A Novel High-Throughput Screening Tool in Saccharomyces Cerevisiae
Authors:   , Coorey Namal 
Published: November 2021

<p>The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indicative of TORC1 pathway inhibition and translational elongation inhibition, respectively. This was consistent with literature as rapamycin is an inhibitor of TORC1 and it mimics nutrient starvation response and cycloheximide inhibits eukaryotic translational elongation. These results validated the utility of PD-DMA as a hypersensitive genome-wide deletion reagent for chemical genetic profiling. Following validation of the PD-DMA, biochemical assays were performed on latrunculin-A, a less well characterised marine natural product and Plakortide-T a marine natural product with novel activity. These inhibitory drugs were shown to be PDR substrates with biological activity at low nanomolar concentrations. Latrunculin-A, was ~28 fold more potent in the PDR-deficient strain. In contrast, plakortide-T was biologically active only in the PDR-deficient background and the PDR mediated efflux did not involve the major efflux transporters. DNA barcode microarray experiments performed with latrunculin-A identified several hypersensitive deletion mutants consistent with cytoskeletal disruption specifically, actin microfilament assembly. Latrunculin-A is known to bind monomeric G-actin and inhibit actin polymerisation. However, in novel findings tubulin cytoskeleton disassembly was also shown to be mediated by latrunculin-A. Plakortide-T belongs to a class of compounds that disrupts calcium homeostasis. DNA barcode microarray experiments performed identified 56 deletion mutants hypersensitive to Plakortide-T, however, none were involved in calcium homeostasis and the deletion mutants were not over represented in any of the GO terms. Plakortide-T caused hypersensitivity in several deletion mutants of genes encoding for mitochondrial proteins. This activity however, did not generate reactive oxygen species as increased oxidation of free thiols or induction of the oxidative stress response was not observed. Plakortide-T was shown to induce an increase in cytosolic calcium detected by the nuclear localisation of Crz1p, a transcription factor activated in response to increased cytosolic calcium. This activation was dependent on functional calcineurin which further validates this response is an increase in cytosolic calcium.</p>