In vitro and in vivo antitumor activity of the phosphatidylinositol-3-kinase inhibitor, wortmannin.

The microbial product wortmannin has previously been shown to be a potent inhibitor of phosphatidylinositol-3-kinase. In view of the potential role of this enzyme in transduction of mitogenic signals, we determined the cytotoxic activity of wortmannin against several human tumor cell lines in vitro. The most sensitive lines included GC3 colon carcinoma, IGROV1 ovarian carcinoma, and CCRF-CEM leukemia (IC-50s ranging from 0.7-2.1 microM). The cytotoxicity of wortmannin was decreased approximately 10-fold by serum-free conditions. Wortmannin was generally less active in low passage human breast cancer cell lines that overexpress either epidermal growth factor receptor or Her2/neu. Wortmannin was also tested for in vivo antitumor activity against seven murine tumor and ten human tumor xenograft models. Activity (> 60% inhibition of tumor growth) was observed in only the C3H mammary carcinoma and the human BxPC-3 pancreatic carcinoma xenograft. In vivo antitumor activity did not correlate with in vitro sensitivity to wortmannin cytotoxicity.

Advances with phospholipid signalling as a target for anticancer drug development.

The phosphatidylinositol-3-kinases (PtdIns-3-kinase) are a family of enzymes involved in the control of cell replication. One member of the family, the mammalian p110/p85 PtdIns-3-kinase, is a potential target for anticancer drug development because of its role as a component of growth factor and oncogene activated signalling pathways. There are a number of inhibitors of this PtdIns-3-kinase, the most potent being wortmannin (IC50 4 nM). Wortmannin inhibits cancer cell growth and has shown activity against mouse and human tumor xenografts in mice. Other inhibitors of the PtdIns-3-kinase are halogenated quinones which also inhibit cancer cell growth and have some in vivo antitumor activity. Some D-3-deoxy-3-substituted myo-inositol analogues and their corresponding PtdIns analogues have been synthesized. They may act as myo-inositol antimetabolites in the PtdIns-3-kinase pathway and they can inhibit cancer cell growth.

Atomic structure of the trypsin-A90720A complex: a unified approach to structure and function.

BACKGROUND: A90720A is a potent serine proteinase inhibitor produced by the terrestrial blue-green alga Microchaete loktakensis. Most of its structure has been defined by spectroscopic and degradative studies, but the configurations of several stereochemical centers are unknown, and its mode of inhibition of serine proteinases is not understood. We therefore examined the structure of the compound in a complex with trypsin. RESULTS: We have crystallized the bovine trypsin-A90720A complex and determined its three-dimensional structure at 1.90 A resolution using single crystal X-ray diffraction. The structure of the bound inhibitor is clearly shown in the electron density. The structure defines the absolute stereostructure of A90720A, establishes its bound conformation and illuminates its mode of inhibition. CONCLUSIONS: A90720A interacts with trypsin in a substrate-like manner through an extensive series of hydrogen bonds, hydrophobic interactions and steric complementarity. The compound uses a mixture of peptidal and nonpeptidal elements to imitate the canonical conformation of the exposed binding loop of 'small' proteinase inhibitors.

A multisample assay for inhibitors of phosphatidylinositol phospholipase C: identification of naturally occurring peptide inhibitors with antiproliferative activity.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol specific phospholipase C (PtdInsPLC) has been developed. Three naturally occurring peptide inhibitors of PtdInsPLC have been identified, myroridin K, streptothricin B and edeine, with IC50 values of 8.3, 6.7 and 16.1 microM, respectively. All three peptides inhibited colony formation of HT-29 human colon adenocarcinoma cells, with IC50 values of 7.2, 3.9 and 13.0 microM, respectively. The compounds also inhibited the growth of other human cancer cells in culture. One of the peptides, myroridin K, has previously been reported to have in vivo antitumour activity. It is possible that inhibition of PtdInsPLC is responsible for the cell growth inhibition and antitumour properties of the peptide compounds.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.

Phosphatidylinositol-3-kinase is an important enzyme for intracellular signaling. The microbial product wortmannin and some of its analogues have been shown to be potent inhibitors of phosphatidylinositol-3-kinase. The 50% inhibitory concentration for inhibition by wortmannin is 2 to 4 nM. Kinetic analysis demonstrates that wortmannin is a noncompetitive, irreversible inhibitor of phosphatidylinositol-3-kinase, with inactivation being both time- and concentration-dependent. Wortmannin has previously been reported to be an inhibitor of myosin light chain kinase but with an inhibitory concentration of 0.2 microM. Wortmannin was found not to be an inhibitor of phosphatidylinositol-4-kinase, protein kinase C, or protein tyrosine kinase. Wortmannin inhibited the formation of phosphatidylinositol-3-phosphates in intact cells. The results of the study suggest that wortmannin and its analogues may have utility as pharmacological probes for studying the actions of phosphatidylinositol-3-kinase.

Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues.

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genus Helenium, caused a marked potentiation of the increases in intracellular free Ca2+ concentration ([Ca2+]i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca2+ partly attenuated the increased [Ca2+]i responses caused by helenalin. The increased [Ca2+]i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca2+]i responses. No change in resting [Ca2+]i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca2+]i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca2+]i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca2+ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca2+]i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca2+ from these stores.

A83016F, a new member of the aurodox family.

A new member of the aurodox family of antibiotics, A83016F, has been isolated from an unidentified actionmycete designated A83016. The structure and relative stereochemistry of A83016F were elucidated by NMR examination of the parent compound and its diacetate derivative. A83016F exhibits only weak antimicrobial activity.

Hypotensive properties of the protein kinase inhibitor, staurosporine, in normotensive and spontaneously hypertensive rats.

The protein kinase inhibitor, staurosporine, was examined for hemodynamic actions in conscious rats and for its effects on isolated vascular smooth muscle. In conscious normotensive rats, staurosporine, 0.1 to 1.0 mg/kg intravenous, produced a dose-related decrease in blood pressure. Doses of 0.1 and 0.5 mg/kg produced reflex tachycardia, while heart rate decreased following 1.0 mg/kg. Higher doses of staurosporine (5 and 10 mg/kg), although not significantly altering heart rate, caused a precipitous drop in blood pressure and all rats died within 48 hours of treatment. Spontaneously hypertensive rats were significantly more sensitive than normotensive rats to the blood pressure lowering effects of staurosporine. The depressor activity of staurosporine was also observed in cord-stimulated pithed rats indicating that hypotension is a peripherally- rather than centrally-mediated effect of staurosporine. In ring preparations of rat thoracic aorta, staurosporine (3 nM to 0.1 mM) produced a concentration-dependent inhibition of serotonin-induced contraction, consistent with its ability to lower blood pressure by an effect at a peripheral vascular site. These results provide the first documentation of the potent cardiovascular activity of staurosporine in the conscious rat and support the contention that protein kinases, in particular protein kinase C, may be important in the regulation of vascular tone.

Synthesis and structure-activity relationships of new 9-N-alkyl derivatives of 9(S)-erythromycylamine.

A series of new 9-N-alkyl derivatives of 9(S)-erythromycylamine has been synthesized by reductive alkylation of erythromycylamine with aliphatic aldehydes and sodium cyanoborohydride. Alternative syntheses employing hydrogenation methods have also been developed. These new 9-N-alkyl derivatives possess excellent antimicrobial activity in vitro and in vivo, especially when administered orally to treat experimental infections in mice. From structure-activity studies, 9-N-(1-propyl)erythromycylamine (LY281389) was selected as the most efficacious derivative. These methods have also been extended to the synthesis of some 9-N,N-dialkyl derivatives of erythromycylamine.

Studies of the antitumor activity of (2-alkoxyalkyl)- and (2-alkoxyalkenyl)phosphocholines.

Analogues of the synthetic antitumor phospholipid ALP (1-octadecyl-2-methyl-sn-glycero-3-phosphocholine; alkyl lysophospholipid) in which the 1-ether oxygen atom has been removed have been prepared and evaluated for in vitro and in vivo anticancer activity. Compounds are presented in which the saturated long chain varies in length from 8 to 25 carbon atoms. Sites of unsaturation are also incorporated into the framework in some examples. In particular, rac-(2-ethoxyeicosyl)phosphocholine (10) displays the best in vivo activity of the chemical series against a variety of transplanted tumors and activates murine peritoneal macrophages to express tumor cytotoxicity in vitro. However, 10 does not offer the wide spectrum of antitumor activity we feel necessary to warrant further study.

Cyclic platelet-activating factor analogues derived from 2-deoxy-D-erythro-pentose.

A method for the synthesis of chiral cyclic analogues of platelet-activating factor (PAF) is reported. Treatment of suitably substituted derivatives of 2-deoxy-D-erythro-pentose with phosphorus oxychloride, followed by choline p-toluenesulfonate generates cyclic phospholipids in good yield. Further chemical modification produces other compounds including optically active gamma-butyrolactones such as 2-deoxy-5-O-hexadecyl-3-O-phosphocholyl-D-erythro-pentono-1, 4-lactone and 2-deoxy-3-O-hexadecyl-5-O-phosphocholyl-D-erythro-pentono-1, 4-lactone. All phospholipids were poor antagonists of PAF-induced aggregation of human platelets, and two analogs were poor agonists. The chemistry presented should be useful for the syntheses of other conformationally restricted analogues of PAF.