Recent Theoretical Insights into the Oxidative Degradation of Biopolymers and Plastics by Metalloenzymes.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.

N-Acetylcysteine Antagonizes NGF Activation of TrkA through Disulfide Bridge Interaction, an Effect Which May Contribute to Its Analgesic Activity.

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300-345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

First-Principles Calculations on Ni,Fe-Containing Carbon Monoxide Dehydrogenases Reveal Key Stereoelectronic Features for Binding and Release of CO2 to/from the C-Cluster.

In view of the depletion of fossil fuel reserves and climatic effects of greenhouse gas emissions, Ni,Fe-containing carbon monoxide dehydrogenase (Ni-CODH) enzymes have attracted increasing interest in recent years for their capability to selectively catalyze the reversible reduction of CO2 to CO (CO2 + 2H+ + 2e- ⇌ CO + H2O). The possibility of converting the greenhouse gas CO2 into useful materials that can be used as synthetic building blocks or, remarkably, as carbon fuels makes Ni-CODH a very promising target for reverse-engineering studies. In this context, in order to provide insights into the chemical principles underlying the biological catalysis of CO2 activation and reduction, quantum mechanics calculations have been carried out in the framework of density functional theory (DFT) on different-sized models of the Ni-CODH active site. With the aim of uncovering which stereoelectronic properties of the active site (known as the C-cluster) are crucial for the efficient binding and release of CO2, different coordination modes of CO2 to different forms and redox states of the C-cluster have been investigated. The results obtained from this study highlight the key role of the protein environment in tuning the reactivity and the geometry of the C-cluster. In particular, the protonation state of His93 is found to be crucial for promoting the binding or the dissociation of CO2. The oxidation state of the C-cluster is also shown to be critical. CO2 binds to Cred2 according to a dissociative mechanism (i.e., CO2 binds to the C-cluster after the release of possible ligands from Feu) when His93 is doubly protonated. CO2 can also bind noncatalytically to Cred1 according to an associative mechanism (i.e., CO2 binding is preceded by the binding of H2O to Feu). Conversely, CO2 dissociates when His93 is singly protonated and the C-cluster is oxidized at least to the Cint redox state.

Redox Potentials of Small Inorganic Radicals and Hexa-Aquo Complexes of First-Row Transition Metals in Water: A DFT Study Based on the Grand Canonical Ensemble.

The potentials of redox systems involving nitrogen, oxygen, and metal ions of the first-row transition series have been computed according to the general approach of the grand canonical ensemble, which leads to the equilibrium value of the reduction potential via a (complete) sampling of configuration space at a given temperature. The approach is a single configuration approach in the sense that identical molecular structures are sampled for both the oxidized and reduced species considered in water solution. In this study, the solute and a cluster of 11-12 water molecules are treated explicitly at the same level of theory and embedded in a continuum solvent. The molecular energies are computed in the framework of the density functional theory. Our approach is basically different from the approach based on the ThermoDynamic Cycle involving gas-phase calculations of the electron affinity of the oxidized species, corrected by the differential hydration energy (obtained from continuum solvent models only) between oxidized and reduced forms. The calculated redox potentials are in agreement with the available experimental data much closer than other results so far presented in the literature. Our results are very satisfactory also in the case of the 3+/2+ redox states of the first-row transition metals, i.e., systems with a high positive charge for which enhanced effects of the solvent are expected.

Reactivation of the Ready and Unready Oxidized States of [NiFe]-Hydrogenases: Mechanistic Insights from DFT Calculations.

