1,721,126 research outputs found
Pharmacological and biotechnological in vitro approaches unveil the role of GPR17 signaling in regulating the timing of oligodendroglial differentiation
No full textBackground and Purpose - In the adult central nervous system there are many oligodendrocyte precursor cells (OPCs) that serve as the primary source of remyelinating cells in demyelinated lesions. Knowledge of the mechanisms regulating OPC maturation is needed to unveil novel pharmacological targets in demyelinating diseases. The G-protein-coupled membrane receptor GPR17, activated by both uracil nucleotides and cysteinyl-leucotrienes [1], has recently emerged as an important player in oligodendrogliogenesis [2,3]. It has been previously reported that GPR17 presence is restricted to NG2+-OPCs at early differentiation stages and is completely segregated from that of myelin proteins [4]. Here, we used purified primary OPCs from rat cortical parenchyma to assess the functional consequences of GPR17 modulation by either pharmacological or biotechnological approaches on the differentiation program of these cells.
Methods and Results - OPCs were exposed to the GPR17 agonists UDP-glucose, UDP and LTE4 for 48 hours. The degree of OPC differentiation was assessed on fixed cultures by immunostaining with an antibody against Myelin basic protein (MBP), a marker of mature oligodendrocytes. Data show that all these agonists increase the proportion of MBP+ cells compared to controls, suggesting acceleration of cell maturation by promoting receptor activation. Secondly, transfection experiments with fluorescent plasmids, enabling either silencing or over-expression of GPR17 were performed to univocally correlate the expression of this receptor with cell shape changes and phenotype acquisition during oligodendroglial maturation. Preliminary results show that suppression of GPR17 expression at early differentiation stages reduces the number of MBP+cells in culture, indicating that its silencing impairs the normal program of OPC differentiation.
Conclusions - Globally, these data point at GPR17 as a key regulator of oligodendrogliogenesis and at GPR17 ligands as extrinsic local regulators of OPCs under physiological conditions and during myelin repair.
References
[1] P. Ciana, M. Fumagalli, M.L. Trincavelli, C. Verderio, P. Rosa, D. Lecca, S. Ferrario, C. Parravicini, V. Capra, P. Gelosa, U. Guerrini, S. Belcredito, M. Cimino, L. Sironi, E. Tremoli, G.E. Rovati, C. Martini, M.P. Abbracchio, The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinil-leukotrienes receptor. EMBO J, 19, 4615-2627, 2006.
[2] D. Lecca, M.L. Trincavelli, P. Gelosa, L. Sironi, P. Ciana, M. Fumagalli, G. Villa, C. Verderio, C. Grumelli, U. Guerrini, E. Tremoli, P. Rosa, S. Cuboni, C. Martini, A. Buffo, M Cimino, M.P. Abbracchio, The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PloS One, 10, e3579, 2008.
[3] Y. Chen, H. Wu, S. Wang, H. Koito, J. Li, F. Ye, J. Hoang, S.S. Escobar, A. Gow, H.A. Arnett, B.D. Trapp, N.J. Karandikar, J. Hsieh, Q.R. Lu, The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination. Nature Neuroscience, 12, 1398-1406.
[4] M. Fumagalli, S. Daniele, D. Lecca, P.R. Lee, C. Parravicini, R.D. Fields,P. Rosa, F. Antonucci, C. Verderio, M.L Trincavelli, P. Bramanti, C. Martini, M.P. Abbracchio, Phenotypic changes, signaling pathway, and functional correlates of GPR17-expressing neural precursor cells during oligodendrocyte differentiation. The Journal of biology chemistry,12, 10593-10604
Molecular modelling in pharmacology: selected examples
No full textComputational chemistry and biology are helpful in understanding protein structure and the relationships between structure and biological activity.
In particular, to develop a new drug, medicinal chemists and pharmacologists are interested in understanding and predict drug action at a molecular level, especially if the action of the drug is unknown or poorly understood. In these cases, the molecular modelling should reduce some of the work in the development of drug compounds.
Here, we present two examples of homology modelling applied to pharmacology that obtained a great success.
