Figure 1.

Identification of geldanamycin as a potent KDM4B inhibitor

(A) The KDM4B TR-FRET demethylation functional assay: As a KDM4B substrate, the Biotin-linked H3K9me3 peptide is converted to the product Biotin-H3K9me2 peptide, which is bound by both the Terbium-labeled anti-H3K9me2 antibody and AF488-streptavidin and brings the donor fluorophore Tb and acceptor fluorophore AF488 into close proximity. A high TR-FRET signal in the form of light emission at 520 nm from the acceptor fluorophore AF488 is generated when the donor fluorophore Tb was excited at 340 nm. (B = biotin, SA = streptavidin, Tb = Terbium).

(B) Fold ratio between positive and negative controls from the validation assay. The positive control is the group without KDM4B protein (mimic 100% inhibition) and the negative control is the group with 750 nM in-house KDM4B protein (mimic 0% inhibition).

(C) Activity scatterplot of all compounds tested in the TR-FRET assay (Green: positive control; Blue: primary hits; Black: inactive compounds; Red: negative control).

(D) Z-prime factor from the screen. The Z-prime factor value was calculated using the Equation 1 for each plate based on the method reported in the literature(Zhang et al., 1999). Each dot represents Z-prime factor value for one assay plate.

(E) The chemical structure of geldanamycin.

(F) Representative TR-FRET dose-response inhibitory activity curve of geldanamycin against KDM4B. Red dots are activity values of geldanamycin at its respective concentrations in the assay plate of NT00162940. Green dots are activity values of geldanamycin at its respective concentrations in the assay plate of NT00162941. Blue curve is the dose-response curve derived from the average inhibitory activities or pooled activities of geldanamycin at its corresponding concentrations.

(G) Representative HTRF dose-response inhibitory activity curve of geldanamycin against KDM4B. Red dots are activity values of geldanamycin at its respective concentrations in the assay plate of NT00162904. Green dots are activity values of geldanamycin at its respective concentrations in the assay plate of NT00162905. Blue curve is the dose-response curve derived from the average inhibitory activities or pooled activities of geldanamycin at its corresponding concentrations.

(H) Representative background TR-FRET dose-response curve of geldanamycin without KDM4B protein. Red dots are activity values of geldanamycin at its respective concentrations in the assay plate of NT00162907. Green curve is the dose-response curve derived from the inhibitory activities or pooled activities of geldanamycin at its corresponding concentrations.

See also Figures S1 and S2.