Phenotypic HTS multiplex for antifungal efflux pump inhibitors with Validation compound Set
Assay Implementation: J Jacob Strouse, Susan Young, Stephanie Chavez, Dominique Perez, Matthew Garcia, Travis Houston, Keon Ahghar, Terry Foutz, Anna Waller, Annette Evangelisti, Mark Carter, Virginia Salas ..more
BioActive Compounds: 129
UNM CMD Assay Overview:
Assay Support: 1 R03 MH087406-01A1
Project Title: Identification of broad-spectrum antifungal efflux pump inhibitors
PI: Richard Cannon
Screening Center PI: Larry Sklar / UNMCMD
Chemistry Center PI: Craig Lindsley
Assay Implementation: J Jacob Strouse, Susan Young, Stephanie Chavez, Dominique Perez, Matthew Garcia, Travis Houston, Keon Ahghar, Terry Foutz, Anna Waller, Annette Evangelisti, Mark Carter, Virginia Salas
Assay Background and Significance:
Fungal infections, exemplified by oral and invasive candidiasis, cause considerable morbidity and mortality in the immunocompromised. Treatment of patients with fungal infections is severely hampered by the development of antifungal drug resistance [Cannon, et al 2009]. The goal of this project is to address this health need by discovering novel chemical compounds that reverse antifungal drug resistance by inhibiting the drug efflux pump molecules in the fungal cell membrane, overexpression of which is the major cause of resistance in clinical isolates of most Candida species [Niimi, et al. 2004].
Utilizing a a panel of yeast strains expressing efflux pumps from the following clinically relevant fungi: Candida albicans, Candida glabrata, and Candida krusei [Lamping and Cannon, 2010], this approach analyzes three 'sentinel' strains in a phenotypic HTS which measures efflux of the fluorescent pump substrate Nile Red. Follow-up analysis on confirmed HTS hits will be conducted in single-plex assays using each strain individually to identify the target detected in the primary HTS.
Yeast cultures (Saccharomyces cerevisiae strain AD1-8u background), expressing efflux pumps from Candida albicans, namely AD/CaCDR1; AD/CaCDR2 and AD/CaMDR1, and the control strain containing the empty pABC3 cassette without a heterologous efflux pump gene, AD/pABC3, (all supplied by the assay provider) are incubated at 30 degrees C with shaking (250 rpm) until an optical density (OD 600) of 1.5 to 2.0 is obtained. All cultures are adjusted to be at an equivalent OD of 0.6. The cells are collected by centrifugation and washed with PBS, then re-suspended as a tri-plex mixture in PBS with 10 microM Nile Red (Invitrogen USA). The mixture is placed at 4 degrees C to equilibrate with efflux pumps effectively turned off. Wells containing 5 microL of PBS with 2 percent glucose and 20 microM compound are plated during this time. Enniatin B is used as an on-plate positive control at 25 microM. Vector (AD/pABC3, stained in the same protocol as the tri-plex) is used as an off-plate control. The compound/buffer wells receive 5 microL of stained tri-plex mixture. In-well reagent summary: 5 microM Nile Red, 10 microM compound, 1 percent glucose. The assay plate is incubated inverted 30 degrees C for 1 hr. Accumulation of Nile Red is measured by sample delivery via HyperCyt (IntelliCyt, USA) to a Cyan (Beckman Coulter, USA) flow cytometer. Samples are excited with a 488 nm laser and a PE-Texas Red filter is used to detect the fluorescence signal from the Nile Red substrate.
The data are exported into a Microsoft Excel spreadsheet template, and the PERCENT_RESPONSE is calculated for each well as follows:
PERCENT_RESPONSE = 100((MFItest-MFIneg.control)/(MFIpos.control-MFIneg.control))
where MFI is median fluorescence intensity of cells with test compounds, positive or negative control wells.
RAW_MEDIAN = median fluorescence intensity of cells per well
Cutoff for determining compound as active is based on compound having response value larger than 4 standard deviations of the overall response values measured for the entire Validation compound set, i.e., 0.02 + 4*0.13 = 0.42.
The PUBCHEM_ACTIVITY_SCORE is determined by multiplying response value with 100. Meaning Active compounds have PUBCHEM_ACTIVITY_SCORE greater than 42.
** Test Concentration.
Data Table (Concise)