Alessandro Piergentili

Imidazoline receptors: Qualitative structure-activity relationships and discovery of tracizoline and benazoline. Two ligands with high affinity and unprecedented selectivity☆

The observation that all the attempts to characterize imidazoline (I) receptors have been carried out with non-selective or poorly selective ligands prompted us to undertaken research aimed at developing selective ligand(s). In previous work using, as a starting point, cirazoline I, a potent alpha 1-adrenergic receptor agonist that also binds to I receptors, we showed that removal of the cyclopropyl ring (2) retains high affinity for I2 receptors while reducing alpha 1-adrenergic agonist activity. However, it was felt that this residual, albeit modest, alpha 1-adrenergic agonist activity might diminish the usefulness of compound 2, and we now report on our continuing efforts in this field. Starting from compound 2, we first eliminated the alpha 1-agonist component by isosteric replacement and then, by means of conformational restrictions on compound 7, succeeded in discovering tracizoline (9) and benazoline (12). These two new ligands with high affinity (pKi value 8.74 and 9.07, respectively) and unprecedented selectivity with respect to both alpha 2- (I2/alpha 2 7,762 and 18,621) and alpha 1- (I2/alpha 1 2,344 and 2,691) adrenergic receptors, are valuable tools in the study of I receptor structure and function. In addition, the large number of derivatives studied has allowed us to establish congruent qualitative structure-activity relationships and identify some structural elements governing affinity and selectivity.

Agonists and Antagonists Targeting the Different α2-Adrenoceptor Subtypes

Chemical and biological strategies have provided evidence for alpha(2)-receptor heterogeneity, to date classified in three different subtypes, alpha(2A), alpha(2B), and alpha(2C). These are widely distributed throughout the body and mediate numerous effects; therefore, the potential therapeutic indications of agonists and antagonists are numerous. Nevertheless, the lack of subtype-selectivity of the well-known compounds represents a major limit for their use. SAR studies may help to design new and more selective drugs.

Α2-Adrenoreceptors Profile Modulation. 4.1 From Antagonist to Agonist Behavior

The goal of the present study was to modulate the receptor interaction properties of known alpha 2-adrenoreceptor (AR) antagonists to obtain novel alpha 2-AR agonists with desirable subtype selectivity. Therefore, a phenyl group or one of its bioisosteres or aliphatic moieties with similar steric hindrance were introduced into the aromatic ring of the antagonist lead basic structure. The functional properties of the novel compounds allowed our previous observations to be confirmed. The high efficacy of 7, 12, and 13 as alpha 2-AR agonists and the significant alpha 2C-AR subtype selective activation displayed by 11 and 15 demonstrated that favorable interactions to induce alpha 2-AR activation were formed between the pendant groups of the ligands and the aromatic cluster present in transmembrane domain 6 of the binding site cavity of the receptors.

Favourable involvement of α2A-adrenoreceptor antagonism in the I₂-imidazoline binding sites-mediated morphine analgesia enhancement.

Aim of the present study was to obtain novel α(2)-adrenoreceptor (α(2)-AR) antagonists, possibly endowed with subtype-selectivity. Therefore, inspired by the non subtype-selective α(2)-AR antagonist idazoxan, we designed 1,4-dioxane derivatives bearing an aromatic area in position 5 or 6 and the imidazoline nucleus in position 2. Among the novel molecules 1-6, compound 2, with a trans stereochemical relationship between 5-phenyl and 2-imidazoline groups, was able to antagonize the sole α(2A)-subtype. Moreover, 2 showed an affinity at I(2)-imidazoline binding sites (I(2)-IBS) comparable to that at α(2A)-AR. In in vivo studies 2 strongly increased morphine analgesia. This interesting behaviour appeared to be induced by the favourable involvement of α(2A)-AR antagonism in the I(2)-IBS-mediated morphine analgesia enhancement.