Phosphatidylcholine-sterol acyltransferase (LCAT) is a glycoprotein of 416 residues, synthesized by the liver and secreted in plasma. It catalyzes the transacylation of the sn−2 fatty acid of lecithin to the free 3−OH group of cholesterol, generating cholesterol esters and lysolecithin. LCAT shares the Ser/Asp−Glu/His triad with lipases, esterases and proteases, but the low level of overall sequence homology between LCAT and these enzymes makes standard modelling procedures unsuitable. For this reason, to build an LCAT model, we implemented a combined approach that included folding recognition, secondary structure prediction, and ‘chimeric’ homology modeling. In detail, the ab initio model was used as scaffold to merge the two best homology templates identified, Paucimonas lemoignei depolymerase for the N-terminus and Candida antarctica lipase A for the C-terminus. In this way, we built an accurate LCAT structure with a well-defined binding site. Then, we performed a high-throughput virtual screening exploration of LCAT pocket with a large database of chemical compounds. The best compounds identified during the HTS were tested in vitro and demonstrated the ability to modulate LCAT activity.
G-protein coupled receptors (GPCRs) responding to signalling molecules are key transducers in cell-to-cell communication. Malfunctioning of GPCRs invariably leads to disease conditions; for this reason, they represent the target of more than 70% of currently marketed drugs. The rational design of new (ant)agonists targeting GPCRs strictly depends on the resolution of their atomistic structure and their appropriate in silico molecular modeling. However, the low homology degree of GPCRs with the available crystallized templates still represents a serious limitation. For several years, bovine rhodopsin (bRh) has been the only available high-resolution crystal structure of a GPCR, thus representing the unique possible template. The recent publication of new GPCR structures (human A2A adenosine receptor, human β2-adrenergic, turkey β1-adrenergic receptor, squid rhodopsin, human dopamine D3 receptor, human CXC chemokine type 4 receptor) has allowed the construction of more accurate and predictive models of GPCRs, which has represented a significant advancement toward a more rational drug design. A molecular modeling approach based on multiple templates has been applied to GPR17, a previously orphan GPCR that has recently emerged as a promising therapeutic target to foster recovery in diseases characterized by dysfunction of myelin, the oligodendroglial sheath that, by wrapping nerve terminals, ensures impulse transmission and communication between neurons. The obtained “chimeric” model of GPR17 has been then submitted to an efficient pipeline including ii) a high-throughput virtual screening with more than 130,000 lead-like compounds, and ii) a wet pharmacological validation of the top scoring chemical structures. This integrate strategy allowed us to successfully identify 5 agonists or partial agonists that had never been expected a priori to act on a GPCR, and behaved as extremely more potent ligands than GPR17 endogenous activators. Considering the relevance of the role proposed for GPR17 in demyelinating diseases, this new 5 ligands may represent an advancement toward the design of new pharmacological approaches aimed at restoring the myelin sheath integrity
Synthesis and molecular modeling of purine ribonucleotides as potential ligands of the human G protein-coupled receptor 17 (GPR17)
No full textGPCRs (G Protein-Coupled Receptors) are important drug targets in medicinal chemistry [1]. The GPR17 receptor, phylogenetically related to both purinergic P2Y and CysLT receptors, is usually over-expressed in the damaged brain tissue and is involved in various disorders characterized by demyelination, such as multiple sclerosis and stroke. Experimental data have shown that it is responsive to both agonists (e.g. nucleotides and their adducts) and antagonists (e.g. Cangrelor and Montelukast) [2]. Therefore, the human GPR17 receptor is a promising therapeutic target for treatment of neurodegenerative diseases [3].
This evidence prompted us to perform docking studies aided by molecular modeling on a homology model (based on P2Y1 receptors). Among the selected molecules, 8-methylaminoinosinic acid (1) and three N2-alkyl/acyl derivatives of guanylic acid (2-4) emerged as the best potential ligands.
As a result, their synthesis was carried out. Compound 1 was obtained by direct phosphorylation of 8-methylaminoinosine, previously prepared by amination of 8-bromoinosine. In the case of 2, position N2 of the purine ring was activated as a bromo derivative and subjected to displacement with n-octylamine. As for 3 and 4, N2-acylations were performed by treatment with a proper acyl chloride or anhydride through a transient protection strategy. Compounds 2, 3 and 4 were obtained as 2’,3’-O-isopropylidene adducts of the corresponding nucleotides.
Binding assays will be carried out by Surface Plasmon Resonance (SPR) [4], which has been demonstrated as a reliable technique for the systematic identification of agonists and antagonists of GPCRs, including GPR17 as recently demonstrated by our group [5].
[1] D. Wacker, R. C. Stevens, B. L. Roth, Cell 2017, 170, 414-427.
