Figure 1. Dose response from ML199 in Radiotracer Incision Assay and structure of the probe. Results showed dose response inhibition upon treatment with ML199.

Figure 2. Potentiation of probe ML199 in the presence or absence of MMS.

Figure 3. Kinetic parameters without and with ML199 or Compound 1. Ten pg of APE1 (~28 pM) was incubated without or with 5 μM of the indicated inhibitor at room temperature for 15 min. Increasing concentrations of radiolabeled abasic DNA substrate (i.e. 5, 10, 25, 50, or 100 nM) were then added, and the reactions were incubated at 37 ºC for 5 min before the addition of stop buffer. Intact substrate was separated from incised product on a 15% polyacrylamide denaturing gel, and the percent conversion was determined by standard phosphorimager analysis. Lineweaver – Burk plots of 1/V versus 1/[S] were used to determine KM and kcat (shown). The plotted data points (averages and standard deviations) were derived from 11 independent values for the no inhibitor reactions, and 5 values for each of the inhibitor points.

Figure 4. Stability of the APE1-DNA substrate complex in the presence of ML199. (A) Representative EMSA. Three hundred ng of APE1 (~0.8 μM) was incubated without inhibitor (final concentration 1% DMSO) or with the indicated inhibitor (1, 3, 10, 30 or 100 μM) for 10 min on ice. One hundred fmol of abasic DNA substrate (10 nM) was then added, and the binding reaction was incubated on ice for an additional 5 min. At that time, samples were subjected to non-denaturing polyacrylamide gel electrophoresis to separate the APE1-DNA complex (“C”) from unbound radiolabeled DNA (“DNA”). Inh = inhibitor. (B) Relative complex formation without (“0”) or with the indicated inhibitor (in μM). Shown is the average and standard deviation of three independent experimental data points, all relative to the APE1 control, without inhibitor.

Figure 5. Inhibition of HeLa whole cell extract AP site incision activity with ML199. Three hundred ng of HeLa whole cell extract was incubated with 0, 50 or 100 uM of the indicated inhibitor at room temperature for 15 min, prior to the addition of 0.5 pmol radiolabeled AP-DNA substrate and subsequent transfer of the reaction mix (final volume of 10 μL) to 37 ºC for 5 min to allow for incision. Following addition of stop buffer and heat denaturation, the reaction products were subjected to 15% polyacrylamide denaturing gel electrophoresis. Shown is a bar graph reporting the relative percent incision activity in comparison to the no inhibitor control, arbitrarily set at 100. The values reported represent the averages and standard deviation of three independent experimental data points.

Table 1. In vitro ADME to date. Green = desirable property, Yellow = less than desirable property, Red = undesirable property. All experiments were conducted by Shanghai Chempartner Co. Ltd. 34* indicates probe molecule ML199.