A novel bacterial l-arginine sensor controlling c-di-GMP levels in Pseudomonas aeruginosa.

Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium.

The in vitro addition of methotrexate and/or methylprednisolone determines peripheral reduction in Th17 and expansion of conventional Treg and of IL-10 producing Th17 lymphocytes in patients with early rheumatoid arthritis.

The aim of our study was to evaluate methotrexate (MTX) and methylprednisolone (MP) effect on peripheral Th17 and Treg subsets in patients with rheumatoid arthritis (RA). We enrolled 15 patients (10 early RA and 5 long-standing disease) with active RA and 10 age-matched healthy donors as controls. Frequencies of Th17 and Treg were quantified using flow cytometry before and after in vitro addition of MTX, MP or both drugs. Our results showed a reduction in the overall Th17 population followed by an increase in Th17 IL-10(+) and Treg, after in vitro treatment of PBMCs with the drugs in patients with early RA. Long-standing disease patients showed a less evident increase in Treg cells and less enhancement of IL-10 Th17 cells. We suggest that the treatment with MTX and MP could ameliorate RA disease activity by normalizing the distribution/imbalance of Th17/Treg and indicate a new regulatory role of IL-17(+) cells in RA patients.

Limits on light-speed anisotropies from Compton scattering of high-energy electrons.

The possibility of anisotropies in the speed of light relative to the limiting speed of electrons is considered. The absence of sidereal variations in the energy of Compton-edge photons at the European Synchrotron Radiation Facility's GRAAL facility constrains such anisotropies representing the first nonthreshold collision-kinematics study of Lorentz violation. When interpreted within the minimal standard-model extension, this result yields the two-sided limit of 1.6×10(-14) at 95% confidence level on a combination of the parity-violating photon and electron coefficients (κ(o+))(YZ), (κ(o+))(ZX), c(TX), and c(TY). This new constraint provides an improvement over previous bounds by 1 order of magnitude.

Diffusion-weighted imaging in breast cancer: relationship between apparent diffusion coefficient and tumour aggressiveness.

AIM: To assess the utility of diffusion-weighted imaging in diagnosing and characterizing breast malignancy. MATERIALS AND METHODS: From April 2006 to April 2009, all consecutive patients with breast cancer undergoing breast magnetic resonance imaging (MRI) and subsequent surgery in our hospital were enrolled in this study. MRI was performed using a 1.5 T MRI unit using a dedicated, bilateral, four-channel breast coil. The MRI protocol included a diffusion sequence acquired using b values of 0 and 1000 s/mm(2). For each malignant lesion the relationships between tumour grade and histology and the relative value of the apparent diffusion coefficient (ADC) were analysed. RESULTS: There were 136 female patients with 162 lesions. Histology revealed 149 invasive carcinomas and 13 ductal carcinomas in situ. There were 34 grade 1, 61 grade 2, and 67 grade 3 lesions. The mean ADC value of all malignant lesions was 1.03×10(-3) mm(2)/s. The mean ADC values for invasive and in situ carcinomas were 1.03×10(-3) mm(2)/s and 1.05×10(-3) mm(2)/s, respectively. The mean ADC values for grade 1, 2, and 3 tumours were 1.25×10(-3) mm(2)/s, 1.02×10(-3) mm(2)/s, and 0.92×10(-3) mm(2)/s, respectively. A statistically significant (p<0.001) inverse correlation was disclosed between the ADC value and the tumour grading. The mean ADC value of the "less aggressive" group of disease (G1 and in situ lesions) was 1.19×10(-3) mm(2)/s, whereas the mean ADC value of the "more aggressive" group (G2-G3 invasive carcinomas) was 0.96×10(-3) mm(2)/s (p<0.001). CONCLUSION: The study confirms the usefulness of diffusion imaging in assessing the aggressiveness of breast tumours. ADC appears to be a promising parameter in the evaluation of the degree of malignancy of breast cancer tissue.

Spirocerca lupi isolated from gastric lesions in foxes (Vulpes vulpes) in Sicily (Italy).

