*3.3. Structural Studies*

Reference crystal structures of PARP-1 and PARP-2 were taken from the PDB database with identifiers 4ZZZ [85] and 3KJD [86], correspondingly. Localization of the ADP fragment of acceptor NAD+ was predicted by a structural alignment of the reference PARP-1/2 crystal structures with the crystal structure of PARP from *Gallus gallus* in complex with NAD+ (PDB identifier 1A26, [78]). The localization of donor NAD+ was predicted by structural alignment with catalytic domain of PARP-1 (PDB identifier 6BHV) [38] in complex with BAD, structural analog of NAD+. In this case C1 carbon of benzamide was manually replaced by nitrogen. Structural alignment was carried out using Maestro (Schrodinger Inc.). Obtained complexes were minimized with Protein Preparation Wizard (Schrodinger Inc.). Preliminary the side chains of amino acid residues containing steric hindrance with BAD were removed and re-added using Prime Module (Schrödinger Inc.).

3D structures of small molecules under study were prepared using LigPrep module (Schrödinger Inc.). Molecular docking was carried out using Glide in the extra-precision mode (XP Score). Small molecule binding pose with the best XP score were selected [87]. Two grids were calculated for the molecular docking study. First grid was calculated based on the center of mass of the acceptor ADP fragment of NAD+ and the second was calculated based on the center mass of the donor NAD+ ligand. Molecular dynamics simulation was carried out using the pmemd module of Amber12 software [88]. The RESP charges were calculated using the ANTECHAMBER module based on ab initio calculations

of electrostatic potentials using theHF/6-31G\* level of theory using Gaussian 03 [89,90]. Amberff99SB and GAFF force fields were used for the simulation of molecular dynamics of protein and small molecules, respectively. Implicit solvent generalized Born (GB) Onufriev was used for MD simulation with mbondi2 radii set [91].

Molecular dynamics protocol included 1000 steps of steepest descent minimization, 100 ps heating from 0K to 300K, and 100 ns production molecular dynamics simulation. Langevin thermostat with a 5 ps−<sup>1</sup> collision rate was used [92]; 1 kcal/mole restraints on protein CA atoms and heavy atoms of the adenosine fragment of NAD+ analogs were applied at all steps of MD simulation.
