Randy T. McConnell

Characterization of an Akt Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.

Regulation of arachidonate metabolism via lipoxygenase and cyclo-oxygenase by 12-HPETE, the product of human platelet lipoxygenase.

Abstract Human platelets metabolize arachidonic acid via lipoxygenase and cyclo-oxygenase. The labile lipoxygenase product, 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), stimulates its own production by increasing lipoxygenase activity. However, 12-hydroxy-eicosatetraenoic acid (12-HETE), the end-product of the lipoxygenase pathway in human platelets, does not share this activity. In addition, 12-HPETE strongly suppresses prostaglandin and thromboxane production by inhibiting platelet cyclo-oxygenase. These effects of arachidonic acid hydroperoxides exhibit isomeric specificity in platelet homogenates; 12-HPETE is more potent than 11-HPETE, which is more potent than 9- and 8-HPETE. Collagen- and arachidonate-induced platelet aggregation is blocked by 12-HPETE.

In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1

Polo-like kinase 1 (PLK1) plays key roles in the regulation of mitotic progression, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis. PLK1 expression and activity are strongly linked to proliferating cells. Many studies have shown that PLK1 expression is elevated in a variety of tumors, and high expression often correlates with poor prognosis. Using a variety of methods, including small-molecule inhibition of PLK1 function and/or activity, apoptosis in cancer cell lines, cell cycle arrest in normal cell lines, and antitumor activity in vivo have been observed. In the present study, we have examined the in vitro biological activity of a novel and selective thiophene benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 (5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}thiophene-2-carboxamide, called compound 1). Compound 1 has low nanomolar activity against the PLK1 and PLK3 enzymes and potently inhibits the proliferation of a wide variety of tumor cell lines. In the lung adenocarcinoma cell line NCI-H460, compound 1 induces a transient G2-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G2-M and show little apoptosis. We also describe a cellular mechanistic assay that was developed to identify potent intracellular inhibitors of PLK1. In addition to its potential as a therapeutic agent for treating cancer, compound 1 is also a useful tool molecule for further investigation of the biological functions of PLK1 and PLK3. [Mol Cancer Ther 2007;6(2):450–9]

Identification of 4-(2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a novel inhibitor of AKT kinase.

Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.

The appearance of phospholipase and cyclo-oxygenase activities in the human promyelocytic leukemia cell line HL60 during dimethyl sulfoxide-induced differentiation

Abstract The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. During differentiation a phospholipase activity, which releases arachidonic acid from membrane phospholipids, is expressed. Similarly, fatty acid cyclo-oxygenase activity increases 10-fold. In addition, there is a 40-fold increase in chemotactic formyl peptide receptor binding and a dramatic increase in glucose oxidation via the hexosemonophosphate shunt. The addition of indomethacin, a potent cyclo-oxygenase inhibitor, to the culture medium reduced the cyclo-oxygenase activity of HL60 cells exposed to dimethyl sulfoxide by 97%. However, the presence of indomethacin did not block the dimethyl sulfoxide induced increases in chemotactic formyl peptide receptor binding and hexosemonophosphate shunt activity.

Chemotactic peptide stimulated endogenous arachidonic acid metabolism in HL60 granulocytes

Summary The human promyelocytic leukemia cell line HL60, which can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide, incorporates large amounts of [1- 14 C]arachidonic acid into membrane phospholipids following overnight incubation. Nanomolar concentrations of the chemotactic peptide, N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, stimulate a rapid, transient release of free arachidonic acid. The released arachidonate is metabolized via both the cyclo-oxygenase and lipoxygenase pathways. Arachidonic acid is also incorporated into triacylglycerols and phosphatidylinositol. The lipoxygenase product 5-S-hydroxy-6,8,11,14-icosatetraenoic acid is rapidly esterified, mainly into triacylglycerols and phosphatidylcholine. In contrast, 12-L-hydroxy-5,8,10-heptadecatrienoic acid and Leukotriene B 4 are not incorporated into cellular lipids.

The lipoxygenase product, 5-hydroperoxy-arachidonic acid, augments chemotactic peptide-stimulated arachidonic acid release from HL60 granulocytes

Abstract The chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys stimulates the release of arachidonic acid from endogenous phospholipids of the human promyelocytic leukemia cell line HL60. This release of unsaturated fatty acid is augmented by the presence of exogenously added lipoxygenase product, 5-hydroperoxy-arachidonic acid (5-HPETE). Other hydroperoxy- and hydroxy-arachidonic acid derivatives are less potent. In addition, saturated fatty acids and hydrogen peroxide do not possess this ability to augment arachidonic acid release. None of the arachidonic acid derivatives are capable of stimulating the release of arachidonic acid from endogenous phospholipids in the absence of the chemotactic peptide.

Specificity of a phosphatase for phospholipid, Ca2+-dependent protein kinase-phosphorylated histone H1 resides in the catalytic subunit.

A protein phosphatase from liver which acts preferentially on histone phosphorylated with phospholipid, Ca2+-dependent protein kinase has been purified and the intrinsic specificity determined to reside in the catalytic subunit of the enzyme complex. Comparison with a preparation of pork heart protein phosphatase suggests that this specificity may be a general property of a class of protein phosphatases. Protein kinase C-phosphorylated histone H1 represents an improved substrate for phosphatase detection providing a five to tenfold greater sensitivity than other substrates including cAMP-dependent protein kinase phosphorylated H1.

Histones H3 and H4 inhibit protein kinase C specifically

The lysine-rich histone H1 is a preferred substrate for the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Histones H3 and H4 are poor substrates but potent inhibitors of the enzyme. The inhibitory effect of H3 and H4 seems to result mainly from a decreased sensitivity of protein kinase C to stimulation by phosphatidylserine (PS). These observations suggest that site-specific phosphorylation of one histone type can be regulated by other histones.