Matteo Masetti

A catalytically silent FAAH-1 variant drives anandamide transport in neurons.

The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, that remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), that lacked amidase activity but bound anandamide with low micromolar affinity and facilitated its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, blocked these effects. We also identified a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, that prevented anandamide internalization in vitro, interrupted anandamide deactivation in vivo and exerted profound analgesic effects in rodent models of nociceptive and inflammatory pain, which were mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.

Revealing DNA Interactions with Exogenous Agents by Surface-Enhanced Raman Scattering

The standard protocols for DNA analysis largely involve polymerase chain reaction (PCR). However, DNA structures bound to chemical agents cannot be PCR-amplified, and therefore any sequence changes induced by external agents may be neglected. Thus, the development of analytical tools capable of characterizing the biochemical mechanisms associated with chemically induced DNA damage is demanded for the rational design of more effective chemotherapy drugs, understanding the mode of actions of carcinogenic chemicals, and monitoring the genotypic toxicology of environments. Here we report a fast, high-throughput, low-cost method for the characterization and quantitative recognition of DNA interactions with exogenous agents based on surface-enhanced Raman scattering spectroscopy. As representative chemical agents, we selected a chemotherapeutic drug (cisplatin) which forms covalent adducts with DNA, a duplex intercalating agent (methylene blue), and a cytotoxic metal ion (Hg(II)) which inserts into T:T mismatches. Rich structural information on the DNA complex architecture and properties is provided by the unique changes of their SERS spectra, which also offer an efficient analytical tool to quantify the extent of such binding.

Covalent Inhibitors of Fatty Acid Amide Hydrolase: A Rationale for the Activity of Piperidine and Piperazine Aryl Ureas

Recently, covalent drugs have attracted great interest in the drug discovery community, with successful examples that have demonstrated their therapeutic effects. Here, we focus on the covalent inhibition of the fatty acid amide hydrolase (FAAH), which is a promising strategy in the treatment of pain and inflammation. Among the most recent and potent FAAH inhibitors (FAAHi), there are the cyclic piperidine and piperazine aryl ureas. FAAH hydrolyzes efficiently the amide bond of these compounds, forming a covalent enzyme-inhibitor adduct. To rationalize this experimental evidence, we performed an extensive computational analysis centered on piperidine-based PF750 (1) and piperazine-based JNJ1661010 (2), two potent lead compounds used to generate covalent inhibitors as clinical candidates. We found that FAAH induces a distortion of the amide bond of the piperidine and piperazine aryl ureas. Quantum mechanics/molecular mechanics ΔE(LUMO-HOMO) energies indicate that the observed enzyme-induced distortion of the amide bond favors the formation of a covalent FAAH-inhibitor adduct. These findings could help in the rational structure-based design of novel covalent FAAHi.

Protein Flexibility in Drug Discovery: From Theory to Computation.

Nowadays it is widely accepted that the mechanisms of biomolecular recognition are strongly coupled to the intrinsic dynamic of proteins. In past years, this evidence has prompted the development of theoretical models of recognition able to describe ligand binding assisted by protein conformational changes. On a different perspective, the need to take into account protein flexibility in structure‐based drug discovery has stimulated the development of several and extremely diversified computational methods. Herein, on the basis of a parallel between the major recognition models and the simulation strategies used to account for protein flexibility in ligand binding, we sort out and describe the most innovative and promising implementations for structure‐based drug discovery.

Ion Conduction through the hERG Potassium Channel

The inward rectifier voltage-gated potassium channel hERG is of primary importance for the regulation of the membrane potential of cardiomyocytes. Unlike most voltage-gated K+-channels, hERG shows a low elementary conductance at physiological voltage and potassium concentration. To investigate the molecular features underlying this unusual behavior, we simulated the ion conduction through the selectivity filter at a fully atomistic level by means of molecular dynamics-based methods, using a homology-derived model. According to our calculations, permeation of potassium ions can occur along two pathways, one involving site vacancies inside the filter (showing an energy barrier of about 6 kcal mol−1), and the other characterized by the presence of a knock-on intermediate (about 8 kcal mol−1). These barriers are indeed in accordance with a low conductance behavior, and can be explained in terms of a series of distinctive structural features displayed by the hERG ion permeation pathway.

