Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.

The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repair prior to undergoing mitosis. We hypothesized that the G(2) checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G(2) arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 microM PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G(2) arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G(2) checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.

Binding of 14-3-3beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G2-M cell population.

Wee1 protein kinase plays an important regulatory role in cell cycle progression. It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G2-M phase. In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s). Five of the eight positive clones identified encode 14-3-3beta. In vivo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta. The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta. The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection. Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase. Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability.

Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity.

Activation of the Cdc2.cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2. cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2.cyclin complexes.

Myt1: a Wee1-type kinase that phosphorylates Cdc2 on residue Thr14.

Most somatic cell division cycles contain a gap period (G2 phase) between the completion of DNA synthesis and the initiation of mitosis. This delay of mitotic entry is controlled, at least in part, by the repression of Cdc2 kinase activity by the phosphorylation of two conserved residues (Thr14 and Tyr15) within the ATP-binding pocket of the Cdc2 catalytic subunit. The kinases responsible for these two phosphorylation events include the Myt1 and Wee1 kinases, which phosphorylate Cdc2 on Thr14 and Tyr15, respectively. In this discussion, we summarise our current knowledge of the Myt1 kinase and its regulation of Cdc2 kinase activity during the G2-to -M phase transition.

Pharmacological characterization of LY293284: A 5-HT1A receptor agonist with high potency and selectivity.

(-)-LY293284, (-)-4R-6-acetyl-4-(di-n-propylamino)1,3,4,5- tetrahydrobenz[c,d]indole, is a conformationally restricted tryptamine derivative with an acetyl group serving as a protophilic substitution for the hydroxyl in serotonin (5-HT). In ligand displacement studies, LY293284 had a Ki of 0.07 nM for the 5-HT1A receptor but no affinity for other monoaminergic receptors within 3 orders of magnitude. LY293284 was evaluated in in vivo models, which have been used as markers for presynaptic and postsynaptic 5-HT1A receptor activity. LY293284 decreased hypothalamic 5-hydroxyindoleacetic acid levels (ED50, 2.9 micrograms/kg s.c.) and dorsal raphe serotonergic neuron firing rate (ED50, 0.08 micrograms/kg s.c.), which are accepted indices of presynaptic activity. LY293284 also induced a reduction in body temperature in rats (ED50, 3.6 micrograms/kg s.c.), which was blocked by pretreatment with (+/-)-pindolol. Hypothermic responses of rats to 5-HT1A agonists have had both pre- and postsynaptic characteristics in previous studies. The ED50 values for 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in these tests were 15 to 45 times higher than those observed for LY293284. In models for postsynaptic activity, the ED50 for LY293284 for elevating serum corticosterone levels was 9.7 micrograms/kg s.c. and the minimum effective doses to induce lower lip retraction and flat posture were 3 micrograms/kg s.c. For comparison, the same indices obtained for 8-OH-DPAT were 222.4 and 100 micrograms/kg, respectively. The 5-HT syndrome responses induced by LY293284 were also attenuated by pretreatment with (+/-)-pindolol. LY293284 was 10 times more potent than 8-OH-DPAT in a drug discrimination test that used pigeons trained to identify 8-OH-DPAT. In sexual behavior tests with male rats, LY293284 induced a maximal reduction in ejaculatory latency at 0.01 micrograms/kg s.c., which was approximately 10 times higher potency than 8-OH-DPAT. In the pigeon conflict model for anxiolytic activity, LY293284 was 100 times more potent than 8-OH-DPAT in increasing punished responding. In the rat forced swim model for antidepressant-like activity, LY293284 was 30 and 35 times more potent than 8-OH-DPAT in decreasing immobility time and defecation rate. These studies have demonstrated that LY293284 is a highly selective and extremely potent 5-HT1A receptor agonist and represents a useful pharmacological tool for studying 5-HT1A receptor-mediated effects.

Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins.

Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity. We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene. SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants. Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition. These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.

cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2.

cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2. Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts. We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases. cdc25 itself is a very specific protein tyrosine phosphatase. Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates. In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro. Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation.

The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization.

The products of the cdc13+ and cdc2+ genes form a stable complex that displays protein kinase activity in vitro. p63cdc13 is a substrate of p34cdc2, the catalytic subunit of the kinase. The histone H1 kinase activity of cdc2 oscillates during the cell cycle. Activation of the preformed cdc2/cdc13 complex at the G2/M transition requires cdc25+ gene function. Post-metaphase inactivation of the kinase is associated with loss of cdc13, which shares sequence homology with mitotic cyclins and, in common with these proteins, is degraded at each cell division. cdc13 and cdc2 co-localize in the cell nucleus. cdc2 is not degraded during mitosis, but in the absence of cdc13 it is not localized in the nucleus. These observations suggest that the cdc13+-encoded cyclin acts to regulate both the catalytic properties and the localization of the protein kinase of which it is a subunit.

Synthesis of D-oxa tricyclic partial ergolines as dopamine agonists.

A series of hetero fused hexahydro-1,4-benzoxazines has been synthesized and evaluated for dopamine agonist activity. This class of compounds is another example in which an oxygen substitution in the D ring of a partial ergoline or ergoline retains dopaminergic properties. Compound 10, trans-(+/-)-4,4a,5,6,8a,9-hexahydro-5-propyl-2H,7H-pyrazolo[4,3-g] [1,4]benzoxazine, is a D-ring analogue of trans-(+/-)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo[3,4-g]qu ino line (1, LY141865) and also a des-A-ring analogue of 9-oxaergoline. Compounds 10, 2-aminohexahydrothiazolo[1,4]benzoxazine 11, and 2-aminohexahydropyrimido[1,4]benzoxazine 12 possess dopaminergic activity in prolactin inhibition and 6-hydroxydopamine lesioned rat turning assays.

Synthesis of two metabolites of (+)-propoxyphene.

Two minor metabolites of (+)-propoxyphene were prepared in both the (+/-) and (+) forms. The optically pure forms were synthesized by a degradative route from (+)-propoxyphene. The di-N-demethylated metabolite 10a,b had weak analgesic activity while the cyclized oxazine metabolite 7a was inactive.

Various 5-substituted and 2,5-disubstituted 1,3-dioxanes, a new class of analgesic agents.

A series of 5-substituted and 2,5-disubstituted 1,3-dioxanes was prepared and evaluated for analgesic activity in mice and rats. Some of the compounds possessed significant analgesic effects; their structure-activity relationships and chemistry are discussed. These compounds represent a unique series of analgesic agents.

Synthesis of a new metabolite of acetylmethadol.

The primary amine metabolites of alpha-(plus or minus)- alpha-(minus)-acetylmethadol were synthesized. A neutral permanganate oxidation of noracetylmethadol gave a nitroalkane. This unusual oxidation product was readily converted to the primary amine metabolite of acetylmethadol.