MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations.

BACKGROUND AND PURPOSE: The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. EXPERIMENTAL APPROACH: UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs. KEY RESULTS: UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. CONCLUSIONS AND IMPLICATIONS: The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

Epidermal growth factor receptor irreversible inhibitors: chemical exploration of the cysteine-trap portion.

Covalent EGFR irreversible inhibitors showed promising potential for the treatment of gefitinib-resistant tumors and for imaging purposes. They contain a cysteine-reactive portion forming a covalent bond with the protein. Irreversible kinase inhibitors have been advanced to clinical studies, mostly characterized by an acrylamide or butynamide warhead. However, the clinical usefulness of these compounds has been hampered by resistances, toxicity and pharmacokinetic problems. Investigation on the structure-activity and structure-reactivity relationships may provide useful information for compounds with improved selectivity and pharmacokinetic properties. This review focuses on the exploration of the cysteine-trap portions able to irreversibly inhibit EGFR and other erbB receptors.

2-N-acylaminoalkylindoles: design and quantitative structure-activity relationship studies leading to MT2-selective melatonin antagonists.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Structure-property relationships on histamine H3-antagonists: binding of phenyl-substituted alkylthioimidazole derivatives to rat plasma proteins.

The binding of a series of H3-antagonists to rat plasma proteins was investigated by dialysis experiments, with RP-HPLC measurement of the free ligand. The series was composed of 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles having, on the phenyl ring, meta- and para-substituents, with different physico-chemical characteristics. As high protein binding had been proposed as being one of the features limiting brain access for the reference H3-antagonist thioperamide, the title series was employed to test the possibility of achieving lower protein binding by modulation of lipophilicity, while maintaining good receptor affinity. The compounds tested showed quotas of bound drug ranging from 60 to 97.5%, while for thioperamide a 78% bound drug quota was observed at high total concentrations, with a steep increase in bound percentage at lower concentrations. Two of the tested compounds, having a carboxamide substituent, showed lower protein binding compared to thioperamide over a wide range of total concentration, without a significant loss in affinity with respect to the parent compound. A strict dependence of protein binding on lipophilicity was observed, and a QSPR model was derived which could also account for the protein binding observed for thioperamide, while receptor affinity had been reported to be quite insensitive to phenyl ring substitution. It is therefore possible to modulate protein binding of these H3-antagonists, through lipophilicity adjustment, without losing receptor affinity; this finding could help in the design of new compounds with improved brain access.

Synthesis and biological assays of new H3-antagonists with imidazole and imidazoline polar groups.

New histamine H3-receptor antagonists were synthesised and tested on rat brain membranes and on electrically stimulated guinea-pig ileum. The new compounds have a central polar group represented by a 2-alkylimidazole or a 2-thioimidazoline nucleus. The effect of the polar group basicity on the optimal length of the alkyl chain, connecting this group to a 4(5)-imidazolyl ring, was investigated. The best affinity values, obtained by displacement of [3H]-RAMHA from rat brain, were obtained for the 2-alkylimidazole derivatives (2a-f) with tetramethylene chain (pKi 8.03-8.97), having an intermediate basicity between that of the previously reported 2-thioimidazoles (1a-i) and that of 2-alkylthioimidazolines (3a-h). In contrast, a general lowering of affinity (pKi 5.90-7.63) was observed for compounds of the last series (3a-h), with a complex dependence on the terminal lipophilic group and chain length.

Experimental and theoretical analysis of the interaction of (+/-)-cis-ketoconazole with beta-cyclodextrin in the presence of (+)-L-tartaric acid.

1H NMR spectroscopy was used for determining the optical purity of cis-ketoconazole enantiomers obtained by fractional crystallization. The chiral analysis was carried out using beta-cyclodextrin in the presence of (+)-L-tartaric acid. The mechanism of the chiral discrimination process, the stability of the complexes formed, and their structure in aqueous solution were also investigated by 1H and 13C chemical shift analysis, two-dimensional NOE experiments, relaxation time measurements, and mass spectrometry experiments. Theoretical models of the three-component interaction were built up on the basis of the available NMR data, by performing a conformational analysis on the relevant fragments on ketoconazole and docking studies on the components of the complex. The model derived from a folded conformation of ketoconazole turned out to be fully consistent with the molecular assembly found in aqueous solution, as inferred from NOE experiments. An explanation of the different association constants for the complexes of the two enantiomers is also provided on the basis of the interaction energies.

Melatonin receptor ligands: synthesis of new melatonin derivatives and comprehensive comparative molecular field analysis (CoMFA) study.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

H3-receptor antagonists: synthesis and structure-activity relationships of para- and meta-substituted 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles.

