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. EXPERIMENTAL APPROACH: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B2 receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B2 receptors. KEY RESULTS: Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B2 receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B2 receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B2 receptor complex is proposed. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

Development of two multiplex PCR systems for the analysis of 12 X-chromosomal STR loci in a northwestern Italian population sample.

Two multiplex polymerase chain reaction systems for the automated profiling of 12 X-chromosomal short tandem repeat (STR) markers were developed. Multiplex A consisted of DXS6789, DXS6809, GATA172D05, DXS101, DXS8378, and DXS8377. Multiplex B consisted of DXS7132, DXS6800, DXS6801, DXS7424, HPRTB, and DXS10011. The set of amplified X-STRs was designed to include groups of closely linked markers (DXS101-DXS7424 and DXS6789-DXS6801-DXS6809) to generate highly informative haplotypes for kinship testing. A population genetics study of the 12 X-STRs was conducted in a northwestern Italian population sample (n=160, 80 women and 80 men). A diallelic pattern at locus DXS6789 was observed in one man.

Regio- and stereoselective cycloadditions of cyclic nitrones to maleic diamide forced in a peptide: synthesis of potent ligands of human NK-2 receptor.

The regioselectivity and the stereoselectivity induced by relatively small peptidomimetic maleic diamide 1 in cycloaddition reactions with cyclic nitrones 2-5 was studied. The high regio- and stereoselectivity observed, sensibly increased by nonpolar solvents, was the effect of a double-asymmetric induction produced by the nitrone substituent on the pseudopeptidic tether. A new class of potent human tachykinin NK-2 receptor ligands was synthesized.

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.

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.

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.

MEN 11270, A novel selective constrained peptide antagonist with high affinity at the human B2 kinin receptor.

We investigated the pharmacological profile of MEN 11270, or H-D-Arg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10 alpha), a conformationally constrained derivative of the B2 kinin receptor antagonist Icatibant. MEN 11270 bound with high-affinity to the B2 kinin receptor constitutively expressed by WI38 human fibroblasts, inhibiting 3H-bradykinin (BK) with a pKi value of 10.3 +/- 0.08 (n = 5). The rank order of affinity of several peptide and nonpeptide antagonists was also assessed: Icatibant (pKi = 10.6) approximately MEN 11270 (pKi = 10.3) approximately B9430 (pKi = 10.0) > B9858 (pKi = 8.0) > FR173657 (pKi = 7.6) > WIN64338 (pKi = 7.2) > Lys-[des-Arg9, Leu8]-BK (pKi < 6) > [des-Arg9,Leu8]-BK (pKi < 5). MEN 11270 showed a low affinity in inhibiting 3H-Lys-[des-Arg9]-BK binding at the human B1 kinin receptor constitutively expressed by the same cells (pKi 6.0 +/- 0.33; n = 3). MEN 11270 showed no binding affinity (pIC50 < 5.5) at 29 different receptors and ion channels. In the human umbilical vein contraction assay, MEN 11270, shifted the concentration-response curve to BK to the right in a concentration-dependent manner (pA2 8.14 +/- 0.22, n = 7). The Schild plot was linear (slope 0.95 +/- 0.11), consistent with a competitive antagonism. In the same bioassay, MEN 11270 (10 microM) did not affect the concentration-response curve to the B1 agonist Lys-[des-Arg9]-BK nor the contractile responses elicited by noradrenaline or serotonin. These findings indicate MEN 11270 as an antagonist at the human B2 kinin receptor, with potency and selectivity comparable to those of the linear peptide antagonist, supporting the hypothesis that a constrained C-terminal beta-turn conformation preserves a high affinity for the interaction of Icatibant with the B2 kinin receptor.

Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists.

Structural comparison of NK2 receptor agonists and antagonists.

The conformational space of two NK2 receptor agonists and a new potent antagonist has been sampled by the simulated annealing technique. Low-energy conformers were obtained, which were compared with respect to their key residues, namely phenylalanine, leucine and methionine. The hypothesis is that they share part of the binding site on the receptor.

Three-dimensional QSAR of human immunodeficiency virus (I) protease inhibitors. 1. A CoMFA study employing experimentally-determined alignment rules.