Fruitful Adrenergic α2C-Agonism/α2A-Antagonism Combination to Prevent and Contrast Morphine Tolerance and Dependence(1),†

The functional in vitro study of the enantiomers of imidazolines 4-7 highlighted the role played by the nature of the ortho phenyl substituent in determining the preferred α(2C)-AR configuration. Indeed, the (S) enantiomers of 4-6 or (R) enantiomer of 7 behave as eutomers and activate this subtype as full agonists; the corresponding distomers are partial agonists. Because in clinical pain management with opioids α(2C)-AR agonists, devoid of the α(2A)-AR-mediated side effects, may represent an improvement over current therapies with clonidine like drugs, 4 and its enantiomers, showing α(2C)-agonism/α(2A)-antagonism, have been studied in vivo. The data suggest that partial α(2C)-activation is compatible with effective enhancement of morphine analgesia and reduction both of morphine tolerance acquisition and morphine dependence acquisition and expression. On the contrary, full α(2C)-activation appears advantageous in reducing morphine tolerance expression. Interestingly, the biological profile displayed by 4 (allyphenyline) and its eutomer (S)-(+)-4 has been found to be very unusual.

Ligand binding to I2 imidazoline receptor: the role of lipophilicity in quantitative structure-activity relationship models.

A series of 2-trans-styryl-imidazoline (tracizoline) congeners were designed and tested to develop 2-D and 3-D QSAR models for their binding to imidazoline (I2) receptor. The important role of lipophilicity was assessed by classical 2-D QSAR study (Hansch approach) and by comparative molecular field analysis (CoMFA) with the inclusion of the molecular lipophilicity potential (MLP), as an additional descriptor, besides standard steric and electrostatic fields. Results from these studies were compared to those obtained in a previous modeling study of I2 receptor ligands and integrated into a new, comprehensive model, based on about sixty I2 receptor ligands. This model revealed, at the three-dimensional level, the most significant steric, electrostatic, and lipophilic interactions accounting for high I2 receptor affinity.

Novel Highly Potent and Selective σ1 Receptor Antagonists Related to Spipethiane

Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel potent sigma(1) ligands. sigma(1) affinity and sigma(1/)sigma(2) selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing sigma(1) receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the sigma(1) antagonist profile of this series of compounds. In particular, due to its high sigma(1) affinity (pK(i) = 10.28) and sigma(1)/sigma(2) selectivity ratio (29510), compound 9 might be a novel valuable tool for sigma receptor characterization and a suitable template for the rational design of potential therapeutically useful sigma(1) antagonists.

Roles of Wnt/β-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats.

Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt-β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt-β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200mg/kg/day for 8days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n=8, P<0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P<0.01). At 25mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.

Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

BackgroundThere is evidence that calcium (Ca2+) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha1D-adrenergic receptor (alpha1D-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha1D-AR to cross-talk with TRPV1 in PCa cell lines.MethodsThe expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha1D-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha1D-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca2+ flux, IP3 production, western blot and MTT assay.ResultsThe levels of the alpha1D-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha1D-AR and TRPV1 expression levels was found. Moreover, alpha1D-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha1D-AR- and TRPV1-dependent protons release and Ca2+ flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha1D-AR/TRPV1 double knockdown.ConclusionsWe demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

Might adrenergic alpha2C-agonists/alpha2A-antagonists become novel therapeutic tools for pain treatment with morphine?

The imidazoline nucleus linked in position 2 via an oxyethylene bridge to a phenyl ring carrying an ortho substituent of moderate steric bulk provided alpha(2)-adrenergic (AR) ligands endowed with significant alpha(2C)-agonism/alpha(2A)-antagonism. Similar behavior was displayed by cirazoline (12). For their positive morphine analgesia modulation (due to alpha(2C)-AR stimulation) and sedation overcoming (due to alpha(2A)-AR antagonism), 8 and 11 might be useful as adjuvant agents in the management of pain with morphine.