Chiara Bianca Maria Platania

Homology Modeling of Dopamine D2 and D3 Receptors: Molecular Dynamics Refinement and Docking Evaluation

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D3 (hD3) receptor has been recently solved. Based on the hD3 receptor crystal structure we generated dopamine D2 and D3 receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD3 and hD2L receptors was differentiated by means of MD simulations and D3 selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental Ki was obtained for hD3 and hD2L receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.

Dopamine D3 Receptor Is Necessary for Ethanol Consumption: An Approach with Buspirone

Mesolimbic dopamine (DA) controls drug- and alcohol-seeking behavior, but the role of specific DA receptor subtypes is unclear. We tested the hypothesis that D3R gene deletion or the D3R pharmacological blockade inhibits ethanol preference in mice. D3R-deficient mice (D3R−/−) and their wild-type (WT) littermates, treated or not with the D3R antagonists SB277011A and U99194A, were tested in a long-term free choice ethanol-drinking (two-bottle choice) and in a binge-like ethanol-drinking paradigm (drinking in the dark, DID). The selectivity of the D3R antagonists was further assessed by molecular modeling. Ethanol intake was negligible in D3R−/− and robust in WT both in the two-bottle choice and DID paradigms. Treatment with D3R antagonists inhibited ethanol intake in WT but was ineffective in D3R−/− mice. Ethanol intake increased the expression of RACK1 and brain-derived neurotrophic factor (BDNF) in both WT and D3R−/−; in WT there was also a robust overexpression of D3R. Thus, increased expression of D3R associated with activation of RACK1/BDNF seems to operate as a reinforcing mechanism in voluntary ethanol intake. Indeed, blockade of the BDNF pathway by the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and strongly decreased the striatal expression of D3R. Finally, we evaluated buspirone, an approved drug for anxiety disorders endowed with D3R antagonist activity (confirmed by molecular modeling analysis), that resulted effective in inhibiting ethanol intake. Thus, DA signaling via D3R is essential for ethanol-related reward and consumption and may represent a novel therapeutic target for weaning.

Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles

Addition of one or more surfactant agents is often necessary for the production of nanostructured lipid and polymeric systems. The removal of residual surfactants is a required step for technological and toxicological reasons, especially for peculiar applications, such as the ophthalmic field. This study was planned to assess the technological properties of some surfactants, commonly used for the production of lipid nanoparticles, as well as their ocular safety profile. Stable and small-size solid lipid nanoparticles were obtained using Dynasan(®) 114 as the lipid matrix and all the tested surfactants. However, from a toxicological point of view, the nanocarriers produced using Kolliphor(®) P188 were the most valuable, showing no irritant effect on the ocular surface up to the highest tested surfactant concentration (0.4%, w/v). The SLN produced using Cremophor(®) A25 and Lipoid(®) S100 were tolerated up to a surfactant concentration of 0.2% by weight, while for Tween(®) 80 and Kolliphor(®) HS 15 a maximum concentration of 0.05% can be considered totally not-irritant.

Fortified Extract of Red Berry, Ginkgo biloba, and White Willow Bark in Experimental Early Diabetic Retinopathy

Diabetic retinopathy is a complex condition where inflammation and oxidative stress represent crucial pathways in the pathogenesis of the disease. Aim of the study was to investigate the effects of a fortified extract of red berries, Ginkgo biloba and white willow bark containing carnosine and α-lipoic acid in early retinal and plasma changes of streptozotocin-induced diabetic rats. Diabetes was induced by a single streptozotocin injection in Sprague Dawley rats. Diabetics and nondiabetic (control) rats were treated daily with the fortified extract for the ten days. Retina samples were collected and analyzed for their TNF-α and VEGF content. Moreover, plasma oxidative stress was evaluated by thiobarbituric acid reacting substances (TBARS). Increased TNF-α and VEGF levels were observed in the retina of diabetic rats. Treatment with the fortified extract significantly lowered retinal cytokine levels and suppressed diabetes-related lipid peroxidation. These data demonstrate that the fortified extract attenuates the degree of retinal inflammation and plasma lipid peroxidation preserving the retina in early diabetic rats.

Current drug treatments targeting dopamine D3 receptor

Dopamine receptors (DR) have been extensively studied, but only in recent years they became object of investigation to elucidate the specific role of different subtypes (D1R, D2R, D3R, D4R, D5R) in neural transmission and circuitry. D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D2R and D4R) differ in signal transduction, binding profile, localization in the central nervous system and physiological effects. D3R is involved in a number of pathological conditions, including schizophrenia, Parkinsons disease, addiction, anxiety, depression and glaucoma. Development of selective D3R ligands has been so far challenging, due to the high sequence identity and homology shared by D2R and D3R. As a consequence, despite a rational design of selective DR ligands has been carried out, none of currently available medicines selectively target a given D2-like receptor subtype. The availability of the D3R ligand [(11)C]-(+)-PHNO for positron emission tomography studies in animal models as well as in humans, allows researchers to estimate the expression of D3R in vivo; displacement of [(11)C]-(+)-PHNO binding by concurrent drug treatments is used to estimate the in vivo occupancy of D3R. Here we provide an overview of studies indicating D3R as a target for pharmacological therapy, and a review of market approved drugs endowed with significant affinity at D3R that are used to treat disorders where D3R plays a relevant role.

Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam

Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D3R). We previously reported that D3R null mice (D3R(-/-)) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D3R(-/-) than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D3R affect GABAA subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D3R(-/-) mice exhibited tolerance to diazepam (0.5mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D3R(-/-) mice; conversely, WT exhibited tolerance to diazepam after a 14-21 day-treatment. Analysis of GABAA α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D3R(-/-) than in WT. Diazepam treatment did not modify α6 expression in D3R(-/-), but progressively increased α6 expression in WT, to the level of untreated D3R(-/-) after 14-21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D3R(-/-); such expression level, however, slowly declined below control levels, by 14-21 days. Following a 7 day-treatment with the selective D3R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D3R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABAA subunit that has been linked to diazepam insensitivity. Modulation of GABAA receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D3R.

P2X7 receptor antagonism: Implications in diabetic retinopathy

ABSTRACT Diabetic retinopathy (DR) is the most frequent complication of diabetes and one of leading causes of blindness worldwide. Early phases of DR are characterized by retinal pericyte loss mainly related to concurrent inflammatory process. Recently, an important link between P2X7 receptor (P2X7R) and inflammation has been demonstrated indicating this receptor as potential pharmacological target in DR. Here we first carried out an in silico molecular modeling study in order to characterize the allosteric pocket in P2X7R, and identify a suitable P2X7R antagonist through molecular docking. JNJ47965567 was identified as the hit compound in docking calculations, as well as for its absorption, distribution, metabolism and excretion (ADME) profile. As an in vitro model of early diabetic retinopathy, human retinal pericytes were exposed to high glucose (25 mM, 48 h) that caused a significant (p < 0.05) release of IL‐1&bgr; and LDH. The block of P2X7R by JNJ47965567 significantly (p < 0.05) reverted the damage elicited by high glucose, detected as IL‐1&bgr; and LDH release. Overall, our findings suggest that the P2X7R represents an attractive pharmacological target to manage the early phase of diabetic retinopathy, and the compound JNJ47965567 is a good template to discover other P2X7R selective antagonists.

Effects of topical indomethacin, bromfenac and nepafenac on lipopolysaccharide-induced ocular inflammation.

To evaluate the effects of topical non‐steroidal anti‐inflammatory drugs (NSAIDs) on retinal vascular leakage, and inflammatory markers in endotoxin‐induced uveitis (EIU) in rats.

Molecular features of interaction between VEGFA and anti-angiogenic drugs used in retinal diseases: a computational approach.

Anti-angiogenic agents are biological drugs used for treatment of retinal neovascular degenerative diseases. In this study, we aimed at in silico analysis of interaction of vascular endothelial growth factor A (VEGFA), the main mediator of angiogenesis, with binding domains of anti-angiogenic agents used for treatment of retinal diseases, such as ranibizumab, bevacizumab and aflibercept. The analysis of anti-VEGF/VEGFA complexes was carried out by means of protein-protein docking and molecular dynamics (MD) coupled to molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculation. Molecular dynamics simulation was further analyzed by protein contact networks. Rough energetic evaluation with protein-protein docking scores revealed that aflibercept/VEGFA complex was characterized by electrostatic stabilization, whereas ranibizumab and bevacizumab complexes were stabilized by Van der Waals (VdW) energy term; these results were confirmed by MM-PBSA. Comparison of MM-PBSA predicted energy terms with experimental binding parameters reported in literature indicated that the high association rate (Kon) of aflibercept to VEGFA was consistent with high stabilizing electrostatic energy. On the other hand, the relatively low experimental dissociation rate (Koff) of ranibizumab may be attributed to lower conformational fluctuations of the ranibizumab/VEGFA complex, higher number of contacts and hydrogen bonds in comparison to bevacizumab and aflibercept. Thus, the anti-angiogenic agents have been found to be considerably different both in terms of molecular interactions and stabilizing energy. Characterization of such features can improve the design of novel biological drugs potentially useful in clinical practice.

TGF-β1 prevents rat retinal insult induced by amyloid-β (1–42) oligomers

To set up a retinal degenerative model in rat that mimics pathologic conditions such as age-related macular degeneration (AMD) using amyloid-β (Aβ) oligomers, and assess the effect of TGF-β1. Sprague-Dawley male rats were used. Human Aβ1-42 oligomers were intravitreally (ITV) injected (10µM) in the presence or in the absence of recombinant human TGF-β1 (1ng/μl ITV injected). After 48h, the animals were sacrificed and the eyes removed and dissected. The apoptotic markers Bax and Bcl-2 were assessed by western blot analysis in retina lysates. Gene-pathway network analysis was carried out in order to identify pathways involved in AMD. Treatment with Aβ oligomers induced a strong increase in Bax protein level (about 4-fold; p<0.01) and a significant reduction in Bcl-2 protein level (about 2-fold; p<0.05). Co-injection of TGF-β1 triggered a significant reduction of Bax protein induced by Aβ oligomers. Bioinformatic analysis revealed that Bcl-2 and PI3K-Akt are the most connected nodes, for genes and pathways respectively, in the enriched gene-pathway network common to AMD and Alzheimer disease (AD). Overall, these data indicate that ITV injection of Aβ1-42 oligomers in rat induces molecular changes associated with apoptosis in rat retina, highlighting a potential pathogenetic role of Aβ oligomers in AMD. Bioinformatics analysis confirms that apoptosis pathways can take part in AMD. Furthermore, these findings suggest that human recombinant TGF-β1 can prevent retinal damage elicited by Aβ oligomers.