Developing FGFR4 inhibitors as potential anti-cancer agents via in silico design, supported by in vitro and cell-based testing.

Fibroblast growth factor receptor-4 (FGFR4) is a tyrosine kinase with a range of important physiological functions. However, it is also frequently mutated in various cancers and is now generating significant interest as a potential therapeutic target. Unfortunately, biochemical characterization of its role in disease, and further evaluation as a drug target is hampered by lack of a specific inhibitor. We aimed to discover new inhibitors for FGFR4 ab initio using a strategy combining in silico, in vitro and cell-based assays. We used the homologous FGFR1 to calculate docking scores of a chemically-diverse library of approximately 2000 potential kinase inhibitors. Nineteen potential inhibitors and ten randomly- selected negative controls were taken forward for in vitro FGFR4 kinase assays. All compounds with good docking scores significantly inhibited FGFR4 kinase activity, some with sub-micromolar (most potent being V4-015 with an IC(50) of 0.04 µM). Four of these compounds also demonstrated substantial activity in cellular assays using the FGFR4- overexpressing breast carcinoma cell line, MDA-MB453. Through immunoblot assays, these compounds were shown to block the phosphorylation of the FGFR4 adaptor protein, FGFR substrate protein-2α (FRS2α). The most potent compound to date, V4-015, suppressed proliferation of MDA-MB453 cells at sub-micromolar concentrations, activated the pro-apoptotic caspases 3/7 and inhibited cellular migration. While achieving complete selectivity of this compound for FGFR4 will require further lead optimization, this study has successfully identified new chemical scaffolds with unprecedented FGFR4 inhibition capacities that will support mechanism of action studies and future anti-cancer drug design.

Imatinib-ULS-lysozyme: a proximal tubular cell-targeted conjugate of imatinib for the treatment of renal diseases.

The anticancer drug imatinib is an inhibitor of the platelet-derived growth factor receptor (PDGFR) kinases, which are involved in the pathogenesis of fibrotic diseases. In the current study we investigated the delivery of imatinib to the proximal tubular cells of the kidneys and evaluated the potential antifibrotic effects of imatinib in tubulointerstitial fibrosis. Coupling of imatinib to the low molecular weight protein lysozyme via the platinum (II)-based linker ULS yielded a 0.8:1 drug-carrier conjugate that rapidly accumulated in the proximal tubular cells upon intravenous and intraperitoneal administration. The bioavailability of intraperitoneally administered imatinib-ULS-lysozyme was 100%. Renal imatinib levels persisted for up to 3 days after a single injection of imatinib-ULS-lysozyme. Compared with an equal dose imatinib mesylate, imatinib-ULS-lysozyme resulted in a 30- and 15-fold higher renal exposure of imatinib, for intravenous and intraperitoneal administration respectively. Imatinib-ULS-lysozyme could not be detected in the heart, which is the organ at risk for side-effects of prolonged treatment with imatinib. The efficacy of imatinib-ULS-lysozyme in the treatment of tubulointerstitial fibrosis was evaluated in the unilateral ureteral obstruction (UUO) model in mice. Three days UUO resulted in all signs of early fibrosis, i.e. an increased deposition of matrix and production of profibrotic factors. Although a moderately increased activity of PDGFR-ß was observed, the profibrotic phenotype could not be inhibited with imatinib mesylate or with imatinib-ULS-lysozyme. Further evaluation of imatinib mesylate and imatinib-ULS-lysozyme is therefore warranted in an animal model of renal disease in which the activation of PDGFR-ß is more pronounced.

Seroprevalence and risk factors associated with Visna/Maedi virus in semi-intensive lamb-producing flocks in northwestern Spain.

A cross-sectional study was carried out to determine Visna/Maedi virus (VMV) seroprevalence and risk factors in semi-intensive lamb-producing flocks as a prelude to establishing a monitoring program in northwestern (NW) Spain. A total of 15,155 serum samples were taken from 78 commercial flocks and were submitted to an indirect VMV ELISA. Association between potential risk factors and seroprevalence at the flock level was assessed using a multivariable logistic regression model. A Generalized Estimating Equations (GEE) model and Exhaustive Chi-squared Automatic Interaction Detector (CHAID) were used to determine the seropositivity against VMV at the individual animal level. Individual seropositivity was 24.8% while 52.6% of the flocks examined had a true seroprevalence ≥1%. Flock size and introduction of new animals in the flock were significantly associated with seropositivity at the flock level. Flock size, sheep-goat contact, type of housing of lambs prior to weaning and age were significantly associated with individual VMV seropositivity. Confinement of lambs in preweaning lamb groups and high sheep-goat contact, regardless of the low number of goats per flock, were risk factors associated with individual VMV seropositivity, suggesting that these two factors are crucial for VMV control in semi-intensive lamb-producing flocks. These factors should be considered for developing more efficient strategies that will reduce the rate of VMV transmission.