Combining dyad protonation and active site plasticity in BACE-1 structure-based drug design.

The ability of the BACE-1 catalytic dyad to adopt multiple protonation states and the conformational flexibility of the active site have hampered the reliability of computational screening campaigns carried out on this drug target for Alzheimers disease. Here, we propose a protocol that, for the first time, combining quantum mechanical calculations, molecular dynamics, and conformational ensemble virtual ligand screening addresses these issues simultaneously. The encouraging results prefigure this approach as a valuable tool for future drug discovery campaigns.

An automated docking protocol for hERG channel blockers.

A docking protocol aimed at obtaining a consistent qualitative and quantitative picture of binding for a series of hERG channel blockers is presented. To overcome the limitations experienced by standard procedures when docking blockers at hERG binding site, we designed a strategy that explicitly takes into account the conformations of the channel, their possible intrinsic symmetry, and the role played by the configurational entropy of ligands. The protocol was developed on a series of congeneric sertindole derivatives, allowing us to satisfactorily explain the structure-activity relationships for this set of blockers. In addition, we show that the performance of structure-based models relying on multiple-receptor conformations statistically increases when the protein conformations are chosen in such a way as to capture relevant structural features at the binding site. The protocol was then successfully applied to a series of structurally unrelated blockers.

Mechanistic insights into Pin1 peptidyl-prolyl cis-trans isomerization from umbrella sampling simulations

The peptidyl‐proyl isomerase Pin1 plays a key role in the regulation of phospho(p)‐Ser/Thr‐Pro proteins, acting as a molecular timer of the cell cycle. After recognition of these motifs, Pin1 catalyzes the rapid cis‐trans isomerization of proline amide bonds of substrates, contributing to maintain the equilibrium between the two conformations. Although a great interest has arisen on this enzyme, its catalytic mechanism has long been debated. Here, the cis‐trans isomerization of a model peptide system was investigated by means of umbrella sampling simulations in the Pin1‐bound and unbound states. We obtained free energy barriers consistent with experimental data, and identified several enzymatic features directly linked to the acceleration of the prolyl bond isomerization. In particular, an enhanced autocatalysis, the stabilization of perturbed ground state conformations, and the substrate binding in a procatalytic conformation were found as main contributions to explain the lowering of the isomerization free energy barrier. Proteins 2014; 82:2943–2956.

Collecting and Assessing Human Lactate Dehydrogenase-A Conformations for Structure-Based Virtual Screening

Human lactate dehydrogenase-A (LDHA) is emerging as a promising anticancer target. Up to now, structure-based investigations for identifying inhibitors of this enzyme have not explicitly accounted for active site flexibility. In the present study, by combining replica exchange molecular dynamics with network and cluster analyses, we identified reliable LDHA conformations for structure-based ligand design. The selected conformations were challenged and validated by retrospective virtual screening simulations.

Protein dynamics of the HIF-2α PAS-B domain upon heterodimerization and ligand binding.

Hypoxia-Inducible Factor (HIF) transcription factors are heterodimeric proteins involved in the regulation of oxygen homeostatis. Their upregulation has been related to several tumors with a remarkably poor clinical outcome. The recent discovery of a druggable cavity in the HIF-2α PAS-B domain has opened an unprecedented opportunity for targeting the HIF-2α transcription factor in view of pharmaceutical strategies. Coincidentally, a novel compound able to selectively disrupt the HIF heterodimerization with a submicromolar activity has been reported. In this work, we investigated the molecular mechanisms responsible for the inhibition by comparing the dynamical features of the HIF-2α PAS-B monomer and the HIF-2α PAS-B/HIF-1β PAS-B complex, in the ligand-bound and -unbound states. Plain and biased Molecular Dynamics were used to characterize the differential conformational changes both structurally and energetically.