[2] P. Ciana, M. Fumagalli, M.L. Trincavelli, C. Verderio, P. Rosa, D. Lecca, S. Ferrario, C. Parravicini, V. Capra, P. Gelosa, U. Guerrini, S. Belcredito, M. Cimino, L. Sironi, E. Tremoli, G.E. Rovati, C. Martini and M.P. Abbracchio, EMBO J 2006, 25, 4615-4627.
[3] G. Marucci, D. Dal Ben, C. Lambertucci, A. Marti Navia, A. Spinaci, R. Volpini and M. Buccioni, Expert Opin. Ther. Pat. 2019, 29, 85-95.
[4] D.-S. Wang, S.-K. Fan, Sensors 2016, 16, 1175-1192.
[5] D. Capelli, C. Parravicini, G. Pochetti, R. Montanari, C. Temporini, M. Rabuffetti, M. L. Trincavelli, S. Daniele, M. Fumagalli, S. Saporiti, E. Bonfanti, M. P. Abbracchio, I. Eberini, S. Ceruti, E. Calleri, S. Capaldi, Front. Chem. 2020, 7, 910
With or without you - Proteomics with or without major plasma/serum proteins
Full text linkThe first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels
Forced unbinding of GPR17 ligands from wild-type and R255I mutant receptor models
No full textBackground:
GPR17 is a “hybrid” GPCR that responds to two unrelated ligand families, extracellular nucleotides and cysteinyl-LTs. Its in vivo blockade reduces progression of cerebral ischemic damage, highlighting GPR17 as a novel therapeutic target for ischemia.
Methods:
To explore the binding mode of the ”purinergic” and “leukotrienic” components of the receptor, we study the binding and the forced unbinding of two GPR17 ligands, the endogenous agonist UDP and the receptor antagonist pranlukast, from the wild-type and a mutant (R255I) models of GPR17, by means of docking and molecular dynamics simulations (MD) techniques .
Results:
MD suggests that GPR17 nucleotides binding pocket is enclosed between the helical bundle and covered by EL2. The driving interaction involves R255 and the phosphate moieties of nucleotides. This hypothesis was also supported by steered MD experiments. These showed that the energy required to unbind UDP was higher for the WT receptor than for R255I.
MD showed three possible binding sites for pranlukast. In one of its preferential docking conformation pranlukast places tetrazole group close to R255 and phenyl rings into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No significant differences between wild-type and mutant receptors were found for the unbinding of pranlukast from receptor.
Conclusion:
MD suggest a crucial role for R255 in nucleotides binding for GPR17. Aromatic interactions were likely to play a predominant role in recognition of the leukotrienic ligand pranlukast, suggesting that two different binding sites are present in GPR17
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
In silico identification of small molecules engaging the TNFR2-TNF-α interaction: a novel approach for targeting demyelinating diseases
No full textTumor necrosis factor alpha (TNF-α) is a cytokine secreted by macrophages, involved in immune and proinflammatory responses, cellular proliferation and differentiation. TNF-α exists in two isoforms: a soluble one, that participates to pathological mechanisms of demyelinating and neurodegenerative diseases preferably via TNF receptor 1 (TNFR1), and a transmembrane form, which can mediate neurorepair and remyelination via TNFR2.
Due to the protective role of TNFR2 in central nervous system (CNS), our aim was to identify ligands able to selectively enhance TNFR2::TNF-α engagement for promoting its reparative effects.
First, we characterized in silico the differences between TNFR1 and TNFR2 structures. We found that the interaction surfaces of both receptors show opposite electrostatic potential surfaces, suggesting that the selective engagement of the two TNF-α isoforms to TNFR1 or TNFR2 may depend on these electrostatic differences. Then, we tested a large library of commercially available drug-like compounds against the TNFR2::TNF-α complex, through virtual high-throughput screening. We identified 20 compounds with high affinity for the complex. Moreover, each TNFR2::TNF-α complex in association with the investigated ligands showed a G binding free energy gain with respect to the complex alone, suggesting that all the 20 compounds may enhance the affinity of TNF-α for TNFR2.
Finally, to assess if these compounds could be efficiently delivered to the brain, their ability to target CNS was predicted in silico by computing significant pharmacokinetic descriptors. Five out of the 20 selected compounds were characterized by a potential CNS activity.
To date, all the available approaches targeting the TNFR1/2: TNF-α axis for promoting TNFR2 neuroprotection are based on biotechnological drugs. On this basis, our data pave the way for the development of a new therapeutic strategy for demyelinating diseases based on TNFR2 engagement by small molecules with drug-like properties
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