Protostrongylid infection in meat sheep from Northwestern Spain: prevalence and risk factors.

2093 Faecal samples from 74 commercial meat ovine flocks were collected and examined by the Baermann-Wetzel method for protostrongylid infection. The risk of being infected by lungworms was evaluated with a data mining classification tree (CHAID), and the intensity of infection with a general linear model (GLM). 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2-12.9). Only two species were found, Muellerius capillaris (97.9%) and Neostrongylus linearis (5.4%). 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces. All of them held animals infected by M. capillaris and seven presented mixed infections with N. linearis. Average larval output in infected sheep was 11.9 (SD 30.91). This study showed that protostrongylid prevalence in sheep for meat production was determined mainly by a positive interaction with Dictyocaulus filaria infection; other factors that have influenced over protostrongylid infection were age, introducing external animals in the flocks, mixed management with goats and animal density in pastures. Treatment effects on prevalence were only observed in flocks that did not introduce ewes. The lowest protostrongylid prevalence has been reported in flocks without D. filaria infection and without contact with goats.

Signalling inhibitors against Mycobacterium tuberculosis--early days of a new therapeutic concept in tuberculosis.

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains.

Assessment of water pollution in the Antuã River basin (Northwestern Portugal).

The Antuã River, located in northwestern Portugal, drains a region with a high population density and a strong economic dynamism. These factors, together with a lack of facilities for appropriate treatment of domestic and industrial sewage, are putting increasing pressure on water resources. In this context, the aim of the present study was to identify point sources of pollution and to assess the surface water quality in the Antuã basin by monitoring physicochemical variables. A total of 40 point sources of wastewater, including some with a high pollution load, were detected in the most populated and industrialized areas of the São João da Madeira and Oliveira de Azeméis municipalities. These sources explained the strong degradation of water quality observed in the upper and medium Antuã River and in one of its tributaries, where maxima of 49 mg l(-1) for biochemical oxygen demand, 29 mg l(-1) for Kjeldahl nitrogen and 3.7 mg l(-1) for total phosphorus, were found after five surface water monitoring campaigns. Despite the relevance of pollution problems, a considerable water quality improvement, promoted by favourable reaeration conditions, was observed in the final stretch of the river, giving evidence of a great self-depuration capacity. However, the Antuã is a significant contributor of nutrients to the Ria de Aveiro, the coastal lagoon where the river meets the Atlantic Ocean.

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

Analysis of polyanionic macromolecular carrier poly-(N-vinylpyrrolidone-co-maleic acid) and its bioconjugats by capillary electrophoresis.

Anionic carrier poly(N-vinylpyrrolidone-co-maleic acid) and its conjugates, prepared with coupling of 2-cyano-3-hydroxy-5-amino-2-pentenoyc(4-trifluoromethyl anilide) or (6', 7'-dimethyl-l'-quinoxalinyl)-4-(2' amino) acetanilide to the carrier, were analyzed by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in the following buffers: 0.25 N triethylammonium phosphate (TEAP); sodium dodecyl sulfate (SDS; 25-150 mM) in TEAP (pH 2.25-6.30); 0.1 N Na-borate buffer (pH range 7-11) and SDS (25-150 mM) in Na-borate buffer (pH range 7-11). The presence of strong carboxyl groups (dissociated even at pH 2.25) on the polymer chain was proved by the CZE method. It was also proved by potentiometric titration that carboxyls with a wide range of acidity were on the polymer chain. CZE was able to differentiate among the analytes possessing carboxyl groups of different acidic strengths at pH 2.25. These components were not distinguished by CZE at high pH values (11.0). Interaction between the analyte and SDS affected the separation at this pH, and hence good resolution was obtained by MEKC. Informative separations were achieved both for the carrier and the conjugates in TEAP buffer at pH 2.25 by the CZE method. Optimal separation was achieved in borate buffer containing 75 mM SDS at pH 11.0 for the carrier and at pH 7.7 for the conjugates in MEKC.