Spirocerca lupi (Rudolphi 1809) is a cosmopolitan nematode of dogs and wild carnivores. In the past it has been reported in Italy, mainly in southern regions and in Sicily, where the parasite was observed in foxes in 2005. The parasite typically produces nodular masses in the oesophagus and thoracic aorta. During the 2003-2004 hunting season, the authors investigated a total of 55 foxes (Vulpes vulpes) hunted or killed by car accidents in the provinces of Palermo and Agrigento. All the foxes were subjected to necropsy and 6 (9.16%) had S. lupi nodules located exclusively in the gastric wall. The nature of the nodules was determined by opening them and detecting the nematodes inside, which were identified as S. lupi. Some of the nodules were characterized anatomopathologically and histopathologically. The formation of the parasitic nodule in the stomach only suggests a deviation from the route commonly followed by the nematode to reach the oesophagus, the elective anatomical site for completion of its lifecycle. This survey gives a contribution to the epidemiology of this parasite which is severely outdated in Italy and highlights some distinctive features of the life cycle and parasite migration.

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

Isolation and characterization of unusual Mycoplasma spp. from captive Eurasian Griffon (Gyps fulvus) in Sicily.

Mycoplasmas have been isolated from birds of prey during clinical examinations, but their significance to the health of raptors is unclear. We report the isolation and characterization of four mycoplasmas found in the upper respiratory tract of four sick Eurasian Griffon (Gyps fulvus) that were housed in a Sicilian rehabilitation center at Ficuzza, near Palermo in Sicily, before reintroduction into the wild. These included Mycoplasma gallinarum, an unidentified mycoplasma highly similar to Mycoplasma glycophilum, and two unidentified mycoplasmas with similarities to Mycoplasma falconis and Mycoplasma gateae.

Discovering Selective Diguanylate Cyclase Inhibitors: From PleD to Discrimination of the Active Site of Cyclic-di-GMP Phosphodiesterases.

One of the most important signals involved in controlling biofilm formation is represented by the intracellular second messenger 3',5'-cyclic diguanylic acid (c-di-GMP). Since the pathways involved in c-di-GMP biosynthesis and breakdown are found only in bacteria, targeting c-di-GMP metabolism represents an attractive strategy for the development of biofilm-disrupting drugs. Here, we present the workflow required to perform a structure-based design of inhibitors of diguanylate cyclases, the enzymes responsible for c-di-GMP biosynthesis. Downstream of the virtual screening process, detailed in the first part of the chapter, we report the step-by-step protocols required to test the positive hits in vitro and to validate their selectivity, thus minimizing possible off-target effects.

[Diastolic heart failure]. / Lo scompenso cardiaco diastolico: aspetti clinici, fisiopatologici e terapeutici.

Until recently, the development of heart failure was related exclusively to the acute or chronic impairment in systolic function. Currently, the concept of heart failure not sustained primarily by a significant reduction in contractility has been clearly defined by several epidemiological and pathophysiological observations. This condition, defined as 'diastolic heart failure' or 'heart failure with preserved systolic function' can be related to different cardiac diseases with a higher prevalence in the elderly. Afterload mismatch situations, such as hypertension or aortic stenosis, as well as hypertrophic cardiomyopathy or pericardial diseases, determine this common clinical syndrome more frequently. Currently, the treatment of diastolic heart failure is still empirical, as there are few and inconclusive data coming from evidence-based medicine.

Quantitative expression of the human kallikrein gene 9 (KLK9) in ovarian cancer: a new independent and favorable prognostic marker.

Many members of the human kallikrein gene family were found to be differentially expressed in various malignancies and some are useful cancer diagnostic/prognostic markers. KLK9 is a newly discovered human kallikrein gene that is expressed in several tissues including thymus, testis, spinal cord, salivary gland, ovary, and skin. Like other kallikreins, the KLK9 gene was found to be regulated by steroid hormones in cancer cell lines. Our purpose is to examine whether quantitative analysis of KLK9 expression has prognostic value in ovarian cancer. We studied the expression of KLK9 by quantitative reverse transcription-PCR in 168 consecutive ovarian tumors of different stages, grades, and histological types, and correlated the expression with clinicopathological parameters, response to chemotherapy, and patients' survival. We found that KLK9 expression was significantly higher in patients with early disease stages (I or II; P = 0.044) and in patients with optimal debulking (P = 0.019). Kaplan-Meier survival curves demonstrated that patients with KLK9-positive tumors have substantially longer progression-free and overall survival (P < 0.001 and P = 0.016, respectively). When the Cox proportional hazard regression analysis was applied to subgroups of patients, KLK9 expression was found to be a significant predictor of progression-free survival in the subgroup of patients with low-grade tumors [hazard ratio (HR), 0.13; P = 0.0015], early stage (HR, 0.099; P = 0.031); and those with optimal debulking (HR, 0.26; P = 0.012). After adjusting for other known prognostic variables, KLK9 retained its independent prognostic value in all of these subgroups of patients. A negative correlation was found between the expression levels of CA125 and KLK9 (rs, 0.350; P = 0.002). Our results indicate that KLK9 is under steroid hormone regulation in ovarian and breast cancer cell lines. Immmunohistochemically, human kallikrein protein (hK9) was localized in the cytoplasm, but not in the nuclei, of the epithelial cells of ovarian cancer tissues. We conclude that KLK9 is a potential new independent favorable prognostic marker for early stage, low-grade, optimally debulked ovarian cancer patients.

