2.2.3. Determination of the Type of Inhibition

We conducted kinetic studies of the inhibition of PARP-1 by the most effective inhibitors — NAD+ mimetics. For studies we selected compounds **4IU, 10IU, 11IU**. For comparison, kinetic parameters of the autopoly(ADP-ribosyl)ation in the presence of compound **10A**, which more effectively inhibits PARP-2, were determined. The 3-aminobenzamide (**3-AB**) is a well known PARP inhibitor [74,75] and has been taken for comparison.

The type of inhibition was determined based on at what stage of the reaction the inhibitor binds to the enzyme (before or after the substrate binding) and where it binds (at the substrate binding site or elsewhere). There are four types of inhibition: competitive, whereby an inhibitor binding occurs at the substrate-binding site; noncompetitive, where an inhibitor binds at the allosteric center; mixed with inhibitor binding at both the active and allosteric center; or uncompetitive under an inhibitor binding to the enzyme-substrate complex. Determination of the type of inhibition is necessary to confirm the computer simulation data and to plan further modifications of the inhibitor structure. The type of inhibition can be determined from the plots of the reaction parameters, such as maximum rates, Vmax and the apparent Michaelis constant, Km, values, versus inhibitor concentration [76]. So, under conditions of competitive inhibition, the Vmax value does not change, and the Km value does grow. Noncompetitive inhibition leads to a decrease of Vmax and retention of the Km values. Mixed inhibition is a superposition of the first two, and is characterized by a decrease in the reaction rate and an increase in Km values. In the case of uncompetitive inhibition, both values increase.

To determine the type of inhibition of the studied compounds, the activity of PARP-1 was measured at presence of three different inhibitor concentrations at nine NAD+ substrate concentrations by the fluorescence anisotropy technique [77]. Based on the obtained data, dependence graphs of the initial reaction rate on the substrate concentration were constructed, and reaction parameters such as maximum reaction rate (Vmax) and apparent Michaelis constant (Km) values were found (Tables S3 and S4, Supporting). To determine the type of inhibition, we plotted the dependence graph of Vmax and Km values on the inhibitor concentration using OriginPro 8.6.0 software. (Figure 3). As can be seen from Table S3 and Figure 3, the Km values have been grown, and the maximum reaction rate remains unchanged; that is, compounds **10IU** and **11IU** are competitive inhibitors for PARP-1 catalyzed reaction. Therefore, the results of modeling of the binding type of these compounds in the enzyme's active center are legitimate. Compound **4IU** may be characterized as a mixed inhibitor, although its influence on Km and Vmax values is weak. Compound **10A** turned out to be a mixed inhibitor because the Km values grow and the Vmax values decreased. These results indicate that compounds containing adenine and iodouracil differ in their binding mode.

**Figure 3.** Graphs of Km (left) and Vmax (right) values versus inhibitor concentration. **3-AB**, a commercially-available PARP-1 inhibitor [75], was used as a positive control.

#### 2.2.4. PARP-3 Inhibition

The effects of 16 compounds MorXppA **4** and **10** on the auto(ADP-ribosyl)ation of PARP-3 and ADP-ribosylation of DNA by PARP-3 were studied. The results are summarized in Figure 4. None of the compounds affected DNA modification (Figure 4A). In the auto(ADP-ribosyl)ation reaction, also none of the compounds showed significant inhibitory activity except **10IU** used in 1 mM concentration (Figure 4B).

**Figure 4.** Basic activity of PARP-3 in DNA (**A**) and protein ADP-ribosylation (**B**) and the influence of the inhibitors on these reactions. (**A**) Activity of PARP-3 on [32P]-labeled one-window gapped DNA substrate in the absence (lanes 1–4) or presence of inhibitors in the different concentrations (lanes 5–32) on the upper panel. The reactions were performed using increasing concentration of NAD+. Lane c corresponds to initial electrophoretic mobility of the DNA substrate. The chart on the bottom panel is reflected of the reaction yield of the ADP-ribosylated DNA in percentage terms. (**B**) Activity of PARP-3 on gap1 DNA substrate in the absence (lanes 1–3) or presence of inhibitors in the different concentrations (lanes 4–18) on the upper panel. The reactions were performed using increasing concentration of NAD+ in the presence of [32P]-labeled NAD+. Lane c corresponds to reaction mixture without PARP-3. The chart on the bottom panel is reflected of the reaction yield of the ADP-ribosylated PARP-3 in the presence of inhibitor normalized on the yield of the autoribosylation of PARP-3 in percentage terms. The ticks on the chart mark the bars with the 0.5 mM of inhibitor in the experiment.