Comparative molecular field analysis (CoMFA), a three-dimensional, quantitative structure-activity relationship (QSAR) paradigm, was used to examine the correlations between the calculated physicochemical properties and the in vitro activities of a series of human immunodeficiency virus (HIV-1) protease inhibitors. The training set consisted of 59 molecules from five structurally-diverse transition-state isostere classes: hydroxyethylamine, statine, norstatine, keto amide, and dihydroxyethylene. The availability of X-ray crystallographic data for at least one representative from each class bound to the protease provided information regarding not only the active conformation of each ligand but also, via superimposition of protease backbones, the relative positions of each ligand with respect to one another in the active site of the enzyme. Once aligned, these molecules served as templates on which additional congeners were field-fit minimized. Additional alignment rules were derived from minimizations of the ligands in the active site of the semirigid protease. The predictive ability of each resultant model was evaluated using a test set comprised of molecules containing a novel transition-state isostere: hydroxyethylurea. Crystallographic studies (Getman, D.P.; et al. J. Med. Chem. 1993, 36, 288-291) indicated an unexpected binding mode for this series of compounds which precluded the use of the field-fit minimization alignment technique. The test set molecules were, therefore, subjected to a limited systematic search in conjunction with active-site minimization. The conformer of each molecule expressing the lowest interaction energy with the active site was included in the test set. Field-fit minimization of neutral molecules to crystal ligands and active-site minimizations of protonated ligands yielded predictive correlations for HIV-1 protease inhibitors. The use of crystallographic data in the determination of alignment rules and field-fit minimization as a molecular alignment tool in the absence of direct experimental data regarding binding modes is strongly supported by these results.

Environmental aspects of pesticide use in Italian agriculture.

Pesticides used in agriculture accumulate in organisms and reach man through complex metabolic pathways. Accurate analysis of pesticides presence in each step of the food chain is necessary because of the high number of these substances, their potential toxicity and the presence of amplified toxic effects due to synergic interactions. In the same way it is necessary to evaluate the environmental effects of the whole pesticides cycle, from industrial production until final disposal of used containers and residuals. Furthermore recent introduction of new categories of chemicals which are not strictly classifiable as pesticides (photosynthetic activity stimulators, aesthetic aspect enhancers) and not yet sufficiently studied in their metabolic behaviour, increase the problem complexity. Complete studies about these complex problems and about the effects on ecosystems have not yet been carried out in Italy, due to ambiguous laws and regulations, inadequate data collection methods and decisional influence of the chemical industry on agriculture policy.

New benzofuran-imidazoles as antimycotic agents. I. Synthesis and characterization.

With the aim of obtaining new drugs having antimycotic activity together with antibacterial and fewer side effects, we synthesized ten new benzofuran-2-yl-imidazoles.

New benzofuran-imidazoles as antimycotic agents. II. In vitro studies.

Ten new imidazole derivatives, benzofuran-imidazoles, were studied in vitro to establish their antimycotic activity against 70 fungal strains, in comparison with three known imidazoles and, for the dermatophytes only, griseofulvin. A very high inhibitory activity against dermatophytes was shown by five of the new substances. The other filamentous fungi and yeasts showed a more marked variation in their susceptibility. However a good sensitivity of some isolates of Candida albicans and other yeasts was seen.

Evidence for the involvement of arachidonic acid metabolites in spontaneous and drug-induced contractions of rat urinary bladder.