The apparently simple dihydrogen formation from protons and electrons (2H+ + 2e- ⇄ H2) is one of the most challenging reactions in nature. It is catalyzed by metalloenzymes of amazing complexity, called hydrogenases. A better understanding of the chemistry of these enzymes, especially that of the [NiFe]-hydrogenases subgroup, has important implications for production of H2 as alternative sustainable fuel. In this work, reactivation mechanism of the oxidized and inactive Ni-B and Ni-A states of the [NiFe]-hydrogenases active site has been investigated using density functional theory. Results obtained from this study show that one-electron reduction and protonation of the active site promote the removal of the bridging hydroxide ligand contained in Ni-B and Ni-A. However, this process is sufficient to activate only the Ni-B state. H2 binding to the active site is required to convert Ni-A to the active Ni-SIa state. Here, we also propose a reasonable structure for the spectroscopically well-characterized Ni-SIr and Ni-SU species, formed respectively from the one-electron reduction of Ni-B and Ni-A. Ni-SIr, depending on the pH at which the reaction occurs, features a bridging hydroxide ligand or a water molecule terminally coordinated to the Ni atom, whereas in Ni-SU a water molecule is terminally coordinated to the Fe atom, and the Cys64 residue is oxidized to sulfenate. The sulfenate oxygen atom in the Ni-A state affects the stereoelectronic properties of the binuclear cluster by modifying the coordination geometry of Ni, and consequently, by switching the regiochemistry of H2O and H2 binding from the Ni to the Fe atom. This effect is predicted to be at the origin of the different reactivation kinetics of the oxidized and inactive Ni-B and Ni-A states.

Theoretical investigation of aerobic and anaerobic oxidative inactivation of the [NiFe]-hydrogenase active site.

The extraordinary capability of [NiFe]-hydrogenases to catalyse the reversible interconversion of protons and electrons into dihydrogen (H2) has stimulated numerous experimental and theoretical studies addressing the direct utilization of these enzymes in H2 production processes. Unfortunately, the introduction of these natural H2-catalysts in biotechnological applications is limited by their inhibition under oxidising (aerobic and anaerobic) conditions. With the aim of contributing to overcome this limitation, we studied the oxidative inactivation mechanism of [NiFe]-hydrogenases by performing Density Functional Theory (DFT) calculations on a very large model of their active site in which all the amino acids forming the first and second coordination spheres of the NiFe cluster have been explicitly included. We identified an O2 molecule and two H2O molecules as sources of the two oxygen atoms that are inserted at the active site of the inactive forms of the enzyme (Ni-A and Ni-B) under aerobic and anaerobic conditions, respectively. Furthermore, our results support the experimental evidence that the Ni-A-to-Ni-B ratio strongly depends on the number of reducing equivalents available for the process and on the oxidizing conditions under which the reaction takes place.

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

Copper ion interaction with the RNase catalytic site fragment of the angiogenin protein: an experimental and theoretical investigation.

The angiogenin protein (Ang) is a member of the vertebrate-specific secreted ribonucleases and one of the most potent angiogenic factors known. Ang is a normal constituent of human plasma and its concentration increases under some physiological and pathological conditions to promote neovascularization. Ang was originally identified as an angiogenic tumour factor, but its biological activity has been found to extend from inducing angiogenesis to promoting cell survival in different neurodegenerative diseases. Ang exhibits weak ribonucleolytic activity, which is critical for its biological functions. The RNase catalytic sites are two histidine residues, His-13 and His-114, and the lysine Lys-40. Copper is also an essential cofactor in angiogenesis and influences angiogenin's biological properties. The main Cu(ii) anchoring site of Ang is His-114, where metal binding inhibits RNase activity of the protein. To reveal the Cu(ii) coordination environment in the C-terminal domain of the Ang protein, we report on the characterization, by means of potentiometric, voltammetric, and spectroscopic (CD, UV-Vis and EPR) methods and DFT calculations, of Cu(ii) complexes formed with a peptide fragment including the Ang sequence 112-117 (PVHLDQ). Potentiometric titrations indicated that [CuLH-2] is the predominant species at physiological pH. EPR, voltammetric data and DFT calculations are consistent with a CuN3O2 coordination mode in which a distorted square pyramidal arrangement of the peptide was observed with the equatorial positions occupied by the nitrogen atoms of the deprotonated amides of the Asp and Leu residues, the δ-N atom of histidine and the oxygen atom of the aspartic carboxylic group. Moreover, two analogous peptides encompassing the PVHLNQ and LVHLDQ sequences were also characterized by using thermodynamic, spectroscopic and DFT studies to reveal the role they play in Cu(ii) complex formation by the carboxylate side chain of the Asp and Pro residues, a known breaking-point in metal coordination.