We report the synthesis, octanol/water partition coefficient (log P), dissociation constants (pKa), H3-receptor affinity (pKi in rat brain membranes, [3H]-N alpha-methylhistamine), and H3-antagonist potency (pA2 in guinea ileum, (R)-alpha-methylhistamine) of novel H3-receptor antagonists obtained by introducing a para or meta substituent on the phenyl ring of the lead compound 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole (3a). The substituents were chosen to obtain broad and uncorrelated variation in their lipophilic, electronic, and steric properties. The log P values of the neutral species cover almost 3 orders of magnitude (from 1.40 to 4.11). The pKa,2 values (protonation of the 2-thioimidazole fragment) vary from 3.13 to 4.34, indicating that this fragment, which incorporates the so-called polar group common to many H3-receptor antagonists, is neutral at physiological pH. The compounds had pKi values in a range too narrow (from 7.28 to 8.03) to derive QSAR equations. In one case (3g), a biphasic displacement curve was observed (pKi,1 = 8.53; pKi,2 = 6.90). The pA2 values ranged 2 orders of magnitude (from 6.83 to 8.87) and yielded a QSAR model (PLS) indicating that antagonist potency depends parabolically on lipophilicity and is decreased by bulky para substituents. The compounds of this series, therefore, maintain a fair-to-good affinity for rat brain H3-receptor and a fair-to-good H3-antagonist potency on guinea pig ileum, although varying markedly in their lipophilicity. The series thus appears as a good candidate for pharmacokinetic optimization leading to brain-penetrating H3-receptor antagonists.

Conformationally restrained melatonin analogues: synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

Plasma concentration and brain penetration of the H3-receptor antagonist thioperamide in rats.

Thioperamide is a potent and selective H3-receptor antagonist, whose in vivo effects have been reported after systemic administration. Some questions have arisen about its ability to cross the blood-brain barrier, since different experimental conditions have given different results in rats, namely a low brain/blood ratio at low doses (10 mg/Kg) and a much higher one at higher doses (60 mg/Kg). In this work we demonstrate the dose-dependence of thioperamide pharmacokinetics, measuring its plasma and cerebral levels after i.p. administration of different doses to rats. Both the plasma half-life and brain penetration of thioperamide resulted as being dose-dependent: when administered to 80 g body weight Wistar rats at 10 mg/Kg i.p., the drug has a short half life (120') and a rather poor brain penetration, but increasing the dose (to 20, 40 and 60 mg/Kg) gives rise to a prolongation of its persistence in the blood (up to 600' at highest dose) and a higher brain penetration. Also, the profile of the plasma concentration curve varies from the dose of 10 to that of 20 mg/Kg, passing from a mono-exponential decrease to a more complex one characterized by an apparent distribution phase. The different distribution processes can be interpreted in the light of thioperamide protein binding and affinity for lipophilic tissues: protein binding can prevent brain penetration (but not distribution to other tissues) at lower doses, while at higher doses the free plasma fraction increases and it can allow passive distribution to lipophilic tissues such as brain tissues. A re-distribution from these tissues and plasma is probably responsible for the strong increase in half-life at high doses.

In vitro characterization of potency, affinity and selectivity of H3-antagonists: from thioperamide to thioperamide unrelated imidazole derivatives.

This paper summarizes the findings obtained for three different series of original compounds designed as potential H3-antagonists starting from thioperamide structure. The compounds were tested in functional and binding assays to estimate their potency, affinity and selectivity for histamine H3 receptors. Among them, many non-thiourea/isothiourea derivatives acted as selective H3 competitive antagonists and, particularly, 4(5)-[2-[4(5)-cyclohexylimidazol-2-ylthio]ethyl] imidazole (dIII) proved to be the most potent H3 blocker vs (R)-alpha-methylhistamine in electrically-stimulated ileum. This imidazole derivative, devoid of thiourea dependent toxic effects, with high affinity displaced biphasically [3H]-N alpha-methylhistamine bound to rat brain H3 sites. Thus, such compound could be proposed as the prototype molecule for the development of new non-thiourea/isothiourea H3-antagonists and as experimental tool to explore the intriguing question of H3 receptor heterogeneity.

Ligands of the histamine H3-receptor: new potent antagonists of the 2-thioimidazole type.

An overview of H3-receptor ligands is presented, with particular attention to antagonists. The protein binding of the classical H3-receptor antagonist thioperamide and its effect on in vivo distribution are discussed. A series of H3-receptor antagonists characterised by the presence of an imidazole ring, a spacer (ethylthio-, ethylamino-, propylthio- or propylamino-chain), a second heterocycle nucleus and a lipophilic group is described. Their H3-receptor antagonist potency has been measured on electrically stimulated guinea-pig intestine, and their affinity for central H3-receptor has been determined by competitive inhibition of [3H]N alpha-methylhistamine binding to rat cortex. Biphasic inhibition curves have been observed in some cases. Compounds endowed with interesting activity belong mostly to the class of 2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole, having a phenyl or a cyclohexyl group.