Insights into the interaction of negative allosteric modulators with the metabotropic glutamate receptor 5: discovery and computational modeling of a new series of ligands with nanomolar affinity.

Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors.

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.

Edaravone inhibits protein carbonylation by a direct carbonyl-scavenging mechanism: focus on reactivity, selectivity, and reaction mechanisms.

The aim of this study was to evaluate the ability of the well-known radical scavenging compound edaravone (EDA) to entrap and detoxify reactive carbonyl species (RCS) derived from lipid peroxidation [4-hydroxy-trans-2-nonenal (HNE), acrolein and glyoxal], as well as its ability to prevent RCS-induced protein carbonylation, by using hemoglobin (Hb) modified by HNE as an in vitro model. Through a combined HPLC and high-resolution mass spectrometric approach, we confirmed the ability of EDA to scavenge precursors for either advanced glycation or lipoxidation end products (EAGLEs), such as glyoxal, and demonstrated for the first time that EDA is also a potent quencher of alpha,beta-unsaturated aldehydes (providing mass spectral characterization of the adducts), being significantly more active than a series of well-known RCS sequestering agents. Direct infusion analysis of the intact protein and nano LC-ESI-MS/MS analysis of the tryptic digest, carried out on an LTQ-Orbitrap hybrid mass spectrometer, were used to study the modifications occurring on Hb after exposure to increasing HNE concentrations, providing evidence for Cys93 (Hb beta-chain) involvement in covalent attachment, and to demonstrate the ability of EDA dose-dependently to inhibit Hb carbonylation. Computational studies allowed us to elucidate the mechanism of EDA-RCS interaction and to explain the preferential site of HNE adduction to Hb. The same combined approach indicated that EDA is not a selective RCS scavenger, being able to react also with nontoxic, physiologically relevant aldehydes, such as pyridoxal.

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.

Mono- or diphenylpyridazines connected to N-(2,4-difluorophenyl)-N'-heptylurea as acyl-CoA:cholesterol acyltransferase inhibitors.

Mono- and diphenylpyridazine ureido derivatives, structurally related to DuP 128, were synthesized and tested for their inhibitory activity against ACAT isolated from rat liver microsomes. Several compounds displayed ACAT inhibition in the micromolar range. The amino derivatives 4a-c were also tested against hACAT-1 and hACAT-2 isoforms. They retained the same trend shown in the previous assay. Modeling studies on representative terms were performed. Significant similarities between the geometrical features of the model DuP 128 and the most active pyridazine derivatives were observed.