Excited state properties of diiron dithiolate hydrides: implications in the unsensitized photocatalysis of H2 evolution.

Density functional theory (DFT) and time-dependent DFT (TDDFT) have been used to investigate how visible light photons can excite an asymmetrically substituted diiron hydride, [Fe2(pdt)(µ-H)(CO)4dppv](+) (1(+), dppv = cis-1,2-C2H2(PPh2)2; pdt = 1,3-propanedithiolate), as well as the symmetric species [Fe2(pdt)(µ-H)(CO)4(PMe3)2](+) (2(+)), which are the first photocatalysts of proton reduction operating without employing sensitizers (Wang, W.; Rauchfuss, T. B.; Bertini, L.; Zampella, G.; J. Am. Chem. Soc., 2012, 134, 4525). Theoretical results illustrate that the peculiar reactivity associated to the excited states of 1(+) and 2(+) is compatible with three different scenarios: (i) it can arise from the movement of the hydride ligand from fully bridging to semibridging/terminal coordination, which is expected to be more reactive toward protons; (ii) reactivity could be related to cleavage of a Fe-S bond, which implies formation of a transient Fe penta-coordinate species that would trigger a facile turnstile hydride isomerization, if lifetime excitation is long enough; (iii) also in line with a Fe-S bond cleavage is the possibility that after excited state decay, a highly basic S center is protonated so that a species simultaneously containing S-H(δ+) and Fe-H(δ-) moieties is formed and, once reduced by a suitable electron donor, it can readily afford H2 plus an unprotonated form of the FeFe complex. This last possibility is consistent with (31)P NMR and IR solution data. All the three possibilities are compatible with the capability of 1(+) and 2(+) to perform photocatalysis of hydrogen evolving reaction (HER) without sensitizer. Moreover, even though it turned out difficult to discriminate among the three scenarios, especially because of the lack of experimental excitation lifetimes, it is worth underscoring that all of the three pathways represent a novelty regarding diiron carbonyl photoreactivity, which is usually associated with CO loss. Results provide also a rationale to the experimental observations which showed that the simultaneous presence of donor ligands (dppv in the case of 1(+)) and a H ligand in the coordination environment of diiron complexes is a key factor to prevent CO photodissociation and catalyze HER. Finally, the comparison of photoexcitation behavior of 1(+) and 2(+) allows a sort of generalization about the functioning of such hydride species.

Speciation of copper-peptide complexes in water solution using DFTB and DFT approaches: case of the [Cu(HGGG)(Py)] complex.

The DFTB and DFT methods are applied to the study of different forms of the [Cu(HGGG)(Py)] complex in water, with the aim of identifying the most stable isomer. The DFTB calculations were possible thanks to a careful parametrization of the atom-atom repulsive energy terms for Cu-H, Cu-C, Cu-N, and Cu-O. The speciation process is carried out by computing different DFTB-steered molecular dynamics (SMD) trajectories, each of which ends in a well-defined different form. The last frame of each trajectory is subjected to geometry optimization at both DFTB and DFT levels, leading to a different isomer. From the corresponding energy values, a rank of relative stability of the isomers can be established. The computational protocol developed here is of general applicability to other metal-peptide systems and represents a new powerful tool for the study of speciation of metal-containing systems in water solution, particularly useful when the full characterization of the compound cannot be carried out on the basis of experimental results only.

Probing the effects of one-electron reduction and protonation on the electronic properties of the Fe-S clusters in the active-ready form of [FeFe]-hydrogenases. A QM/MM investigation.

A QM/MM investigation of the active-ready (H(ox)) form of [FeFe]-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H(2) formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in H(ox) is unlikely, and in any case it would not trigger electron transfer from the accessory Fe(4)S(4) clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed.

Mechanistic and physiological implications of the interplay among iron-sulfur clusters in [FeFe]-hydrogenases. A QM/MM perspective.

