Complexes are in agreement with the crucial NMR experimental findings about the binding mode of these inhibitors.The rigid D-Glu mimetics of second generation sulfonamide inhibitors form stable electrostatic interactions with the D-Glu-binding site, which is supported by their large effects on the CPSs of methyl ICG-001 groups near the D-Glu-binding site.The C6 arylalkyloxy substituents are stabilized in the uracil-binding pocket with a number of stable electrostatic and hydrophobic interactions. This is in agreement with their pronounced effects on the CSPs of methyl groups near the uracil binding site.The C6 alkyloxy substituents are flexible in the uracil-binding site, forming weaker hydrophobic interactions; the CSPs of methyl groups near the uracil binding site are significantly lower.The naphthalene ring rotations are supported by the NOE patterns of bound ligands.The type of substitution of rigid D-Glu mimetic significantly effects the electrostatic interactions of the sulfonamide group with the central domain. This is supported by the pronounced effects of 6b on the CPSs belonging to the central domain residues. MurD conformational changes have to date been given insufficient attention in the process of MurD inhibitor optimization. MD simulations show the complex dynamic behavior of these MurD�Cinhibitor complexes, where the interactions are affected both by movements of the protein domains and by the flexibility of the ligand. The differing degrees of conformational flexibility of the ligands were also predicted on the basis of the NOE patterns. The sulfonamide inhibitors studied span from the C-terminal domain to the N-terminal domain and also interact with the central domain. The distances between the C-terminal and Nterminal domains fluctuate. Therefore, the bound ligands are exposed to Niraparib stretching forces that tend to pull either the D-Glu mimetic part or the C6 substituent out of the binding site. Stronger interactions in one domain tend to weaken the interactions in the other domains. This needs to be considered in the optimization of these sulfonamide inhibitors through the design of new compounds that have improved interactions not only with one but with both the C-terminal and N-terminal domains of MurD. Our data also suggest that inhibitors that can span from the C-terminal domain to the N-terminal domain should not be highly rigid to allow them to adapt to the conformational changes of the MurD protein. Such compounds might also benefit from having slightly longer linkers between the naphthalene rings and the aromatic rings of the C6 substituent. These data represent upgraded knowledge that will now be useful for the rational structure-based design of new improved MurD inhibitors. STD ligand epitope mappingwas performed with an 8389 Hz spectral width, with 16384 data points, a saturation time of 350 ms, a relaxation delay of 11.35s, and 3000 to 8000 scans. The spectra were recorded at a protein/ligand ratio of 1:100. Selective saturation was achieved by a train of 50 ms long Gaussshaped pulses, separated by 1 ms delays. Water was suppressed via excitation sculpting. The on-resonance selective saturation of MurD was applied at 0.21 ppm. The off-resonance irradiation was applied at 30 ppm for the reference spectrum. Subtraction of the on-resonance and off-resonance spectra was performed internally via phase cycling. The transferred NOESYspectra were acquired at a protein/ligand ratio of 1:45, 
with an 8389 Hz spectral width, with 4096 data points in t2, 32�C48 scans, 256�C356 complex points in t1, a mixing time of 250 ms, and a relaxation delay of 1.5 s. The residual water signal was suppressed using excitation sculpting, and adiabatic pulseswere applied for suppression.