Biomedical Engineering Reference
In-Depth Information
including neoplastic, cardiovascular, and neurodegenerative diseases. Recent find-
ings point to DNA replication stress downstream of growth signaling by ROS and
other molecules as an equally important factor in aging and the development of these
diseases. The screen provided by W. Burhans is designed to identify compounds that
mimic caloric restriction by downregulating growth signaling pathways, thus redu-
cing the accumulation of superoxide anions and promoting a tighter growth arrest in
G1. The primary readout for this phenotypic screen is the measurement of superoxide
using the fluorogenic compound dihydroethidium (DHE).
Lessons Learned : The cell incubation is 1 week long during which time yeast cells
grow and settle, and liquid from the wells evaporates as well. The plates are covered
and placed in a humidified incubator and then diluted and resuspended for HTS flow
cytometry. The data analysis involves the detection of populations of cells with high
and low level of staining.
4.4.16 Identification of Broad-Spectrum Antifungal Efflux Inhibitors:
R03MH08740601A1 (Phenotypic Cell-Based Multiplex)
Fungal infections, exemplified by oral and invasive candidiasis, cause morbidity and
mortality in an ever-increasing human population: the immunocompromised. Treat-
ment of patients with fungal infections is severely hampered by the development of
antifungal drug resistance. The goal of the project, submitted by R. Cannon, is to
address this health need by discovering novel therapeutic agents that overcome
clinical drug resistance by inhibiting a major cause of antifungal resistance, the drug
efflux pump molecules in the fungal cell membrane. Lead compounds will be
identified by high-throughput screening in which the strains express individual fungal
efflux pumps that will be the targets for the proposed HTS. The yeast cell-based
multiplex approach developed involves the use of three “sentinel” strains in an HTS
that measures efflux of the fluorescent pump substrate Nile red and follow-up
microtiter fluorescent assays with the other panel strains to identify pump inhibitors.
Lessons Learned : The strains provided by R. Cannon highly overexpress the pumps
of interest interfereing with the use of stains to color code the cells. The profiling of
fluorescence substrates at the UNMCMD revealed that since Nile red is pumped by all
the strains [58], mixtures of strains allow detection of transporter inhibition reflecting
one, two, or three strains in the primary assay. However, dose-response studies on
individual strains with active compounds will be required to identify the strains that
respond.
4.5 OPERATIONAL ISSUES
When we entered the pilot phase of the MLI, we had experience with a single
target and screening
10,000 compounds in 96-well plates. Once we transitioned to
384-well plate multiplexes, we can create up to 50,000-100,000 screening data
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