Paeciloquinones A, B, C, D, E and F: new potent inhibitors of protein tyrosine kinases produced by Paecilomyces carneus. I. Taxonomy, fermentation, isolation and biological activity.

Paeciloquinones A to F as well as versiconol have been isolated as inhibitors of protein tyrosine kinase from the culture broth of the fungus Paecilomyces carneus P-177. The novel anthraquinones inhibit epidermal growth factor receptor protein tyrosine kinase in the micromolar range. Two compounds, paeciloquinones A and C, are potent and selective inhibitors of the v-abl protein tyrosine kinase with an IC50 of 0.4 microM. Dependent on the fermentation conditions, partially different sets of paeciloquinones may be produced. An HPLC method allows separation of all major active components.

M3 muscarinic receptors on murine HSDM1C1 cells: further functional, regulatory, and receptor binding studies.

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50 = 9 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) were full agonists which stimulated PI turnover by 13.3 +/- 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin (5-2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 microM) PI turnover. The half-maximal effect of TPA was produced at 1.8 +/- 0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd = 0.19 +/- 0.04 nM) and moderate capacity (Bmax = 201 +/- 22 fmol/mg protein). The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity.

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.

Development of high-throughput radioligand binding assays for interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF-alpha) in isolated membrane preparations.

Cytokine binding has been studied in a variety of intact cells, and in isolated receptor preparations. Each approach is associated with limitations with regard to screening large numbers of samples on a repetitive basis. In order to provide a more reproducible system of screening for compounds which modify IL-1 alpha and TNF-alpha binding, we have developed isolated membrane preparations for studying agents which can alter the association of these ligands with their receptors. These results demonstrate IL-1 alpha binding to BALB/c 3T3 cell membranes and TNF-alpha binding to HeLa S3 cell membranes, and indicate that this is a viable approach to high-throughput screening.

Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin.

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.

On the interaction of gallamine with muscarinic receptor subtypes.

The interaction of gallamine with muscarinic receptor subtypes was examined using radioligand binding studies. In competition studies using [3H]N-methylscopolamine [( 3H]NMS), gallamine displayed high affinity for the rat cardiac and guinea-pig uterine M2 muscarinic receptors and for the atypical muscarinic receptor present in chicken heart. Gallamine displayed low affinity for rat glandular and human 1321 N1 astrocytoma cell M3 receptors and also for the M4 receptors of NG108-15 and PC12 cells. The compound displayed intermediate affinity for M1 receptors of rat cortex labeled using [3H]pirenzepine. The interaction of gallamine with the M1 and M2 receptors appeared to be competitive at the low concentrations required to determine affinity estimates. Thus, gallamine inhibited the binding of [3H]pirenzepine to M1 receptors and [3H]NMS to M2 receptors at concentrations that were 263- and 23-fold lower, respectively, than those required to decrease radioligand dissociation kinetics. Furthermore, gallamine, at a concentration that inhibited between 63 and 71% of specific radioligand binding, had no effect on the observed rate of association of the radioligand with either the M1 or the M2 receptor. At the M3 glandular receptor, there was little separation between the concentrations of gallamine that produced inhibition of binding and those that decreased the association and dissociation rates of [3H]NMS. It is therefore difficult to determine if the inhibition of binding seen in competition studies on the M3 receptor was produced through a competitive or an allosteric mechanism. Despite its possible allosteric properties at the M3 receptor, gallamine can be used to detect heterogeneity of muscarinic receptor subtypes in several tissues and therefore represents a useful tool for defining muscarinic receptor subtypes.

Antagonist characterization of atypical beta adrenoceptors in guinea pig ileum: blockade by alprenolol and dihydroalprenolol.

