Additional stabilization is achieved through stable cation-p interactions between their phenyl rings and the positively charged guanidino group of Arg37. The same interactions are also observed in the cocrystal structure of compound 1b. The introduction of substituted benzoic acid derivatives as glutamic acid mimetics in the second-generation sulfonamide inhibitors allows p�Cp stacking interactions with the Phe422 phenyl ring. This might contribute to increased binding affinities compared to the D-Glu-containing compounds. Another important difference between the binding modes of the most potent compound from the first-generation 1b and of the most potent compound from the second-generation 6b that could contribute to the 10-fold difference in their inhibitory activities lies in interactions with the central domain residues. Only indirect interactions of the ligand sulfonyl group across the water molecule with the residues Asn138 and Ser159 are observed in the crystal structure of the 1b�CMurD complex. The MD simulations show that the direct hydrogen bond of compound 1b with Asn138 is formed much less frequently compared to the case of compound 6b. These observations are supported by NMR data. The CSPs�� patterns reveal a significantly increased effect of compound 6b on the central domain signals with regard to the compound 1b. The MD data reveal complex dynamic behaviors of these ligand�CMurD complexes and show that these influence the ligand�C enzyme contacts. As well as the rotation of ligand segments at the MurD binding site, as revealed by transferred NOESY, slight opening/closing movements of the protein domains are seen in MD trajectories. Movements of protein domains can adversely affect ligand binding through effects on the conformation and flexibility of the bound ligand, the stability of the ligand�Cenzyme interactions, and the binding-site adaptability. These movements should not be confused with the open and closed conformations of the MurD protein that have 
been reported in the literature, where the Cterminal domain has a drastically different position. The most pronounced fluctuations are evident from the distances between the geometric centers of the C-terminal and N-terminal domains. The Regorafenib variations between the minimal and maximal distances can be up to 5.5 A ?. An additional, longerMD run of selected ligands 6a and 6b clearly indicate that the fluctuations of distance between these geometric centers are less pronounced when the most potent inhibitor 6b is bound to MurD. This might be the consequence of better binding interactions that tend to hold the domains together. Visual inspection of the trajectories reveals that the movements of the C-terminal and N-terminal domains have important roles in ligand binding. Sulfonamide inhibitors span from the N-terminal domain to the C-terminal domain. Opening movements tend to weaken the interactions either with the uracil binding pocket or with the D-Glu binding site. The C6-alkyloxy-substituted compoundsgenerally have weaker interactions with the uracil binding pocket compared to the C6-arylalkyloxy-substituted compounds. Consequently, the alkyl WZ8040 EGFR/HER2 inhibitor chains can even be pulled out of the uracil binding pocket during domain movement. The C6-alkyloxy-substituted compounds are also shorter than the C6-arylalkyloxy-substituted compounds. Therefore, the alkyl chain has more freedom to move in the uracil binding pocket. This is accompanied by conformational changes of the ligand. During domain movement, a rotation of the mimetic ring for compounds 3a, 4a, and 5a is observed around the hinges formed by the carboxyl groups that switches their conformation between extended and bent form.