Protein interfaces
A protein and compound technology, applied in the field of protein interface
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Embodiment 1
[0238]Example 1: Method for identifying disruptors of protein-protein interactions.
[0239] To achieve stabilization of short peptides, terminator sequences and untranslated regions (UTRs) or short proteins (such as sORF1) are used. The N-terminal rule for protein stability was used so that the peptide sequence began with a residue that minimized proteolysis (eg, Met, Gly, Ala, Ser, Thr, Val, or Pro).
[0240] PPI integration plasmid (plasmid 1; Figure 7 ) contains two restriction sites enabling the integration of the two proteins constituting the PPI of interest. The plasmid encodes AD (such as Dof1; Figure 8 (below)) and DBD (such as TetR; Figure 8 (above)) fusions to each protein driven by a strong promoter and terminator such as ADH1 or by an inducible promoter such as GAL1. Each protein fusion sequence is tagged with FLAG or HA. The plasmid also contains bacterial selection and reproduction markers (ie ori and AmpR), and yeast replication and selection markers (i...
Embodiment 2
[0254] Example 2: Treatment of SSP-25 cells (cholangiocarcinoma) with compounds disclosed herein.
[0255] To assess the growth inhibitory properties of the compounds disclosed herein, the growth inhibition of SSP-25 cells at 5 μM or 10 μM of Compounds 1, 3 and 5 was tested using the MTT assay relative to water mock treatment after 48 hours of cell growth. figure 1 It was shown that compounds 1 and 3 had dose-dependent effects on the growth of SSP-25, while compound 5 had the strongest inhibitory effect independent of concentration.
[0256] Figure 13 Shows the results of Compound 5 treatment, which was shown by xCELLigence TM Instrumentally measured acute cell death kinetics in SSP-25 cells. First, 5000 SSP-25 cells were inoculated and used xCELLigence TM The instrument measures the current impedance through the plate in real time to measure adhesion and growth for 20 hours. At the 20 h mark, 30 μM Compound 5 or PBS control was added to SSP-25 cells, which were cultured...
Embodiment 3
[0257] Example 3: Treatment of PC3 cells (mCRPC cells) with compounds disclosed herein.
[0258] Figure 14 Shows the results of Compound 5 treatment, which was shown by xCELLigence TM Instrumentally measured acute cell death kinetics in mCRPC cells. First, 5,000 PC3 cells were plated, and the xCELLigence TM The instrument measures the current impedance through the plate in real time to measure adhesion and growth for 24 hours. At the 24 hour mark, 60 μM Compound 5, 60 μM Compound 6 or PBS control were added to PC3 cells grown in 100 μM DMEM containing 10% FBS. Observe acute death kinetics in real time and allow cells to continue growing. At 84 hours, 100 μM more compound 5 was added to the pretreated cells. Repeat again at the 124 hour mark. This protocol successfully eliminated more than 75% of PC3 cells compared to compound 6 and PBS control.
[0259] Figure 14 Compound 5 was shown to be very effective on PC3 cells while compound 6 was not. In addition, increased ...
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