IL-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelium.

Interleukin-15 (IL-15) is a recently described cytokine with IL-2-like stimulating activities on T lymphocytes and natural killer (NK) cells. IL-15 mediates its function through the beta- and gamma-chains of the IL-2 receptor. In this work, we have investigated the effect of IL-15 on the directional migration of NK cells in chemotaxis assays and on the ability of NK cells to bind to vascular endothelium. IL-15 (10-20 ng/mL) had chemotactic effects on freshly isolated resting NK cells as well as on long-termed IL-2-cultured NK cells. A checkerboard experiment demonstrated that migration in response to IL-15 was observed only in the presence of a positive gradient (chemotaxis). Overnight treatment of freshly isolated NK cells with IL-15 (10-20 ng/mL) augmented their binding to cultured endothelial cells (EC) in vitro, especially to resting EC. IL-15-activated NK cells bound to resting and tumor necrosis factor-activated EC by use of LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways, essentially as untreated NK cells do. The fact that IL-15 increased NK cell binding to ICAM-1-transfected NIH-3T3 fibroblasts, together with the finding that IL-15 did not increase binding to extracellular matrix proteins, where the major molecules involved are VLA proteins, indicated that IL-15 primarily stimulates LFA-1-dependent adhesion. By increasing NK cell adhesion to vascular endothelium and migratory response, IL-15 is an important determinant of NK cell recruitment in tissues.

Chest and breast MRI: the added value of a fast imaging for a new diagnostic approach in the planning of augmentation surgery in patients with thoracic asymmetries.

OBJECTIVE: In breast augmentation surgery, breast symmetry depends on the breast tissue, implants and chest wall. Any asymmetry of the anterior thoracic wall can influence the breast shape. If breast asymmetry is detected in the preoperative evaluation, a chest wall deformity should be suspected. Until now, very few reports describe the use of MRI to objectively assess breast and chest measurements with the aim of providing customized augmentation. This study describes the use of MRI to evaluate breast and chest wall asymmetry, and considers the feasibility of preoperative measurements which are useful for performing an objective preoperative evaluation. PATIENTS AND METHODS: Between April 2012 and February 2013, 13 patients underwent chest/breast MRI scan. Scans were performed on a 1.5 T scanner using a single T1 FSE non-suppressed axial sequence, without contrast administration. Acquisitions included the breast and chest wall. Specific measurements were obtained to assess the overall shape of the chest wall and breast, as well as any asymmetry. RESULTS: All patients showed some degree of left-right side asymmetry on specific thoracic, breast and implant measurements. MRI provided detailed and objective data. CONCLUSIONS: Preliminary findings revealed the value of breast/chest wall MRI in the planning of augmentation surgery. MRI is a valuable technique in young women because there is no use of ionizing radiation. Scans allow surgeons to determine the best surgical approach and obtain reproducible and better aesthetic results.

IFN-beta partially counteracts inhibition of natural killer activity induced by some antitumor agents.

We investigated whether recombinant human (rHu-IFN-beta) (IFN-beta) could counteract the inhibition of natural killer (NK) activity caused by antitumor agents. Peripheral blood lymphocytes (PBL) were incubated with different antitumor agents alone or in combination with IFN-beta for 3 days and then tested in a cytotoxicity assay against the K562 cell line. The following drugs were used, all of which caused a dose-dependent inhibition of NK activity: etoposide, camptothecin, doxorubicin, cis-DDP, tallimustine, and L-PAM. Concomitant treatment with (1000 U/ml) IFN-beta counteracted the inhibitory effect of etoposide and camptothecin but had no consistent effect on the inhibition mediated by the other drugs. Mean values of inhibition of NK activity at 1 microM camptothecin was 48%+/-3.4% and with IFN-beta was 10%+/-4.9%. With 100 microM etoposide, mean value of inhibition was 78%+/-3.3%, and with IFN-beta, it was 18%+/-1.5%. Cell viability, assessed by vital dye exclusion, and drug uptake, assessed with radiolabeled etoposide, were similar in cells treated with or without IFN-beta. The protective effect of IFN-beta on NK function was rather selective, as IFN-beta did not counteract the drug-mediated inhibition of PBL proliferation when stimulated by phytohemagglutinin (PHA). Other cytokines, IFN-alpha, IFN-gamma, and interleukin-2 (IL-2), had similar protective effect, although IFN-beta, was slightly more potent. On the other hand, IL-6, a cytokine sharing some properties with IFNs was ineffective. Camptothecin inhibited the expression of mRNA for granzyme B, a lytic protein involved in lymphoid-mediated cytotoxicity. Combined treatment with IFN-beta restored-at least in part-the transcription of granzyme B mRNA. These results show that the immunosuppressive effect of some antitumor agents could be partly counteracted by treatment with IFN-beta.

