6019)
Assay plate---Black 384-well plate (Greiner 781086)
Compound dilution plate---Polypropylene 384-well V-bottom plate (Greiner 781280)
Compound storage plate---Polypropylene 1536-well V-bottom plate (Greiner 782270)
C. albicans biofilms were grown in 384-well plates for 24 hr at 37 C. Media was removed and replaced by media containing clotrimazole. Compounds were transferred by pintool into a separate plate containing media and clotrimazole (50 ug/mL). The mixture of media, clotrimazole and test compound was transferred into the biofilm-containing plates and biofilms were incubated for a further 48 hr at 37 C. Media was removed, Alamar Blue was added, and incubated for 2 hr at 37 C. Fluorescence was read on a Perkin Elmer Envision microplate fluorimeter (excitation 535 nm, emission 590 nm).
1. Thaw frozen C. albicans stock. Dilute into RPMI media to OD (600) = 0.1.
2. Dispense C. albicans in media (30 uL) into assay plate using Multidrop.
3. Centrifuge and incubate assay plate for 24 hr at 37 C.
4. Add 25 uL of RPMI media and clotrimazole (50 ug/mL) to compound dilution plate
5. Transfer 2.5-mM compound in DMSO (114 nL) from all four quadrants of a 1536-well plate into 384-well compound dilution plate using pintool (384-pin, slotted, 100 nL).
6. Remove media from assay plate by inverting plate and flicking.
7. Add 10 uL of RPMI media and clotrimazole (50 ug/mL) to assay plate.
8. Transfer compound in media (20 uL) from compound dilution plate to assay plate.
9. Centrifuge and incubate assay plate for 48 hr at 37 C.
10. Remove media from assay plate by inverting plate and flicking
11. Add 30 uL of 1% Alamar Blue in phosphate buffered saline to assay plate
12. Centrifuge and incubate for 2 hrs at 37 C.
13. Read fluorescence (Excitation 535/ Emission 590)
As HTS protocol except for step 5:
5. Transfer 10-mM compound in DMSO (2 x 114 nL) from 384-well polypropylene storage plate into 384-well compound dilution plate using pintool (384-pin, slotted, 100 nL).
Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds in columns 3-22, controls (biofilm, no compound) in columns 2 and 24, and blanks (biofilm & lethal mixture of 50 ug/mL clotrimazole and 200 ug/mL chlorhexidine) in columns 1 and 23. HTS percent inhibition was calculated for each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% Inhibition = 100*(1-((signal mean-blank mean)/ (control mean-blank mean)))
Activity scores were calculated as follows:
For percent inhibition >30.3 in primary HTS and >100 on retest:
Score = 40
For percent inhibition >30.3 in primary HTS and between 100 & 0 on retest:
Score = 0.4 x percent inhibition on retest
For percent inhibition >30.3 in primary HTS and compound unavailable for retest:
Score = 0.3 x Percent inhibition in primary HTS
For percent inhibition >30.3 in primary HTS and <0 on retest:
Score = 0
For percent inhibition between 30.3 & 0 in primary HTS:
Score = 0.4 x Percent inhibition in primary HTS
For percent inhibition <0 in primary HTS:
Score = 0
Activity outcome is reported as follows:
(1) Percent inhibition >30.3 in primary HTS, >30 on retest = active
(2) Percent inhibition >30.3 in primary HTS, <30 on retest = inactive
(3) Percent inhibition >30.3 in primary HTS, compound unavailable for retest = inconclusive
(4) Percent inhibition <30.3 in primary HTS = inactive
ANALYSIS OF SCREENING RESULTS
Primary HTS plate statistics were as follows:
Screening concentration = 7.6 uM (each compound in 4-compound mixture)
30.4 uM (total concentration of mixture)
Number of plates = 104
Mean Z-factor = 0.6
Mean control percent CV = 8.85
Maximum control percent CV = 19.3
A hit cut-off of 30.3% inhibition was selected. Based on this cutoff, a hit rate of 0.27% was observed (assuming that observed activity originated from only one compound in each 4-compound mixture):
Hits (>30.3% inhibition): 327 wells equivalent to 1280 compounds
Inactives (<30.3% inhibition): 33,280 wells equivalent to 121,267 compounds
Retest plate statistics were as follows:
Screening concentration = 61 uM
Number of plates = 4
Mean Z-factor = 0.37
Mean control percent CV = 10.0
Maximum control percent CV = 13.4
A hit cut-off of 30% inhibition was selected. Based on this cutoff, the retest rate of all 1280 compounds was 1.5%. However, given the likelihood that activity observed in the primary HTS originated from only one compound in each 4-compound mixture, the retest rate becomes 5.8% based on the 327 mixtures activity in the primary HTS:
Hits (>30% inhibition): 19
Inactives (<30% inhibition): 1261
This assay was submitted to the PCMD (Scott Diamond, Director; University of Pennsylvania) by Kim Lewis (PI) and Michael Lafleur, Northeastern University, Boston MA. Assay development and HTS was carried out by Edinson Lucumi and data were submitted by Andrew Napper.
Please direct correspondence to Andrew Napper (email@example.com).
** Test Concentration.
Data Table (Concise)