Alessandro Giolitti

Antiproliferative and differentiating activities of a novel series of histone deacetylase inhibitors.

Histone deacetylases are promising molecular targets for the development of antitumor agents. A novel series of histone deacetylase inhibitors of the hydroxamic acid type were synthesized for structure-activity studies. Thirteen tricyclic dibenzo-diazepine, -oxazepine, and -thiazepine analogues were studied and shown to induce variable degrees of histone H3/H4 and tubulin acetylation in a cellular model of myeloid leukemia sensitive to all-trans retinoic acid (ATRA). Multiparametric correlations between acetylation of the three substrates, tumor cell growth inhibition, and ATRA-dependent cytodifferentiation were performed, providing information on the chemical functionalities governing these activities. For two analogues, antitumor activity in the animal was demonstrated.

Molecular determinants of peptide and nonpeptide NK-2 receptor antagonists binding sites of the human tachykinin NK-2 receptor by site-directed mutagenesis.

A series of 14 mutants on nine selected residues of the human tachykinin NK(2) receptor was produced and stably transfected into CHO cells to investigate the binding of the peptide MEN 11420 and the nonpeptide SR 48968 antagonists. The main interactions found for MEN 11420 were with Thr171, Tyr206, Tyr266 and Phe270. In the case of SR 48968 crucial residues were Tyr266 and Tyr289. While some overlapping of the binding sites exists, the binding modes suggested by this study appear not to allow structural correlation, and therefore general SAR, between these two antagonists.

A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis

Binding affinity at the [3H]‐BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild‐type or point‐mutated human B2 receptors (hB2R) expressed in CHO cells. The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F. As the wild‐type receptor‐binding affinity of FR190997 was 40‐fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK. Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400‐ and 150‐fold, and that of FR190997 was reduced by 400‐ and 25‐fold, at the W86A and F259A mutant B2 receptors, respectively. Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103‐fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant. L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high‐affinity antagonist in blocking the IP production induced by BK. The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand. Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.

Bradykinin B2 and GPR100 receptors: a paradigm for receptor signal transduction pharmacology

The aim of the present report was to investigate the ligand selectivity of the human orphan G‐protein‐coupled receptor GPR100 (hGPR100), recently identified as a novel bradykinin (BK) receptor, as compared with that of the human B2 receptor (hB2R) stably transfected in Chinese hamster ovary cells. BK was able to inhibit the cAMP production induced by forskolin with a potency 100‐fold lower at the hGPR100 (pEC50=6.6) than that measured at the hB2R (pEC50=8.6). Both effects were inhibited by the B2 receptor antagonist Icatibant (1 μM). The nonpeptide B2 receptor agonist FR190997 (8‐[2,6‐dichloro‐3‐[N‐methylcarbamoyl)cinnamidoacetyl]‐N‐methylamino]benzyloxy]‐2‐methyl‐4‐(2‐pyridylmethoxy)quinoline) did inhibit the forskolin‐induced cAMP production (pEC50=7.7) at the hB2R, whereas it was not able to exert any effect at the hGPR100. The human insulin‐like peptide relaxin 3 did inhibit the cAMP production at the hGPR100 (pEC50=7.3) at a greater extent than BK, and was devoid of any effect at the hB2R. FR190997 and relaxin 3 responses at the hB2R and hGPR100, respectively, were not inhibited by Icatibant (1 μM). These data indicate FR190997 and relaxin 3 as selective agonists for hB2R and hGPR100, respectively, and support the concept that different agonists may specifically bias the conformational states of a receptor to result in a final common G protein coupling, which is differentially recognized by antagonists.

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor

BACKGROUND AND PURPOSE Icatibant is a well‐known kinin B2 receptor antagonist currently used for angiooedema attacks. MEN16132 is a non‐peptide B2 receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists.

Multifaceted Approach to Determine the Antagonist Molecular Mechanism and Interaction of Ibodutant ([1-(2-Phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) at the Human Tachykinin NK2 Receptor

Ibodutant (MEN15596, [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK2 receptor (NK2R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK2R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-{[Asn(β-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β-5β)}) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK2R, ibodutant potency measured toward concentration-response curves to neurokinin AaspKB was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [3H]nepadutant and [3H]saredutant binding sites. Determined Kon and Koff values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK2Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK2R complex is proposed.

Multifaceted approach to determine the antagonist molecular mechanism and interaction of ibodutant at the human tachykinin NK2 receptor

Ibodutant (MEN15596, [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK2 receptor (NK2R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK2R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-{[Asn(β-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β-5β)}) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK2R, ibodutant potency measured toward concentration-response curves to neurokinin AaspKB was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [3H]nepadutant and [3H]saredutant binding sites. Determined Kon and Koff values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK2Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK2R complex is proposed.

Ala scan analogues of HOE 140. Synthesis and biological activities.

The role of the amino acids contained in the sequence of HOE 140 (H-DArg(1)-Arg(2)-Pro(3)-Hyp(4)-Gly(5)-Thi(6)-Ser(7)-DTic(8)-Oic(9 )-Arg(10)-OH), a potent and selective bradykinin B(2) receptor peptide antagonist, has been investigated by the replacement of each original residue (one by one) with Ala. The resulting set of decapeptides has been tested for the B(2) antagonist activity as well as for competition with the binding of [3H]BK to plasma membranes of the human umbilical vein (hUV). Positive correlations have been established between data obtained with the bioassay and with the binding in the hUV (same species, same tissue) and also between the two bioassays, the guinea-pig ileum (GPI) and the hUV (different species, different tissue). The structure-activity study has shown that the replacement of any of the residues that constitute HOE 140 with Ala is accompanied by a decrease of potency of at least 1 log unit. The analogues can be divided into three groups, with Ala(1) and Ala(7) showing affinities lower than HOE 140 by a factor of 10, Ala(4) and Ala(10) by a factor of 100 and Ala(2), Ala(5), Ala(6), Ala(8) and Ala(9) by a factor higher than 100 (100-1000). To verify the effect of chirality, the DAla(5) and DSer(7) analogues were synthesized and it was found that the substitution with a D-residue in position 5 is not tolerated while that in position 7 is favourable. The DSer(7) derivative is the most potent analogue found in this study: it shows potency as high as that of HOE 140 in the bioassays.

Interaction of linear and cyclic peptide antagonists at the human B2 kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.

New monocyclic and acyclic hNK‐2 antagonists retaining the β‐turn feature. X‐ray and molecular modelling studies

The human tachykinin NK-2 (hNK-2) receptor is considered a promising target for relevant pathologies at the respiratory, gastrointestinal and genitourinary level. With the aim of reducing the complexity of existing peptide antagonists, two series of hNK-2 receptor antagonists were designed, with the support of modelling, and synthesized. The X-ray structure determination of two compounds, each belonging to one of the two series, allowed the experimental validation of the initial rationale. In addition, it has been found that the two series share a beta-turn structure, a key feature for binding the hNK-2 receptor.