In previous work we showed that benzophenanthridines do

In previous work we showed that benzophenanthridines do not affect the surface expression of GlyT1 (Jursky and Baliova, 2011). The results from the surface biotinylation studies reported in Fig. 3 show that the surface expression of the Y370F mutant is not significanly affected by the alkaloids in the time window investigated here. This indicates that the increased inhibition observed in the Y370F mutant is unlikely to be caused by alkaloid-stimulated physical removal of the transporter from the membrane. To investigate the structure-inhibitory activity relationship of the benzophenanthridines, and thereby infer some information about how they might bind to GlyT1, we compared the structures of the sanguinarine and chelerythrine molecules downloaded from the PubChem Substance database (Fig. 4A). From this figure it is clear that positions 7 and 8 in sanguinarine are derivatized in chelerythrine by methoxy groups. This modification does not eliminate its inhibitory activity against GlyT1, however, which indicates that the active pharmacophore is rather delineated by the opposite region of the benzophenanthridine ring. With this information, we performed molecular docking of chelerythrine into the inward-open GlyT1 model using autodock Vina. Because of the potentially significant flexibility of tryptophan 376 (Fig. 1), all ten GlyT1 models produced by Modeller were investigated. Given that the interacting chelerythrine should be in the vicinity of both cysteine 475 and tyrosine 370 and that the common triple benzophenanthridine ring should be oriented into the interaction cavity, the most probable position of alkaloids bound to GlyT1 should be similar to the one shown on Fig. 4B and C. Vina’s calculated binding energy is −8.8kcal/mol.
Discussion Non-covalent interactions involving aromatic rings play a major role in protein–ligand recognition and they are also very important for drug design. In fact, a large majority of the protein-small molecule complexes studied by crystallography contain bonding interactions between aromatic amino Chidamide synthesis receptor side chains and an aromatic or heteroaromatic ligand. Because of the presence of the planar aromatic side chains of tyrosine, phenylalanine and tryptophan around the GlyT1 cysteine 475, stacking interactions between benzophenanthridines and these residues are highly probable. The above supposition is supported by the crystal structure of chitinase A bound to sanguinarine and chelerythrine, in which the alkaloids make parallel stacking interactions with tryptophan and phenylalanine residues (Pantoom et al., 2011). In the present case, elimination of the aromatic character of sanguinarine as well as modification of aromatic tyrosine 370 also significantly affected the inhibition of GlyT1 by benzophenanthridines. In addition to these interactions, the benzophenanthridines have affinity for mercapto-compounds, including the cysteines present in protein molecules. Unfortunately currently no crystal structures show the exact geometry of this interaction. Even though it has been shown that free cysteine in solution interacts with an alkaloid pseudobase non-covalently, it is presently not clear whether the similar interaction of proteinaceous cysteines is non-covalent (Vespalec et al., 2003) or produces a covalent bond via sulfur nucleophilic attack on the electron deficit carbon 6 of the benzophenanthridine ring (Walterova et al., 1981). We previously showed that the interaction of chelerythrine with GlyT1 is reversible at least, which supports non-covalent interaction in this case (Jursky and Baliova, 2011). The majority of the tyrosine mutations around cysteine 475 retained glycine transport and had no effect on the inhibition of GlyT1 by benzophenanthridines, only tyrosine 370 was different. The molecular docking of benzophenanthridine in the vicinity of cysteine 475 indicated that the ring might be placed at an optimal distance for interactions with both cysteine 475 and tyrosine 370. Both these residues also affect the sensitivity of GlyT1 to alkaloids when they are replaced via mutagenesis. Currently, the types of interactions predominantly involved are not clear. The identity of the present molecular moiety, however, suggests that the interactions might involve T-stacking, cation–π interactions, and hydrogen or π –hydrogen bonding. (Gallivan and Dougherty, 1999, Chelli et al., 2002, Chakrabarti and Bhattacharyya, 2007)