Stefano Della Longa

Glucosylceramide synthase (GCS) is an enzyme that catalyzes the first reaction of ceramide glycosylation in sphingolipid metabolism. It represents a primary target in the pharmacological treatment of some lysosomal storage diseases (LSDs), such as Gaucher and Niemann-Pick syndromes. In this study, starting from the model reported in the AlphaFold Protein Structure Database, the location and conformations of GCS substrates and cofactors have been provided by a step-by-step in silico procedure, by which the functional manganese ion and the substrates have been inserted in the GCS structure through combined molecular docking and full-atomistic molecular dynamics approaches, including metadynamics. A detailed analysis by structural dynamics of the complete model system, i.e., the enzyme anchored to the plasma membrane, containing the manganese ion and the two substrates, has been carried out to identify its complex conformational landscape by means of well-tempered metadynamics. A final structure was selected, in which both substrates were present in the active site of the enzyme at minimum distance, thus giving support to a SNi-type reaction mechanism for catalysis. Asp236, Glu235, and Asp144 are found to interact with the metal cofactor, which is able to trap the phosphates of UDP-glucose, while Gly210, Trp276, and Val208 cooperate to provide its correct orientation. Phe205, Cys207, Tyr237, and Leu284 form a pocket for the polar head of the ceramide, which is transiently placed in position to determine the catalytic event, when His193 interacts with the head of the ceramide, thus anchoring the substrate to the active site.

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt¹,Xaa⁵]N/OFQ(1-13)-NH₂ were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr¹ and Thr⁵ at the N-terminal portion of N/OFQ(1-13)-NH₂ with the noncanonical amino acid Dmt. [Dmt^1,5]N/OFQ(1-13)-NH₂ has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O₂ in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Cebranopadol (CBP) is a novel analgesic agonist of the nociceptin (NOP) and μ-opioid (MOP) receptors, exhibiting high potency and efficacy as antinociceptive and antihypersensitive drug. The binding conformation and the dynamical interactions of CBP with the NOP receptor have been investigated by molecular docking, molecular dynamics (MD) in the microsecond time scale, and hybrid quantum mechanics/molecular mechanics (QM/MM). CBP binds to the NOP receptor with a bidentate chelation of the aspartate D1303,32 by means of both its fluoro-indole and dimethyl nitrogens. Starting from the known crystal structure of the inactive state of the receptor, in complex with the antagonist compound-24 (NOP-C24) the comparative analysis of 1 μs MD trajectories of the NOP-C24 itself, NOP_free and NOP-CBP complex provides new insights on the already known microswitches related to receptor activation, in the frame of the extended ternary complex model. The agonist acts by destabilizing the inactive conformation of the NOP receptor, by inducing a conformational change of M1343,36, which allows W2766,48 to flip around its χ2 dihedral, going in close proximity to the receptor hydrophobic core (T1383,40, P2275,50, F2726,44), which is known to be fundamental for the activation of the ORs receptors. A complete rational picture is also provided for the role of N1333,35 and W276sup>6,48 undergoing critical conformational changes related to an anticooperativity effect, i.e. the well known role of sodium as negative modulator of agonist binding. Finally, the movement of residue Y3197,53( belonging to the NPxxY motif, is also induced by the binding of the agonist in the inactive state, opening a gate for a water channel just as upon receptor activation.

Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D130^3,32 (upper indexes are Ballesteros–Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y131^3,33, M134^3,36, I219^5,43, Q280^6,52 and V283^6,55) in the orthosteric site of NOP, accommodating either the pyridine–pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D130^3,32 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y131^3,33 and M134^3,36. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a conformation very similar to the one assumed by the antagonist JDTic into the K-opioid receptor. The proposed binding geometries fit better the binding pocket environment providing clues for experimental studies aimed to design selective or multifunctional opioid drugs.

The conversion of the prion protein (PrP^C) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrP^C interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrP^C constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrP^C coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.