Giovanni Grazioso

Investigating the Mechanism of Substrate Uptake and Release in the Glutamate Transporter Homologue GltPh through Metadynamics Simulations

A homeostatic concentration of glutamate in the synaptic cleft ensures a correct signal transduction along the neuronal network. An unbalance in this concentration can lead to neuronal death and to severe neurodegenerative diseases such as Alzheimers or Parkinsons. Glutamate transporters play a crucial role in this respect because they are responsible for the reuptake of the neurotransmitter from the synaptic cleft, thus controlling the glutamate concentration. Understanding the molecular mechanism of this transporter can provide the possibility of an exogenous control. Structural studies have shown that this transporter can assume at least three conformations, thus suggesting a pronounced dynamical behavior. However, some intermediate states that lead to the substrate internalization have not been characterized and many aspects of the transporter mechanism still remain unclear. Here, using metadynamics simulations, we investigate the substrate uptake from the synaptic cleft and its release in the intracellular medium. In addition, we focus on the role of ions and substrate during these processes and on the stability of the different conformations assumed by the transporter. The present dynamical results can complement available X-ray data and provide a thorough description of the entire process of substrate uptake, internalization, and release.

Nicotine-modulated subunit stoichiometry affects stability and trafficking of α3β4 nicotinic receptor.

Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two β subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3β4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human α3β4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a ∼5-fold increase of cell surface α3β4 and showed a more modest upregulatory effect also at concentrations as low as 10 μM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(β4)3 stoichiometry; these are less prone than (α3)3(β4)2 receptors to proteasomal degradation and, because of the presence in the β subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3β4 nAChR upregulation that may be relevant also for other nAChR subtypes.

Engineering of α-conotoxin MII-derived peptides with increased selectivity for native α6β2* nicotinic acetylcholine receptors

α6β2∗ Nicotinic acetylcholine receptors are expressed in selected central nervous system areas, where they are involved in striatal dopamine (DA) release and its behavioral consequences, and other still uncharacterized brain activities. α6β2∗ receptors are selectively blocked by the α‐conotoxins MII and PIA, which bear a characteristic N‐ terminal amino acid tail [arginine (R), aspartic acid (D), and proline (P)]. We synthesized a group of PIA‐related peptides in which R1 was mutated or the RDP motif gradually removed. Binding and striatal DA release assays of native rat α6β2∗ receptors showed that the RDP sequence, and particularly residue R1, is essential for the activity of PIA. On the basis of molecular modeling analyses, we synthesized a hybrid peptide (RDP‐MII) that had increased potency (7‐fold) and affinity (13‐fold) for α6β2∗ receptors but not for the very similar α3β2∗ subtype. As docking studies also suggested that E11 of MII might be a key residue engendering α6β2∗ vs. α3β2∗ selectivity, we prepared MII[E11R] and RDP‐MII[E11R] peptides. Their affinity and potency for native α6β2∗ receptors were similar to those of their parent analogues, whereas, for the oocyte expressed rat α3β2∗ subtype, they showed a 31‐ and 14‐fold lower affinity and 21‐ and 3.5‐fold lower potency. Thus, MII[E11R] and RDP‐MII[E11R] are potent antagonists showing a degree of α6β2∗ vs. α3β2∗ selectivity in vivo.—Pucci, L., Grazioso, G., Dallanoce, C., Rizzi, L., De Micheli, C., Clementi, F., Bertrand, S., Bertrand, D., Longhi, R., De Amici, M., Gotti, C. Engineering of α‐conotoxin MII‐derived peptides with increased selectivity for native α6β2* nicotinic acetylcholine receptors. FASEB J. 25, 3775–3789 (2011). www.fasebj.org

Peptidomimetics containing a vinyl ketone warhead as falcipain-2 inhibitors

The design, chemical synthesis, and enzymatic activity evaluation of a set of falcipain-2 inhibitors are reported. These compounds contain a proven peptidomimetic recognition motif based on a benzo[1,4]diazepin-2-one (1,4-BDZ) framework built on a dipeptide sequence, and a Michael acceptor terminal moiety capable of deactivating the cysteine protease active site. Our goal is to modify the P(3) site of this motif in order to identify the structural requirements for the interaction with the target.

