Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein.

Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

Tissue distribution of the human soluble guanylate cyclases.

Soluble guanylate cyclase (sGC) is an important component of the NO signaling pathway. Human sGC isoforms alpha(1), alpha(2), and beta(1) show differential mRNA tissue distributions. alpha(1) and beta(1) are expressed in most tissues; however, the alpha(2) isoform shows a more restricted expression pattern with high levels in brain, placenta, spleen, and uterus only. Both alpha subunits exist as multiple transcripts whereas beta(1) exists as a single message. This study reports for the first time the tissue distribution of human sGC message and demonstrates that sGC isoforms are nonuniformly expressed which may be useful if the enzyme is to be exploited as a therapeutic target.

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells.

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.

Comparison of staurosporine and four analogues: their effects on growth, rhodamine 123 retention and binding to P-glycoprotein in multidrug-resistant MCF-7/Adr cells.

The potent kinase inhibitor staurosporine and its protein kinase C (PKC)-selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF-7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nM, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nM were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P-gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp-mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development.

Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones.

The MCF-7 doxorubicin-resistant cell line MCF-7/Dox has been used extensively for studies of the multidrug resistance phenomenon. Using fluorescence-activated cell sorting (FACS), these cells were separated into two populations on the basis of rhodamine 123 (R123) accumulation. We designated these as low P-glycoprotein (LP-gp) and high P-gp (HP-gp) cells on the basis of their P-gp content. Using the reverse transcriptase polymerase chain reaction technique controlled by homologous internal standards, we analysed levels of MDR1 and MDR2 mRNA in each cell type. LP-gp and HP-gp cells had MDR1 mRNA levels of 2.17 +/- 0.17 and 6.65 +/- 2.29 amol ng-1 total RNA respectively, compared with 0.00088 +/- 0.00005 amol ng-1 in wild-type MCF-7 cells (MCF-7/WT). MCF-7/WT cells additionally contained 0.023 +/- 0.016 amol ng-1 of MDR2 mRNA, which was unchanged in LP-gp cells, but lower than in HP-gp cells, which contained 0.42 +/- 0.08 amol ng-1. Both LP-gp and HP-gp cells contained increased copies of the MDR1 gene. However, the degree of gene amplification did not correlate with the changes in MDR1 mRNA levels, indicating further regulatory levels of gene expression. The level of P-gp detected by MRK 16 correlated with R123 accumulation. HP-gp cells expressed a 10-fold higher level of P-gp1 than LP-gp cells. However, there was only a 3-fold increase in MDR1 mRNA level in HP-gp cells compared with LP-gp cells. These data suggest that some regulation of P-gp1 expression also occurred at the post-translational level. Phosphorylation of P-gp by protein kinase C (PKC)-alpha is necessary for its activity. Our analysis of PKC-alpha, 0 and epsilon isozyme levels, and subcellular distribution, shows a co-regulation of expression with P-gp, suggesting a necessary role for PKC in P-gp regulation.

Comparison between inhibition of protein kinase C and antagonism of calmodulin by tamoxifen analogues.

A variety of analogues of tamoxifen were tested for inhibition of protein kinase C (PKC) activity in MCF-7 breast cancer cells. These results were compared with the calmodulin antagonism exhibited by the analogues as measured by inhibition of calmodulin-dependent cyclic AMP phosphodiesterase. The same structural features that enhanced PKC inhibition also led to an increase in calmodulin antagonism, namely 4-iodination and elongation of the basic side-chain. The most potent analogue has a 4-iodine substituent and eight carbon atoms in its basic side-chain with IC50 values of 38 microM for PKC inhibition and 0.3 microM for calmodulin antagonism, which compares with 92 and 6.8 microM, respectively, for tamoxifen. Some selectivity was achieved with a ring-fused analogue that retained the potency of tamoxifen as a PKC inhibitor, but lacked calmodulin antagonism.

Differential inhibition of cytosolic and membrane-derived protein kinase C activity by staurosporine and other kinase inhibitors.

The hypothesis was tested that 9 kinase inhibitors with diverse specificities for protein kinase C (PKC), including staurosporine and four of its analogues, interfere differently with PKC derived from either the cytosolic or particulate fractions of MCF-7 breast carcinoma cells. GF 109203X inhibited the enzyme identically in either preparation. CGP 41251 and calphostin C inhibited cytosolic PKC more effectively than membrane-derived PKC with ratios of IC50 (cytosolic PKC) over IC50 (membrane-derived PKC) of 0.07 and 0.04, respectively. The other six agents inhibited membrane-derived PKC more potently than cytosolic enzyme. Staurosporine and RO 31 8220 exhibited IC50 ratios of 12.3 and 21.6, respectively. The results suggest that there are dramatic differences between kinase inhibitors in their divergent effects on cytosolic and membrane-derived PKC which should be borne in mind in the interpretation of their pharmacological properties.

Comparison of ability of protein kinase C inhibitors to arrest cell growth and to alter cellular protein kinase C localisation.

Inhibitors of protein kinase C (PKC) such as the staurosporine analogues UCN-01 and CGP 41251 possess antineoplastic properties, but the mechanism of their cytostatic action is not understood. We tested the hypothesis that the ability of these compounds to arrest growth is intrinsically linked with their propensity to inhibit PKC. Compounds with varying degrees of potency and specificity for PKC were investigated in A549 and MCF-7 carcinoma cells. When the log values of drug concentration which arrested cell growth by 50% (IC50) were plotted against the logs of the IC50 values for inhibition of cytosolic PKC activity, two groups of compound could be distinguished. The group which comprised the more potent inhibitors of enzyme activity (calphostin C, staurosporine and its analogues UCN-01, RO 31-8220, CGP 41251) were the stronger growth inhibitors, whereas the weaker enzyme inhibitors (trimethylsphingosine, miltefosine, NPC-15437, H-7, H-7I) affected proliferation less potently. GF 109203X was exceptional in that it inhibited PKC with an IC50 in the 10(-8) M range, yet was only weakly cytostatic. To substantiate the role of PKC in the growth inhibition caused by these agents, cells were depleted of PKC by incubation with bryostatin 1 (1 microM). The susceptibility of these enzyme-depleted cells towards growth arrest induced by staurosporine, RO 31-8220, UCN-01 or H-7 was studied. The drug concentrations which inhibited incorporation of [3H]thymidine into PKC-depleted A549 cells by 50% were slightly, but not significantly, lower than significantly, lower than those observed in control cells. These results suggest that PKC is unlikely to play a direct role in the arrest of the growth of A549 and MCF-7 cells mediated by these agents. Staurosporine is not only a strong inhibitor of PKC but also mimics activators of this enzyme in that it elicits the cellular redistribution of certain PKC isoenzymes. The ability of kinase inhibitors other than staurosporine to exert a similar effect was investigated. Calphostin C, H-7, H-7I, miltefosine, staurosporine, UCN-01, RO 31-8220, CGP 41251 or GF 109203X were incubated for 30 min with A549 cells in the absence or presence of the PKC activator 12-O-tetradecanoyl phorbol-13-acetate. The subcellular distribution of PKC-alpha-, -epsilon and -zeta was measured by Western blot analysis. None of the agents affected PKC-alpha or -zeta.(ABSTRACT TRUNCATED AT 400 WORDS)