Summary /

Inhibition of IDH1 R132H was screened by coupling IDH1 R132H-mediated NADPH consumption to a diaphorase/resazurin-based detection assay. Result showed that ML309 increased the fluorescence from resorufin indicating a dose-response inhibition against the R132H mutant (Figure 1).

Figure 1. Activity of ML309 against wild type (blue squares) and R132H mutant (red circles) IDH1 determined by the primary diaphorase coupled assay. Each point represents the mean±SD from two duplicate experiments.

Summary /

ML309 inhibits the IDH1 reaction and decreases the production of 2-HG in IDH1 R132H without cytotoxic effect (Figure 2).

Figure 2. Cell activity of ML309 showing potent reduction of the oncometabolite, 2-HG (green diamonds) in the U87 MG cell based assay with no sign of cytotoxicity (purple triangles). Each point represents the mean±SD from two duplicate experiments.

Summary /

To determine whether the probe, ML309, was competitive with respect to the substrate α-KG for binding to IDH1 R132H, a mechanism of action study was undertaken (Figure 3). To determine the rate of reaction, the decrease in fluorescence associated with the consumption of the NADPH substrate was monitored directly over time for four concentrations of α-KG and four concentrations of ML309. Result shows the untransformed data, and the large graph shows the Lineweaver-Burk transformation of the data. The lines for the four concentrations of ML309 intersect each other at the y-axis, which indicates that the inhibitor is competitive with respect to the substrate α-KG. Additionally, an estimate of the Ki can be obtained by plotting the inhibitor concentration versus the slopes of the lines in the Lineweaver Burk plot. The Ki obtained from this analysis (156 nM) is similar to the IC50 (96 nM) obtained from the IDH1 R132H diaphorase/resazurin-coupled assay.

Figure 3. Evaluation of substrate competition of ML309 with α-KG in IDH1 R132H. A) Lineweaver-Burk plot demonstrating competitive mode of inhibition with respect to α-KG; inset shows untransformed data; the data point corresponding to the combination of 400 nM [ML309] and 125 μM [α-KG] did not provide signal above background, hence reaction rate could not be determined. B) Lineweaver Burk plot to determine the Ki for ML309.

Summary /

In vitro assessment of ML309 against mouse and human microsomes, human plasma, aqueous solution and 5 mM glutathione  prove that the probe possesses excellent overall stability. Additionally, the >3 hour half life found in the in vivo PK experiment (Figure 4) establish ML309 as molecule stable enough to warrant further in vivo studies.

Figure 4. In vivo PK study of ML309