The present study was undertaken to further characterize the atypical beta adrenoceptor in guinea pig ileum. Tension was developed in isolated segments of ileum using transmural electrical stimulation of enteric cholingeric nerves. The ability of isoproterenol to relax the ileum, via beta-1 adrenoceptor and atypical beta adrenoceptor agonism, was measured. Propranolol (5 x 10(-6) M) and bromoacetylaprenololmetane blocked beta-1 adrenoceptors but, at the concentrations tested, were without affinity at atypical beta adrenoceptors. (-)-Alprenolol and (-)-dihydroalprenolol, however, acted as competitive antagonists at both sites (pA2 values of 8.2 and 8.81 at beta-1 adrenoceptors and 6.47 and 6.43 at atypical beta adrenoceptors, respectively). (-)-Alprenolol also exerted agonistic activity at the atypical beta adrenoceptor. [3H](-)-Dihydroproalprenolol failed to identify beta-1 adrenoceptors or atypical beta adrenoceptors but, instead, bound to a putative lipophilic site unrelated to ileal adrenoceptors. Before this study, nadolol (pA2 = 4.7) was the only documented antagonist at the atypical beta adrenoceptor in guinea pig ileum. Thus, the present results detail two additional pharmacological probes which exhibit about a 100-fold greater affinity than nadolol for the atypical site.

Binding characteristics of the muscarinic receptor subtype of the NG108-15 cell line.

Kinetic, saturation and competition binding studies were conducted on the muscarinic receptor binding sites labeled by [3H]N-methylscopolamine ([3H]NMS) in membranes prepared from NG108-15 cells. The pharmacology of the NG108-15 cell muscarinic receptors was compared to that of the M1 receptors of rat cortex labeled using [3H]pirenzepine, the M2 and M3 receptors of rat heart and submaxillary gland, respectively, labeled using [3H]NMS and the muscarinic receptors of the PC12 cell line also labeled using [3H]NMS. The rate of dissociation of [3H]NMS from the NG108-15 cell muscarinic receptor was similar to that obtained at the M3 receptor and at the muscarinic receptor of the P12 cells but was slower that the dissociation rate obtained at the M2 cardiac muscarinic receptor. The Kd of [3H]NMS in the NG108-15 cells was significantly lower than that obtained at the M2 and M3 receptor but was similar to the Kd obtained in PC12 cells. In competition studies the affinity estimates for AF-DX 116, 4-DAMP, methoctramine and pirenzepine were not consistent with the presence of either an M1, M2 or M3 receptor but were identical to the affinity estimates obtained at the muscarinic receptor of the PC12 cell line. On the basis of these data we conclude that the muscarinic receptor present in the NG108-15 cells is different to the M1, M2 or M3 subtypes already described but is similar to the muscarinic receptor present in the PC12 cell line. Since NG108-15 cells expresses mRNA for the m4 muscarinic receptor gene described by Bonner et al. (1987) we propose that the muscarinic receptors present in this cell line be denoted as M4 receptors.

Affinity of muscarinic receptor antagonists for three putative muscarinic receptor binding sites.

1. A range of muscarinic receptor antagonists were examined for affinity at the M1 muscarinic binding site, present in rat cerebrocortical membranes and the M2 muscarinic binding sites of rat cardiac and submaxillary gland membranes. 2. The results obtained were consistent with the presence of three classes of muscarinic binding site. 3. Both the M1 binding site, labelled by [3H]-pirenzepine ([3H]-Pir) in rat cerebrocortical membranes, and the M2 gland binding site, labelled by [3H]-N-methyl scopolamine ([3H]-NMS) in rat submaxillary gland membranes, displayed higher affinity for pirenzepine, dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and cyclohexylphenyl (2-piperidinoethyl) silanol (CPPS) than did the M2 binding sites of cardiac membranes labelled by [3H]-NMS. 4. The M2 cardiac sites displayed higher affinity for methoctramine, himbacine and AF-DX 116 than did either the M1 binding site of cerebrocortical membranes or the M2 gland binding site present in rat submaxillary gland membranes. 5. The M1 and M2 gland binding sites could only be distinguished by considering the absolute affinity of compounds for these two sites. Thus, all compounds, with the exception of 4-DAMP, displayed between a 2 and 8 fold higher affinity for the M1 than for the M2 gland binding site. There were no antagonists with higher M2 gland than M1 affinity.