Key stereoelectronic properties of Desulfovibrio desulfuricans [FeFe]-hydrogenase (DdH) were investigated by quantum mechanical description of its complete inorganic core, which includes a Fe(6)S(6) active site (the H-cluster), as well as two ancillary Fe(4)S(4) assemblies (the F and F' clusters). The partially oxidized, active-ready form of DdH is able to efficiently bind dihydrogen, thus starting H(2) oxidation catalysis. The calculations allow us to unambiguously assign a mixed Fe(II)Fe(I) state to the catalytic core of the active-ready enzyme and show that H(2) uptake exerts subtle, yet crucial influences on the redox properties of DdH. In fact, H(2) binding can promote electron transfer from the H-cluster to the solvent-exposed F'-cluster, thanks to a 50% decrease of the energy gap between the HOMO (that is localized on the H-cluster) and the LUMO (which is centered on the F'-cluster). Our results also indicate that the binding of the redox partners of DdH in proximity of its F'-cluster can trigger one-electron oxidation of the H(2)-bound enzyme, a process that is expected to have an important role in H(2) activation. Our findings are analyzed not only from a mechanistic perspective, but also in consideration of the physiological role of DdH. In fact, this enzyme is known to be able to catalyze both the oxidation and the evolution of H(2), depending on the cellular metabolic requirements. Hints for the design of targeted mutations that could lead to the enhancement of the oxidizing properties of DdH are proposed and discussed.

DFT characterization of key intermediates in thiols oxidation catalyzed by amavadin.

Amavadin is an unusual octa-coordinated V(IV) complex isolated from Amanita muscaria mushrooms. The outer-sphere catalytic properties of such a complex toward several oxidation reactions are well known. Nevertheless, a remarkable example exists, in which the V(V) (d(0)) oxidized form of amavadin is able to electro-catalyze the oxidation of some thiols to the corresponding disulfides through an inner-sphere mechanism (Guedes da Silva et al. J. Am. Chem. Soc.1996, 118, 7568-7573.) The reaction mechanism implies the formation of an amavadin-substrate intermediate, whose half-life is about 0.3 s. By means of Density Functional Theory (DFT) computations and Quantum Theory of Atoms in Molecules (QTAIM) analysis of the electron density, we have first characterized the stereoelectronic features of the V(IV) (inactive) and V(V) (active) states of amavadin. Then, the formation of the V(V) complex with methyl mercaptoacetate (MMA), which has been chosen as a prototypical substrate, has been characterized both thermodynamically and kinetically. DFT results reveal that protonation of V(V) amavadin at a carboxylate oxygen not directly involved in the V coordination, favors MMA binding into the first coordination sphere of vanadium, by substitution of the amavadin carboxylate oxygen with that of the substrate and formation of an S-HO hydrogen bond interaction. The latter interaction can promote SH deprotonation and binding of the thiolate group to vanadium. The kinetic and thermodynamic feasibility of the V(V)-MMA intermediates formation is in agreement, along with electrochemical experimental data, also with the biological role exerted by amavadin. Finally, the presence of an ester functional group as an essential requisite for MMA oxidation has been rationalized.

An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes.

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in ß4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the ß4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

Isocyanide in biochemistry? A theoretical investigation of the electronic effects and energetics of cyanide ligand protonation in [FeFe]-hydrogenases.

The presence of Fe-bound cyanide ligands in the active site of the proton-reducing enzymes [FeFe]-hydrogenases has led to the hypothesis that such Brønsted-Lowry bases could be protonated during the catalytic cycle, thus implying that hydrogen isocyanide (HNC) might have a relevant role in such crucial microbial metabolic paths. We present a hybrid quantum mechanical/molecular mechanical (QM/MM) study of the energetics of CN(-) protonation in the enzyme, and of the effects that cyanide protonation can have on [FeFe]-hydrogenase active sites. A detailed analysis of the electronic properties of the models and of the energy profile associated with H(2) evolution clearly shows that such protonation is dysfunctional for the catalytic process. However, the inclusion of the protein matrix surrounding the active site in our QM/MM models allowed us to demonstrate that the amino acid environment was finely selected through evolution, specifically to lower the Brønsted-Lowry basicity of the cyanide ligands. In fact, the conserved hydrogen-bonding network formed by these ligands and the neighboring amino acid residues is able to impede CN(-) protonation, as shown by the fact that the isocyanide forms of [FeFe]-hydrogenases do not correspond to stationary points on the enzyme QM/MM potential-energy surface.