Response to second-line weekly cisplatin chemotherapy in ovarian cancer previously treated with a cisplatin- or carboplatin-based regimen.

Response to a second-line weekly cisplatin chemotherapy in ovarian cancer previously treated with cisplatin- or carboplatin-based regimens was analysed in a clinical series observed between 1984 and 1991. Women who achieved pathological complete response or pathological optimal partial remission after first-line cisplatin- or carboplatin-based regimens were treated at recurrence or progression, occurring at least 4 months after first-line treatment, with second-line chemotherapy. A total of 72 women were included in the analysis. Second-line chemotherapy regimens were: cisplatin 1 mg/kg weekly for seven courses plus epirubicin 70 mg/m2 intravenously (i.v.) every 3 weeks for three courses (28 subjects), cisplatin 1 mg/kg plus etoposide 90 mg/m2 i.v. weekly for a total of seven courses (11 subjects) and cisplatin 1 mg/kg weekly for nine courses plus carboplatin 250 mg/m2 every 3 weeks for three courses (33 subjects). Of the 72 women, 22 (31%, 14 clinical, 8 pathological) had a complete response and 28 (39%), a partial response (24 clinical, 4 pathological). The 24-month cumulative survival probability was 63% in women with complete response, 32% in those who had partial response, but all the 22 non-responders died within 24 months from diagnosis of recurrence (log rank test P < 0.05). The frequency of complete response and partial response increased with the interval between first diagnosis and recurrence: among the 33 women who had recurrent disease to < 18 months from first diagnosis, complete response or partial response was obtained in 20 (61%) subjects, this figure was 67% (14 out of 21 women) among subjects who had recurrent disease between 18 and < 36 months from first diagnosis and 89% (16/18) among those who had recurrence > or = 36 months. In comparison with women who had recurrence 4- < 18 months from first diagnosis, the OR of response was 1.3 (95% CI 0.4-4.1) for those who had recurrence between 18 and < 36 and 5.2 (95% CI 1.1-24.3) for those who had recurrence > or = 36 months from surgery (chi 1(2) trend p < 0.05). Survival rate after the end of second line chemotherapy for women who relapsed 4- < 18 months, 18- < 36 or 36 months or more after surgery were, respectively, 24, 20 and 67% (log rank test, P < 0.05). Age at first diagnosis, histology, stage, and grading of the disease at first diagnosis and site of recurrence were not associated with response to second-line therapy.

(2S)-1-(arylacetyl)-2-(aminomethyl)piperidine derivatives: novel, highly selective kappa opioid analgesics.

This paper describes the synthesis and structure-activity relationships as kappa opioid analgesics of a novel class of 1-(arylacetyl)-2-(aminomethyl)piperidine derivatives. The active conformation of the pharmacophore, with a torsional angle (N1C2C7N8) of 60 degrees, was defined with computational studies and 1H NMR. A quantitative structure-activity relationship study of the arylacetic moiety substitution indicated that the presence of an electron-withdrawing and lipophilic substituent in para and/or meta positions is required for good analgesic activity and kappa affinity. The lead compounds (2S)-1-[(3,4-dichlorophenyl)acetyl]-2-(pyrrolidin-1-ylmethyl )piperidine hydrochloride and (2S)-1-[4-(trifluoromethyl)phenyl]acetyl]-2-(pyrrolidin-1-ylmet hyl) piperidine hydrochloride are the most kappa/mu selective (respectively 6500:1 and 4100:1) and among the most potent (Ki kappa 0.24 and 0.57 nM, respectively) kappa ligands identified so far. In the mouse tail flick model of antinociception, compound 14 (ED50 = 0.05 mg/kg sc) was 25 times more potent than morphine and 16 times more potent than the standard kappa ligand U-50488.

