Via a 3D-QSAR pseudoreceptor modeling approach, atomistic binding site models for pharmacologically active 1,4-dihydropyridines (DHPs) were developed. Applying a training set of pure DHP enantiomers a pseudoreceptor model representing the resting state of voltage-gated calcium channels (VGCCs) was generated by correlating experimental versus predicted free energies of binding (DeltaG degrees). For validation further test set derivatives-not used for receptor generation-were predicted yielding root-mean-square (rms) deviation of 0.532 kcal/mol. Selectivity of the resting state model was checked by using the same DHP training set compounds but experimental data for the inactivated channel mode. Although there was found an almost perfect correlation for the training set, the following free relaxation of the corresponding test set applying a Monte Carlo protocol showed rms of 2.033 kcal/mol, clearly demonstrating the lack of any predicting character of the hybrid model. Taking into consideration 19 additional nifedipine analogues, a further verification of the model was performed. This yielded a good correlation for the 12 training set compounds and a satisfactory prediction for the test set molecules with rms of 0.409 kcal/mol. The generation of a pseudoreceptor model depicting the opened/inactivated state of VGCCs required one single additional residue to achieve a rms of 0.848 kcal/mol for the prediction of the test set derivatives. Since all pseudoreceptor models are composed of the same six amino acid residues-Thr, Phe, Gly, Met, Tyr, Tyr-transition from resting to open/inactivated state may be described by one additional hydrogen bond donor interaction (Thr) at the left-hand side of DHPs. Furthermore, a potential charge-transfer interaction for all electron-deficient 4-phenyl DHPs is postulated, because significant correlation between quantum chemically AM1 (R = 0.91) and RHF 6-31G (R = 0.84) computed LUMO energies and experimentally detected DeltaG degrees exp values was found.

4‐Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide‐tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C‐terminal helix H11 in HPPD. We compared gating equilibria in Arabidopsis thaliana (At) and Zea mays (Zm) wild‐type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open–closed transition and discovered a ligand‐mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in At mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new At mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.

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Three different 3D QSAR methods have been applied for a common pharmacophore model of 45 calcium antagonistically active 1,4-dihydropyridines (DHP) in order to find best correlation of interaction fields and biological activity. Analysis for the entire data set yielded r 2 /q 2 cv values in a range starting from 0.821/0.620 (GRID/ GOLPE) over 0.872/0.600 (CoMFA) to 0.908/0.744 (CoM-SIA). The robustness of these models was tested not only via leave-one-out but also by leave-9-out crossvalidations. Furthermore, models were constructed using a subset of 37 DHPs (training set) allowing the prediction of activity for the residual 8 DHPs (test set). The training set yielded r 2 / q 2 cv values starting from 0.826/0.672 (GRID/GOLPE) over 0.872/0.540 (CoMFA) to 0.899/0.662 (CoMSIA). For the test set r 2 pred values from 0.677 (GRID/GOLPE) over 0.639 (CoMFA) to 0.470 (CoMSIA) were calculated. Besides the statistics, each 3D QSAR model yields further information by analysis of the generated contour maps. Consideration of the CoMFA and CoMSIA fields indicates unfavourable steric interactions for bulky moieties in 4'-position. On the other hand, sterical demanding 2'-and 3'-substituents are favourable and the biological activity of DHPs is further increased if these moieties produce a negative electrostatic potential. In contrast, high p-electron density on top of and parallel to the 4-phenyl ring beside the 2'-position is associated with decreasing activity. This could point to repulsive electronic interactions with binding site residues or to the potential of electron-deficient 4-aryl moieties to behave as electron acceptors in a charge transfer (CT) mechanism.

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