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and endogenous arachidonic acid (AA) depletion on spontaneous and drug-induced contractions of the rat urinary bladder have been determined by both in vivo and in vitro experiments. Our results suggest that some AA metabolites (presumably prostaglandins) are involved in the physiologic regulation of the micturition reflex in the rat. In vivo findings indicate that the ability of various NSAIDs to inhibit distension-induced rhythmic contractions is proportional to their anti-inflammatory effectiveness. NSAIDs administration or depletion of endogenous AA at the detrusor muscle level by essential fatty acid-free diet (EFAFD) decreased the responsiveness of the urinary bladder to reflex activation. Topical AA triggered a series of neurogenic rhythmic contractions in the preparation which failed to respond to saline loading. This effect was prevented by NSAID pretreatment. The effect of topical AA was mimicked in NSAID-treated preparations by topical prostaglandins. Both NSAIDs and EFAFD reduced the responsiveness of the rat urinary bladder to acetylcholine and purinergic stimulation in vivo and in vitro. NSAIDs enhanced, while EFAFD reduced, the responsiveness of the isolated bladder to stable cholinomimetics. Responsiveness to KCl was unaffected by NSAIDs or EFAFD. These latter findings indicate that either blockade of AA metabolism along the cyclooxygenase pathway or endogenous AA depletion might alter bladder responsiveness at the postjunctional level. However, because the amplitude of distension-induced rhythmic contractions is unaffected by NSAIDs or EFAFD, it appears unlikely that endogenous prostanoids play a role in excitatory neurotransmission or in tension development during physiological-like activation of the bladder muscle. In vitro findings indicate that both NSAIDs and EFAFD reduce the myogenic contractility and the responsiveness to stretch of bladder muscle. These findings are suggestive that AA metabolites could regulate micturition by enhancing the amplitude of the myogenic contractions of the bladder muscle and, consequently, the discharge of vesical afferents to the central nervous system.

[Clinical experiences with monocomponent insulins]. / Esperienze cliniche con le insuline monocomponenti

The immunogenicity of conventional therapeutical insulin is discussed according to the concepts of Schlichtkrull: the formation of insulin antibodies is not attributable to the pure Sanger's insulin molecule, but to related protein impurities, present in all crystallized pig and ox insulin preparations. The terms of monocomponent insulin, highly purified insulin, and single peak insulin in defined and personal clinical results obtained with Novo Monocomponent Lente Insulin over a period of 3 years are presented. The Hein Christiansen's radioimmunoelectrophoretic method fo estimation of 125I-insulin IgG binding was used to determine insulin antibody levels. It was found that: 1) Newly detected insulin-dependent diabetics, never previously treated with insulin, do not produce insulin antibodies at a significant level; 2) Long-term insulin treated diabetics, transferred to monocomponent treatment, tend to reduce their antibody levels, if initially high, altough with transient recurrent peaks; 3) Stimulation of the immunocompetent system by intercurrent infection does not generally modify the immunological situation. Apart from immunological changes, satisfactory clinical results were observed in cases of high insulin requirement, insulin allergy, insulin lipoatrophy. Present practical indications for monocomponent insulin therapy (Actrapid-Lenta) are proposed.

[Evaluation of the antibody anti-insulin titer using Christiansen's radioimmunoelectrophoresis in clinical diabetology]. / Valutazione del titolo anticorpale anti-insulina colla tecnica radioimmunoelettroforetica di Christiansen in diabetologia clinica.

On the basis of personal experience concerning 2020 consecutive determinations, the radioimmunoelectrophoretic method of Christiansen for 125I-Insulin-Binding to IgG (= IB, significant limits = mU/ml) has been proved as a reliable tool for the evaluation of insulin antibody titer in clinical diabetology. After a critical review of the recent literature about insulin antibodies both without and after exogenous immunization, the following results are presented and discussed. 1) - In 163 diabetic subjects, never previously treated with insulin, the mean value of IB was X = 0,008 mUml (sigma = 0,023 . Sx = 0,002). 2) - In 221 longterm insulin-treated diabetics the mean value of IB was X = 1,50 mU/ml (sigma = 2,15 . Sx = 0,145). 3) -In 46 insulin-dependent diabetics, serial determinations of IB allowed to follow the insulin antibody production during a 5 years treatment with monocomponent insulin )Lente MC). No or slight antibody formation was observed in newly diagnosed patients, never previously treated with insulin. High antibody starting levels showed tendency to a gradual reduction, after switching from conventional insulin treatment to the monocomponent one. These modifications in the IB values have been studied in correlation with the clinical course of conditions possibly referred to an immunologic pathogenesis, such as: brittle diabetes, high insulin requirement, insulin allergy, insulin lipoatrophy, diabetic microangiopathy. No significant variations in IB values were observed after viral infections.