(1S)-1-(aminomethyl)-2-(arylacetyl)-1,2,3,4-tetrahydroisoquinoline and heterocycle-condensed tetrahydropyridine derivatives: members of a novel class of very potent kappa opioid analgesics.

The synthesis and structure-activity relationship (SAR) of a novel class of kappa opioid analgesics, 1-(aminomethyl)-2-(arylacetyl)-1,2,3,4- tetrahydroisoquinolines and (aminomethyl)-N-(arylacetyl)-4,5,6,7-tetrahydrothienopyridines+ ++, are described. These compounds, formally derived by the condensation of a benzene or thiophene ring on the piperidine nucleus of the recently described compounds 1, are from 3 to 7 times more potent as antinociceptive agents and with a longer duration of action than the original lead compounds. A similar N2-C1-C9-N10 pharmacophore torsional angle of approximately 60 degrees was also found for this class of compounds by using X-ray and 1H NMR analyses. The same absolute configuration (S) at the chiral center of the active (-) enantiomers was determined by X-ray crystallographic analysis. A varied degree of kappa receptor selectivity was a feature of this novel class of antinociceptive agents (mu/kappa ratio from 44 to 950 according to the nature of the basic moiety). SAR analysis indicated that the presence of electron-withdrawing and lipophilic substituents in para and/or meta positions in the arylacetic moiety and the pyrrolidino or dimethylamino basic groups are required to optimize biological activity. The lead compounds 28, 30, and 48 are among the most potent antinociceptive agents (ED50 ca. 0.020 microM/kg sc) and kappa ligands (Ki(kappa) ca. 0.20 nM) identified so far.

Selective kappa-opioid agonists: synthesis and structure-activity relationships of piperidines incorporating on oxo-containing acyl group.

This study describes the synthesis and the structure-activity relationships (SARs) of the (S)-(-)-enantiomers of a novel class of 2-(aminomethyl)piperidine derivatives, using kappa-opioid binding affinity and antinociceptive potency as the indices of biological activity. Compounds incorporating the 1-tetralon-6-ylacetyl residue (30 and 34-45) demonstrated an in vivo antinociceptive activity greater than predicted on the basis of their kappa-binding affinities. In particular, (2S)-2-[(dimethylamino)methyl]-1-[(5,6,7,8-tetrahydro-5-oxo-2- naphthyl)acetyl]piperidine (34) was found to have a potency similar to spiradoline in animal models of antinociception after subcutaneous administration, with ED50s of 0.47 and 0.73 mumol/kg in the mouse and in the rat abdominal constriction tests, respectively. Further in vivo studies in mice and/or rats revealed that compound 34, compared to other selective kappa-agonists, has a reduced propensity to cause a number of kappa-related side effects, including locomotor impairment/sedation and diuresis, at antinociceptive doses. For example, it has an ED50 of 26.5 mumol/kg sc in the rat rotarod model, exhibiting a ratio of locomotor impairment/sedation vs analgesia of 36. Possible reasons for this differential activity and its clinical consequence are discussed.

Substituted 1-(aminomethyl)-2-(arylacetyl)-1,2,3,4-tetrahydroisoquinolines: a novel class of very potent antinociceptive agents with varying degrees of selectivity for kappa and mu opioid receptors.

This study describes the synthesis of a series of novel substituted 1-(aminomethyl)-2-(arylacetyl)-1,2,3,4-tetrahydroisoquinolines, and discusses their structure-activity relationships (SARs) using binding affinity for opioid receptors and antinociceptive potency as the indices of biological activity. The introduction of a hydroxy substituent in position 5 of the isoquinoline nucleus generated a compound, 40, which is 2 times more potent than the previously disclosed unsubstituted analogue 39 in mouse models of antinociception. A QSAR analysis of the 5-substitution clearly demonstrates that antinociceptive activity is inversely associated with the lipophilicity of the substituents. The substituted compounds described herein are less selective for the kappa opioid receptors than the unsubstituted isoquinoline 39. For example, the 5-hydroxy-substituted compound 59 shows high affinity for kappa opioid receptors (Ki kappa = 0.09 nM) and a Ki mu/Ki kappa ratio of only 5. However, a multiple linear regression analysis demonstrates a lack of correlation between antinociceptive activity and affinity for the mu opioid receptor. On the other hand, the correlation between binding affinity to kappa opioid receptor and antinociceptive activity was statistically significant.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 1. Identification of the 4-quinolinecarboxamide framework.

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 2. Identification of (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB 223412).

Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.