Figure 1.
Disease model and HTS strategy.
A. Hypothetical molecular basis for PGL tumorigenesis in cells accumulating succinate due to SDH mutations. Putative tumor suppressor functions of Fe/O2/2KG-dependent dioxygenases in Hifα degradation and epigenetic regulation of histone methylation and 5-methylcytosine hydroxylation are subject to Su inhibition in PGL. B. A simple PGL tumor model is provided by haploid yeast carrying sdh2Δ gene disruption. HTS for compounds differentially toxic to sdh2Δ mutant yeast is performed in three stages. I: Identification of compounds inhibitory to the growth of sdh2Δ mutant yeast; II: Assessment of differential growth inhibition of WT vs. sdh2Δ mutant yeast using repurchased compounds identified in stage I; III: confirmation of differential growth inhibition of sdh2Δ mutant yeast vs. WT and an irrelevant nhp6AΔ gene disruption strain carrying the same NatR selectable marker as the sdh2Δ strain.
Table 1.
Saccharomyces cerevisiae strains used in this study.
Figure 2.
Experimental yeast growth curves on (A) galactose medium; (B) glycerol medium; (C) 1∶1 galactose:glycerol medium.
WT (solid black), sdh2Δ (dashed red). nhp6AΔ, (solid grey).
Table 2.
HTS resultsa.
Figure 3.
Example growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of WT relative to sdh2Δ mutant yeast for two compounds.
A. 4466-0038 (12 µM). B. 3448-8292 (50 µM). Solid lines: growth in the absence of drug for WT (black) and sdh2Δ mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.
Figure 4.
Structures and yeast growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of sdh2Δ mutant yeast for two compounds from the LOPAC library.
A. Disulfiram (100 µM). B. Dequalinium (100 µM). Solid lines: growth in the absence of drug for WT (black) and sdh2Δ mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.
Figure 5.
Structures and yeast growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of sdh2Δ mutant yeast for four non-reactive compounds from main library HTS.
A. 7619814 (50 µM). B. 7172827 (50 µM). C. 7783421 (25 µM). D. 4032-1245 (6 µM). Solid lines: growth in the absence of drug for WT (black) and sdh2Δ mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.
Figure 6.
Structures and yeast growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of sdh2Δ mutant yeast for six nitro-alkene compounds (potential Michael acceptors) from main library HTS.
A. 6035147 (12 µM). B. 6988322 (25 µM). C. 7241889 (6 µM). D. 7279172 (50 µM). E. 7289669 (3 µM). F. 7312219 (25 µM). Solid lines: growth in the absence of drug for WT (black) and sdh2Δ mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.
Table 3.
Compounds of interest and characteristics.
Figure 7.
Behavior of representative inhibitor 7279172 on growth of yeast sdh2Δ pdr5Δ mutant before and after rescue by plasmid-borne SDH2.
Growth of WT (black) vs. sdh2Δ pdr5Δ mutant (red) vs. sdh2Δ pdr5Δ mutant rescued by plasmid-borne SDH2 (blue) on (A) galactose:glycerol (1∶1) medium; (B) glycerol medium; or (C) galactose:glycerol (1∶1) medium in the presence of 25 µM 7279172.
Figure 8.
Behavior of representative inhibitor 7279172 on growth of yeast sdh1Δ mutant (no pdr5Δ mutation).
Growth of WT (black) vs. sdh1Δ mutant (red) on (A) galactose:glycerol (1∶1) medium; (B) glycerol medium; or (C) galactose:glycerol (1∶1) medium in the absence (solid) or presence (dashed) of 25 µM 7279172, showing differential sensitivity of sdh1Δ mutant.
Figure 9.
In vitro yeast alcohol dehydrogenase enzyme inhibition assays for ten compounds that differentially inhibit growth of sdh2Δ mutant vs. WT yeast.
Figure 10.
Comparative growth inhibition of WT yeast, sdh2Δ mutant yeast, and sdh2Δ adh1Δ double mutant yeast by various concentrations (0, 10, 20, 40 µM) compound 7279172 as an example showing sensitization of sdh2Δ adh1Δ double mutant yeast to alcohol dehydrogenase inhibitors.
Effects of the test compound on maximal culture saturation (left) and time to saturation (right) are normalized to growth in the absence of the compound.
Figure 11.
Mammalian cells knocked down for SDHB expression are differentially sensitive to the lactate dehydrogenase inhibitor oxamate.
A. Western blot demonstrating stable lentiviral shRNA knockdown of SDHB in HEK293 cells by SDHB-specific shRNA constructs pJ1824 and pJ1825 but not scrambled shRNA. B. Differential 10 mM oxamate inhibition of HEK293 cell growth after SDHB knockdown.