NGF and heart: Is there a role in heart disease?

The review emphasizes the role of NGF, the most representative member of the neurotrophins family, in cardiac physiopathology with a particular focus on healing and sprouting processes occurring after tissue damage. Cardiac and circulating NGF levels dramatically increase following myocardial injury (MI). A very early rise of this neurotrophin is indeed observed soon after MI (hours). Such a rise may lead to sympathetic nerve sprouting which may underlie the later genesis of arrhythmias but may also favor the healing process. At later times (months after), when heart failure develops, the opposite is detected and NGF tissue levels are below the normal range, an event that may in turn participate to defective innervation and cardiac failure. Through a careful analysis of preclinical and clinical studies, this review proposes that time is the key variable when studying these opposite changes in NGF expression observed following MI and attempting to interpret and correlate them with cardiac physiopathology. The examination of the results leads to the speculation that NGF modulation may be a pharmacological target for interventions in specific stages of heart dysfunction following MI.

DFT characterization of the reaction pathways for terminal- to µ-hydride isomerisation in synthetic models of the [FeFe]-hydrogenase active site.

The mechanism of terminal- to µ-hydride isomerisation in models of synthetic complexes resembling the [FeFe]-hydrogenase active site has been elucidated by DFT calculations, revealing that Ray-Dutt reaction pathways are generally favoured, and providing some clues for the rational design of novel synthetic catalysts to produce H(2).

Electrocatalytic dihydrogen evolution mechanism of [Fe2(CO)4(kappa(2)-Ph2PCH2CH2PPh2)(mu-S(CH2)3S)] and related models of the [FeFe]-hydrogenases active site: a DFT investigation.

A DFT study of protonation thermodynamics in H(2)-evolving biomimetic catalysts related to [FeFe]-hydrogenases active site is presented here. Taking as a reference system the electrocatalytic dihydrogen evolution mechanism recently proposed for the synthetic assembly [Fe(2)(CO)(4)(kappa(2)-Ph(2)PCH(2)CH(2)PPh(2))(mu-S(CH(2))(3)S)] (a, which is able to release H(2) after having undergone monoelectron reduction steps and three sequential protonation reactions), we show how the reduction of model complexes to oxidation states lower than those observed in [FeFe]-hydrogenases cofactor leads to a protonation regiochemistry that has no counterpart in the enzymatic mechanism of H(2) production. In particular, double protonation of the metal centers turned out to be disfavored in a by up to 12.5 kcal mol(-1) with respect to alternative protonation paths; as for the regiochemistry of triple protonation, the formation of eta(2)-H(2) adducts is disfavored by at least approximately 25 kcal mol(-1). Structural analysis of the theoretical models also revealed that over-reduction of synthetic complexes, though necessary for observing H(2) evolution from the currently available biomimetic electrocatalysts, can generally impair their structural integrity. Possible approaches for the modulation of protonation regiochemistry are then proposed; in particular, it turned out that a targeted use of sigma-donating ligands showing low basicity can favor double protonation of iron centers.

Near native-state conformational landscape of psychrophilic and mesophilic enzymes: probing the folding funnel model.

In recent years, increased interest has been directed to the study of enzyme adaptation to low temperatures. In particular, a peculiar folding funnel model was proposed for the free energy landscape of a psychrophilic alpha-amylase and other cold-adapted enzymes. In the present contribution, the comparison between the near native-state dynamics and conformational landscape in the essential subspace of different cold-adapted enzymes with their mesophilic counterparts, as obtained by more than 0.1 micros molecular dynamics simulations at different temperatures, allows the folding funnel model to be probed. Common characteristics were highlighted in the near native-state dynamics of psychrophilic enzymes belonging to different enzymatic families when compared to the mesophilic counterparts. According to the model, a cold-adapted enzyme in its native-state consists of a large population of conformations which can easily interconvert and result in high structural flexibility.

Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor.

The application of frontal affinity chromatography-mass spectrometry (FAC-MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC-MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([(35)S]GTPgammaS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.