The most sensi tive strains are hfi1 and spt20, displaying sensitivity at 25 uM CG 1521. Most deletion mutants demonstrate the same sensitivity to CG 1521 as in the initial screen, however, the sensitivity of the hfi1 mutant is enhanced compared to the screen and yeast deletion mutants ngg1 CAL-101 and spt3 show slightly increased sensitivity. To confirm that the sensitivity of the gcn5 strain is due to the loss of GCN5, the sensitivity of the GCN5 complemented strain was compared to the BY4741 wild type and the gcn5 strain. Complementation with GCN5 results in a similar level of resistance as the wild type, highlighting an important role for Gcn5 in modulating the biological response to CG 1521.

To as sess the importance of the acetyltransferase function of Gcn5 in the attenuation of CG 1521 activity, the sensi tivity of the catalytic site mutant Gcn5 E173Q, which has minimal residual catalytic activity, was mea sured in liquid culture. As shown in Table 3, compared to the wild type, the gcn5 mutant is sensitive at 25 and 50 uM CG 1521. The E173Q catalytic site mutant is sen sitive to CG 1521, but to a lesser extent than the gcn5 mutant, suggesting that functions other than the acetyltransferase activity of Gcn5 play a role in the response to CG 1521 and may be sufficient to maintain cell growth. CG 1521 treatment results in G0 G1 delay and deletion of GCN5 increases susceptibility to cell death Based on the reported involvement of Gcn5 in cell cycle, the effects of CG 1521 on cell cycle progression in wild type and gcn5 cells were compared.

The growth inhibitory effect of CG 1521 is more pronounced in the gcn5 strain than in the wild type strain as determined on agar plates and in liquid culture. Cell cycle analysis shows that CG 1521 induces G0 G1 arrest in both strains. Treatment with 50 uM CG 1521 leads to a significant increase in the G0 G1 population after 1 h and 2 h for the wild type and the gcn5 strain re spectively, indicating that the growth arrest is delayed in the gcn5 strain compared to the wild type strain. At 4 h, the G0 G1 population increases by 1. 8 fold after treatment with CG 1521 in both the wild type and the gcn5 strain. The induction of G0 G1 delay by CG 1521 was confirmed by budding index analysis. Treatment with 50 uM CG 1521 reduces the budding index by approximately 50% in both wild type and gcn5 strains by 2 h to 4 h.

As a positive control for G1 arrest, both strains were treated with 5 ug mL factor, which reduces the budding index to approximately 0. 1 after 2 h in both strains. CG 1521 significantly induces cell death in both gcn5 and wild type strain, as measured by propidium idodide uptake using flow cytometry. As shown in Figure 7, the gcn5 strain displays GSK-3 increased susceptibility to CG 1521 induced cell death compared to the wild type strain.