Imidazo[2,1-b]thiazole guanylhydrazones as RSK2 inhibitors.

The activity of a series of imidazo[2,1-b]thiazole guanylhydrazones as inhibitors of p90 ribosomal S6 kinase 2 (RSK2) is described. It was found that a small subset of compounds show both potent inhibition of RSK2 kinase activity and tumor cell growth in vitro. Detailed study of one of the most active compounds indicates a high degree of selectivity for inhibition of RSK2 compared to a spectrum of other related kinases. Selective inhibition of the MCF-7 breast tumor cell line compared to MCF-10A non-transformed cells, as well as selective inhibition of the biomarker GSK3 provides evidence that the compounds can affect the RSK2 target in cells.

Cellular inhibition of checkpoint kinase 2 (Chk2) and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019 [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide].

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment, with a corresponding signature in xenografts.

Belinostat is a hydroxamate-type histone deactylase inhibitor (HDACi), which has recently entered phase I and II clinical trials. Microarray-based analysis of belinostat-treated cell lines showed an impact on genes associated with the G2/M phase of the cell cycle and downregulation of the aurora kinase pathway. Expression of 25 dysregulated genes was measured in eight differentially sensitive cell lines using a novel high-throughput assay that combines multiplex reverse transcriptase-PCR and fluorescence capillary electrophoresis. Sensitivity to belinostat and the magnitude of changes in overall gene modulation were significantly correlated. A belinostat-gene profile was specific for HDACi in three cell lines when compared with equipotent concentrations of four mechanistically different chemotherapeutic agents: 5-fluorouracil, cisplatin, paclitaxel, and thiotepa. Belinostat- and trichostatin A (HDACi)-induced gene responses were highly correlated with each other, but not with the limited changes in response to the other non-HDACi agents. Moreover, belinostat treatment of mice bearing human xenografts showed that the preponderance of selected genes were also modulated in vivo, more extensively in a drug-sensitive tumor than a more resistant model. We have demonstrated a gene signature that is selectively regulated by HDACi when compared with other clinical agents allowing us to distinguish HDACi responses from those related to other mechanisms.

Identification of a Bis-guanylhydrazone [4,4'-Diacetyldiphenylurea-bis(guanylhydrazone); NSC 109555] as a novel chemotype for inhibition of Chk2 kinase.

Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4'-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.

Altered expression of neuronal nitric oxide synthase in the duodenum longitudinal muscle-myenteric plexus of obesity induced diabetes mouse: implications on enteric neurodegeneration.

Type 2 diabetes caused by obesity shows autonomic neuropathy. Molecular mechanism involved in enteric neurodegeneration is not clear. Neuronal nitric oxide synthase (nNOS) is one of the important agents involved in gastrointestinal function. Therefore, expression of nNOS in the duodenum LM-MP of type 2 diabetes model mouse was studied. Real time RT-PCR analysis showed reduction in nNOS expression in male diabetic LM-MP compared to male control. In contrast, female diabetic LM-MP had high level of nNOS mRNA compared to female control. Western blot determination of LM-MP showed reduction in nNOS protein in male diabetic LM-MP and high level of nNOS in female diabetic LM-MP compared to their respective controls. Expression of nNOS observed by Western blot was further confirmed by nNOS immunostaining of the mouse duodenum. TUNEL staining of mouse duodenum showed apoptosis in male diabetic enteric neurons. These studies suggest that nNOS expression in LM-MP varies with gender during early stage of type 2 diabetes. In addition, reduced expression of nNOS is likely to contribute to apoptosis seen in the enteric neurons of male type 2 diabetic mice.

7-hydroxystaurosporine (UCN-01) inhibition of Akt Thr308 but not Ser473 phosphorylation: a basis for decreased insulin-stimulated glucose transport.

7-hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 mumol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 mumol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr308 but not Ser473 phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation.

Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation.

Perifosine is a novel p.o. bioavailable alkylphospholipid. Perifosine has displayed significant antiproliferative activity in vitro and in vivo in several human tumor model systems and has recently entered phase I clinical trials. Recent studies have identified that perifosine could cause cell cycle arrest with induction of p21(WAF1/CIP1) in a p53-independent fashion; however, the basis for that effect is not known. Structurally, perifosine resembles naturally occurring phospholipids. Therefore, we hypothesized that perifosine might perturb pathways related to phospholipids modulated by growth factor action. We demonstrate here that perifosine causes dose-dependent inhibition of protein kinase B/Akt phosphorylation and thus activation at concentrations causing growth inhibition of PC-3 prostate carcinoma cells. Only the myristoylated form of Akt (MYR-Akt), which bypasses the requirement for pleckstrin homology (PH) domain-mediated membrane recruitment, abrogated perifosine-mediated decrease of Akt phosphorylation and cell growth inhibition by perifosine. We demonstrate further that perifosine decreases the plasma membrane localization of Akt, and this is substantially relieved by MYR-Akt along with relief of downstream drug effect on induction of p21(WAF1/CIP1). Perifosine does not directly affect phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent kinase 1, or Akt activity at concentrations inhibiting Akt phosphorylation and membrane localization. Our results demonstrate that Akt is an important cellular target of perifosine action. In addition, these studies show that the membrane translocation of certain PH domain-containing molecules can be greatly perturbed by the alkylphospholipid class of drugs and imply further that the PI3K/Akt pathway contributes to regulation of p21(WAF1/CIP1) expression.

Evidence for the existence of a new receptor for CGRP, which is not CRLR.

Receptors for calcitonin gene-related peptide (CGRP), a neuropeptide known to be the most potent vasodilator, are abundantly expressed in cerebellum. A monoclonal antibody to cerebellar CGRP receptors specifically detects a 66 kDa protein from rat cerebellum and other rat and human tissues, but not from SK-N-MC cells which express calcitonin receptor-like receptor (CRLR), a recently described component of CGRP receptors. In contrast, mRNA expression for CRLR was abundant in SK-N-MC cells, but it was undetectable in rat cerebellum. Furthermore, the antibody could not detect any immunoreactive protein in HEK 293 cells transiently transfected with CRLR and receptor activity-modifying protein 1 (RAMP(1)) indicating the possible existence of another CGRP receptor, which does not involve CRLR. Due to the absence of biochemical or structural data on the existence of a CGRP(2) receptor and the new data provided in this paper, we suggest to identify the two CGRP receptors as CGRP-A and CGRP-B.

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas.

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.

Calcitonin gene-related peptide in pregnancy and its emerging receptor heterogeneity.

Calcitonin gene-related peptide (CGRP) is the most potent vasodilator, and there is a growing body of evidence that this peptide might have multiple other functions. During pregnancy, circulating CGRP levels in rats increase up to the time of delivery, followed by a sharp decline at term and postpartum. In addition, the sensitivity of various vascular beds to CGRP in rats appears to increase with advancing pregnancy. This increased sensitivity might be involved in regulating uteroplacental blood flow, in addition to other vascular adaptations that occur during normal pregnancy. Furthermore, the uterine relaxation response to CGRP is elevated during pregnancy and decreased at term. Sex steroid hormones, estrogens and progesterone, regulate CGRP synthesis and its effects on both myometrial and uterine vascular tissues. These changes in smooth muscle relaxation sensitivity to CGRP appear to be a consequence of changes in CGRP-receptor levels in these tissues. There appear to be two receptors for CGRP: the CGRP-A receptor, a well-characterized receptor consisting of calcitonin receptor-like receptor and receptor activity modifying protein 1, and the CGRP-B receptor. The CGRP system might play a role in the maintenance of normal pregnancy, and a defect in this system might lead to complications.