Effect of gonadotropin-releasing hormone analogs and their conjugates on gonadotropin-releasing hormone receptor--positive human cancer cell lines.

The purpose of the present investigation was to develop new gonadotropin-releasing hormone (GnRH) antagonists and to increase their stability and antitumor effect by conjugation with carrier macromolecules. Antitumor effect was evaluated using clonogenic assay, cell counting for antiproliferation, and sulforhodamine B method. The presence of GnRH-binding sites in human cancer cell lines (MCF-7, MDA-MB-231, Ishikawa, LNCaP) was proved. The direct growth inhibition of tumor cell lines is achieved with relatively high analog concentrations (10(-10)- 10(-5) M). We have developed new GnRH analogs of human and chicken origin. MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)GnRH and the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3, D-Cpa2, Lys5, [beta-Asp(DEA)]6, Gln8, D-Ala10)-GnRH have stronger direct antitumor properties than the agonists. The antagonists inhibited proliferation of GnRH receptor-positive human cancer cell lines by 28 to 38%. GnRH peptide analogs were coupled with macromolecules through biodegradable groups, to enhance their antitumor effects. The antagonists reduced survival of MCF-7 and MDA-MB-231 cells by 38 to 48% and 20 to 41%, respectively. They showed less activity against human endometrial and prostate cancer cells (10-20%). The copolymer (P) as polyanionic carrier molecule reached only 15 to 20% survival reduction in all cell lines. However, the copolymer GnRH antagonist conjugates P-X-1892 and P-X-1544 killed 95 to 98% of cells at doses corresponding to the GnRH analog concentration. These compounds having antitumor activity could be tried for the treatment of prostate, breast, and endometrium cancer.

In vivo studies of the new gonadotropin-releasing hormone antagonist--copolymer conjugates having antitumor activity.

The aim of the study was to test in vivo the gonadotropin-releasing hormone (GnRH) antagonists and their conjugates showing antitumor activities in vitro. The in vivo experiments with the human GnRH antagonist MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH, the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3,D-Cpa2,Lys5,/beta-Asp(DEA)/6,Gln8,D-Al a10)-GnRH, and their copolymer conjugates were carried out on MCF-7 and MDA-MB-231 human breast tumors xenografted in immunosuppressed CBA/Ca HRIJ-T6 female mice and on MXT mouse mammary tumors in BDF1 mice. The P-X-1544 and P-X-1892 conjugates were prepared by coupling the GnRH antagonists to macromolecule copolymer through biodegradable spacers. MI-1544 and its conjugate had strong, whereas MI-1892 and its conjugate had slight, castration effect in rats. All of them showed selective antitumor activity. The conjugates, given daily, inhibited both types of xenografts by 42 to 49%. Their activity was stronger in MXT mammary tumor (72 to 61%). The in vivo effect of GnRH antagonists was largely increased by coupling them to nonbiodegradable macromolecule carriers of polyanionic character. P-X-1544 and P-X-1892 GnRH antagonist-macromolecule conjugates might become important therapeutic agents for the treatment of breast cancer.

Intracerebral implantation of hydrogel-coupled adhesion peptides: tissue reaction.

Arg-Gly-Asp peptides (RGD) were synthesized and chemically coupled to the bulk of N-(2-hydroxypropyl) methacrylamide-based polymer hydrogels. Fourier Transform Infrared Spectroscopy (FTIR) and amino acid analysis confirmed the peptide coupling to the polymer. Activated and control (unmodified) polymer matrices were stereotaxically implanted in the striata of rat brains, and two months later the brains were processed for immunohistochemistry using antibodies for glial acidic fibrillary protein (GFAP), laminin and neurofilaments. RGD-containing polymer matrices promoted stronger adhesion to the host tissue than the unmodified polymer matrices. In addition, the RGD-grafted polymer implants promoted and supported the growth and spread of GFAP-positive glial tissue onto and into the hydrogels. Neurofilament-positive fibers were also seen running along the surface of the polymer and, in some instances, penetrating the matrix. These findings are discussed in the context of using bioactive polymers as a new approach for promoting tissue repair and axonal regeneration of damaged structures of the central nervous system.