Fragmental modeling of hPepT2 and analysis of its binding features by docking studies and pharmacophore mapping.

Over the last years, considerable progress has been made for the identification and characterization of drug transporters, and several modeling studies have been undertaken to predict their effects on ADME profiling. Thus, this study was focused on the peptide transporter hPepT2, which influences the regional pharmacokinetics in brain, the reabsorption from renal tubular fluid and the pulmonary delivery. A reliable model for hPepT2 was generated by fragments based on the resolved structure of the homologue lactose permease LacY and the structure is made available as Supplementary data. The interaction capacities of such a model were explored by docking a set of 75 known ligands. Docking results underlined the predilection of hPepT2 for highly hydrophobic ligands and the key role of ionic interactions elicited by both charged termini. The docking results were further verified developing a pharmacophore model which clarified the key features required for an optimal hPepT2 affinity and confirmed the main factors governing the hPepT2/hPepT1 selectivity. The soundness of the docking results and the agreement with the pharmacophore mapping afford an encouraging validation for the proposed hPepT2 model and suggest that it can be conveniently exploited to design peptide-like molecules with an improved affinity for this transporter.

Comparative modeling of the quaternary structure for the human TRPM8 channel and analysis of its binding features.

The aim of this study was to generate a reliable model for the homotetrameric structure of the human TRPM8 cation channel, a temperature sensor involved in innocuous cold perceptions. The described model was generated using a fragmental strategy and its interaction capacities were explored by docking a representative set of ligands. The analysis of the quaternary structure suggests that the N-terminus possesses a solenoidal topology which could be involved in tetramerization due to its electrostatic characteristics. Again, the tetramer model unveils a precise fitting between the segments of neighbouring monomers affording attractive suggestions for the multifaceted mechanism of channel gating. Docking results are in convincing agreement with mutational analyses and confirm that S4 and S4-S5 linker play a key role in channel activation. Overall, the proposed model could find fertile applications to further investigate the gating mechanism and to design truly selective ligands able to clarify the pathophysiological roles of the TRPM8 channel.

Modelling of full-length human alpha4beta2 nicotinic receptor by fragmental approach and analysis of its binding modes.

The objective of the study was to generate a full-length model for the heteropentameric structure of human alpha4beta2 nicotinic receptor. The monomers structure was derived using a fragmental approach and the pentamer was assembled by protein-protein docking. The reliability of the model was assessed docking a representative set of known nicotinic ligands. Docking results unveiled that the ligand affinity depends on key interactions that the ligand's charged moiety realizes with conserved apolar residues of alpha4 monomer, whereas the H-bond acceptor group interacts with a less conserved and more heterogeneous subpocket, involving polar residues of beta2 subunit. The consistency of docking results and the agreement with the experimental data afford an encouraging validation for the proposed model and emphasize the soundness of such a fragmental approach to model any transmembrane protein.

Docking analyses on human muscarinic receptors: unveiling the subtypes peculiarities in agonists binding.

The study presents a docking analysis for the interaction capabilities of some muscarinic receptors (i.e., M(1), M(2), and M(5)) whose full-length models were previously generated by us. In detail, the docking simulations involved a dataset of 30 agonists, taken from the literature, including a first series of oxathiolane/dioxolane congeners and a second subset of more heterogeneous ligands. The obtained results unveil that it is possible to discriminate among the binding modes of considered muscarinic receptors, developing specific interaction patterns which are significantly different for the arrangement of both polar and hydrophobic interactions. Thus, the M(1) subtype possesses the widest binding site, while the M(2) receptor is characterized by a large but asymmetric region that accommodates the ligand's ammonium head and finally the M(5) binding site is quite similar to that of M(2) subtype being univocally characterized by a second aspartate residue which interacts with the ammonium head. The significant correlations between docking scores and affinity values afford an encouraging validation for the reported binding modes and confirm the key role of molecular flexibility in the ligand recognition of muscarinic receptors.

Muscarinic receptors: A comparative analysis of structural features and binding modes through homology modelling and molecular docking.

Three-dimensional models of the five human muscarinic receptors were obtained from their known sequences. Homology modelling based on the crystallographic structure of bovine rhodopsin yielded models compatible with known results from site-directed mutagenesis studies. The only exceptions were the cytoplasmic loop 3 (CL3) in the five receptors, and the large C-terminal domain in M(1). Here, homology modelling with other closely related proteins allowed to solve these gaps. A detailed comparative discussion of the five models is given. The second part of the work involved docking experiments with the physiological ligand acetylcholine, again yielding results entirely compatible with results from mutagenesis experiments. The study revealed analogies and differences between the five receptors in the residues, and interactions leading to the recognition and binding of acetylcholine.

The approach of conformational chimeras to model the role of proline-containing helices on GPCR mobility: the fertile case of Cys-LTR1.

The homology modeling of GPCRs has benefitted vastly from the availability of some resolved structures, which allow the generation of many reliable GPCR models. However, the dynamic behavior of such receptors has been only minimally examined in silico, although several pieces of evidence have highlighted some conformational switches that can orchestrate the activation mechanism. Among such switches, Pro-containing helices play a key role in determining bending in TM helices and thereby the width of the TM bundle. The approach proposed herein involves the generation of a set of possible models (conformational chimeras) by exhaustively combining the two main conformations (straight and bent) that a Pro-containing helix can assume. This approach was validated by generating conformational chimeras for the Cys-LTR1 receptor, which is involved in contractile and inflammatory processes. The generated chimeras were then used for docking a small set of representative ligands. The results revealed the flexibility mechanisms of Cys-LTR1, showing how the docked agonists vary their stabilizing interactions, shifting from the open to closed state, and how the examined antagonists are able to block the receptor in an open and inactive conformation, thus behaving as inverse agonists. This study emphasizes the promising potential of chimera modeling, confirms the key role of proline residues in receptor activation, and suggests that docking results can be improved by considering the often-overlooked flexibility of receptors.

Modeling of the intestinal peptide transporter hPepT1 and analysis of its transport capacities by docking and pharmacophore mapping.

An early pharmacokinetic screen for peptidomimetic drugs should have the ability to predict molecules with high affinity for intestinal transporters, as peptide-like derivatives are seldom absorbed passively. Hence, the first objective of this study was to generate a reliable model for the structure of the hPepT1 protein, which is the main intestinal transporter involved in the absorption of both dietary peptides and peptidomimetics. The modeling was based on the resolved structure of the homologous bacterial lactose permease LacY using a fragmental strategy. The interaction capacities of the hPepT1 model were explored by docking a set of 50 known ligands. Despite the known predilection of hPepT1 for hydrophobic ligands, docking results unveiled the key role of the polar interactions stabilized by charged termini, especially concerning the ammonium head group. The docking results were further verified by developing a pharmacophore model that confirmed the key features required for optimal hPepT1 affinity. The consistency of the docking results and the agreement with the pharmacophore model afford an encouraging validation for the proposed model and suggest that it can be exploited to design peptide-like molecules with an improved affinity for such a transporter.