Alpha7 Nicotinic Acetylcholine Receptor Agonists: Prediction of Their Binding Affinity Through a Molecular Mechanics Poisson-Boltzmann Surface Area Approach

A group of agonists for the α7 neuronal nicotinic acetylcholine receptors (nAChRs) was investigated, and their free energies of binding ΔGbind were calculated by applying the molecular mechanics Poisson–Boltzmann surface area (MM‐PBSA) approach. This method, based on molecular dynamics simulations of fully solvated protein–ligand complexes, allowed us to estimate the contribution of both polar and nonpolar terms as well as the entropy to the overall free energy of binding. The calculated results were in a good agreement with the experimentally determined ΔGbind values, thereby pointing to the MM‐PBSA protocol as a valuable computational tool for the rational design of specific agents targeting the neuronal α7 nAChR subtypes.

Mechanism of falcipain-2 inhibition by α,β-unsaturated benzo[1,4]diazepin-2-one methyl ester.

Falcipain-2 (FP-2) is a papain-family cysteine protease of Plasmodium falciparum whose primary function is to degrade the host red cell hemoglobin, within the food vacuole, in order to provide free amino acids for parasite protein synthesis. Additionally it promotes host cell rupture by cleaving the skeletal proteins of the erythrocyte membrane. Therefore, the inhibition of FP-2 represents a promising target in the search of novel anti-malarial drugs. A potent FP-2 inhibitor, characterized by the presence in its structure of the 1,4-benzodiazepine scaffold and an α,β-unsaturated methyl ester moiety capable to react with the Cys42 thiol group located in the active site of FP-2, has been recently reported in literature. In order to study in depth the inhibition mechanism triggered by this interesting compound, we carried out, through ONIOM hybrid calculations, a computational investigation of the processes occurring when the inhibitor targets the enzyme and eventually leads to an irreversible covalent Michael adduct. Each step of the reaction mechanism has been accurately characterized and a detailed description of each possible intermediate and transition state along the pathway has been reported.

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors.

A set of racemic spirocyclic quinuclidinyl‐Δ2‐isoxazoline derivatives was synthesized using a 1,3‐dipolar cycloaddition‐based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ2‐Isoxazolines 3 a (3‐Br), 6 a (3‐OMe), 5 a (3‐Ph), 8 a (3‐OnPr), and 4 a (3‐Me) were the ligands with the highest affinity for the α7 subtype (Ki values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM, respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)‐3‐methoxy‐1‐oxa‐2,7‐diaza‐7,10‐ethanospiro[4.5]dec‐2‐ene sesquifumarate 6 a were prepared using (+)‐dibenzoyl‐L‐ or (−)‐dibenzoyl‐D‐tartaric acid as resolving agents. Enantiomer (R)‐(−)‐6 a was found to be the eutomer, with Ki values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.

Synthesis and Molecular Modeling Studies of Derivatives of a Highly Potent Peptidomimetic Vinyl Ester as Falcipain‐2 Inhibitors

Herein we report the synthesis of a set of constrained peptidomimetics endowed with an electrophilic vinyl ester warhead and structurally related to a previously identified lead compound, a potent and irreversible inhibitor of falcipain‐2 (FP‐2). FP‐2 is the main hemoglobinase of the malaria parasite P. falciparum. The new compounds were evaluated for their inhibition against FP‐2, and the results were rationalized on the basis of docking experiments. These studies underscore the pivotal role of both the ester function at the P1′ site and the trifluoromethyl group of the P3 side chain in determining the correct orientation of the Michael acceptor warhead in the catalytic site, and as a consequence, the potency of the inhibitors as well as their reversible or irreversible mode of inhibition.

Design of novel α7-subtype-preferring nicotinic acetylcholine receptor agonists: Application of docking and MM-PBSA computational approaches, synthetic and pharmacological studies

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric alpha7 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the alpha7 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal alpha7 as well as alpha4beta2 nAChRs.

Insights into docking and scoring neuronal α4β2 nicotinic receptor agonists using molecular dynamics simulations and QM/MM calculations

A combined quantum mechanical (QM)‐polarized docking and molecular dynamics approach to study the binding mode and to predict the binding affinity of ligands acting at the α4β2‐nAChR is presented. The results obtained in this study indicate that the quantum mechanical/molecular mechanics docking protocol well describes the charge‐driven interactions occurring in the binding of nicotinic agonists, and it is able to represent the polarization effects on the ligand exerted by the surrounding atoms of the receptor at the binding site. This makes it possible to properly score agonists of α4β2‐nAChR and to reproduce the experimental binding affinity data with good accuracy, within a mean error of 2.2 kcal/mol. Moreover, applying the QM‐polarized docking to an ensemble of nAChR conformations obtained from MD simulations enabled us to accurately capture nAChR‐ligand induced‐fit effects.