Macrocyclic ureas as potent and selective Chk1 inhibitors: an improved synthesis, kinome profiling, structure-activity relationships, and preliminary pharmacokinetics.

A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5-7, 10, 13, 14, 19-21, 25, 27, and 31-34. These compounds significantly sensitize tumor cells to the DNA-damaging antitumor agent doxorubicin in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and camptothecin-induced S checkpoints, indicating that the potent biological activities of these compounds are mechanism-based through Chk1 inhibition. Kinome profiling analysis of a representative macrocyclic urea 25 against a panel of 120 kinases indicates that these novel macrocyclic ureas are highly selective Chk1 inhibitors. Preliminary PK studies of 1a and b suggest that the 14-member macrocyclic inhibitors may possess better PK properties than their 15-member counterparts. An improved synthesis of 2 and 20 by using 2-(trimethylsilyl)ethoxycarbonyl (Teoc) to protect the amino group not only readily provided the desired compounds in pure form but also facilitated the scale up of potent compounds for various biological studies.

Clinical pharmacokinetics of erlotinib in patients with solid tumors and exposure-safety relationship in patients with non-small cell lung cancer.

OBJECTIVE: Our objective was to assess the pharmacokinetics of erlotinib in a large patient population with solid tumors, identify covariates, and explore relationships between exposure and safety outcomes (rash and diarrhea) in patients with non-small cell lung cancer receiving single-agent erlotinib. METHODS: The population pharmacokinetic analysis was performed by use of NONMEM based on 4068 concentration samples from 1047 patients receiving erlotinib as a single agent or in combination with chemotherapy. By use of a 1-compartment model with first-order absorption, the influence of demographic and clinical characteristics on clearance and volume was examined. Spearman rank correlation analyses were performed to test for correlations between maximum grades of rash and diarrhea and erlotinib exposure in non-small cell lung cancer patients treated with single-agent erlotinib. RESULTS: On the basis of the final model developed from patients treated with erlotinib as a single agent, the oral clearance was 3.95 L/h, the oral volume of distribution was 233 L, and the absorption rate was 0.95 h(-1). The median erlotinib half-life based on this patient population was 36.2 hours. Total bilirubin, alpha1-acid glycoprotein, and smoking status were the most important factors affecting clearance. The clearance in current smokers was 24% faster than that in former smokers or those who never smoked. There was a statistically significant correlation between drug exposure and rash (P < .05). However, there was significant overlap in the range of values for patients who had no rash (grade = 0) and those who had any grade of rash. No significant correlation was found between exposure and diarrhea. CONCLUSIONS: The long half-life of erlotinib supports the current once-daily dosing regimen at 150 mg/d. Effects of covariates on erlotinib clearance and correlations with adverse event severity were provided to aid in the detection of a treatment-emergent effect.

A novel rhodopsin kinase in octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein betagamma-subunits.

G protein-coupled receptor kinases (GRKs) play an important role in stimulus-dependent receptor phosphorylation and desensitization of the receptors. Mammalian rhodopsin kinase (RK) and beta-adrenergic receptor kinase (betaARK) are the most studied members among known GRKs. In this work, we purified RK from octopus photoreceptors for the first time from invertebrate tissues. The molecular mass of the purified enzyme was 80 kDa as estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK, octopus RK (ORK) was directly activated by betagamma-subunits of a photoreceptor G protein. We examined the effects of various known activators and inhibitors of GRKs on the activity of the purified ORK and found that their effects were different from those on either bovine RK or betaARK. To analyze the primary structure of the enzyme, we cloned the cDNA encoding ORK from an octopus retinal cDNA library. The deduced amino acid sequence of the cDNA was highly homologous to betaARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in the GRK family, which is distinguished from vertebrate RK.

Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts.

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus.

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay.

1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis.

Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.

Characterization of soluble cyclic adenosine monophosphate-dependent protein kinase isozymes in murine embryonic palatal tissue.

Certain hormonal primary messengers identified in the mammalian palate during its ontogeny transmit information to the interior of the cell via transmembrane signaling systems that control the production of the secondary messenger cyclic adenosine monophosphate. The singular role of intracellular cyclic AMP is to activate cAMP-dependent protein kinases (cAMP-dPK). cAMP-dPK were thus identified and characterized in the developing murine embryonic palate. Incubation of cytosolic fractions of embryonic palatal tissue with cAMP resulted in a dose-dependent increase in the cAMP-dPK activity ratio. A transient elevation of basal cAMP-dPK was seen during the period of palatal ontogeny that corresponded temporally with a previously demonstrated transient elevation of palatal basal cAMP levels. Fractions of embryonic palatal tissue cytosols derived by diethylaminoethyl (DEAE)-Sephacel chromatography were analyzed for phosphotransferase activity and for [3H]-cAMP binding to the regulatory (R) subunits of cAMP-dPK. Such analyses revealed two peaks of activity on day 13 of gestation. Based on the salt concentration at which the material in these peaks eluted from DEAE, its ability to cochromatograph with authentic cAMP-dPK isozymes, its molecular weight as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis, and the ability of the material to be photoaffinity labeled with [3H]-8-azidoadenosine 3',5' cyclic phosphate, types I and II cAMP-dPK were identified. Regulatory subunits of cAMP-dPK were characterized by the binding of [3H]-cAMP to cytosolic fractions of embryonic palatal tissue. Such binding was saturable (Bmax = 1,096 fmol/mg protein) and of high affinity (Kd = 7 nM). Only cAMP and cyclic guanosine monophosphate competed in a dose-related manner with [3H]-cAMP for binding to R subunits of cAMP-dPK. Adenosine, cTMP, and adenosine triphosphate, at doses up to 10(-4) M, did not compete for binding. Temporal analysis of binding data indicated that the number of binding sites transiently decreased during day 13 of gestation. Characterization of cAMP-dPK in tissue derived from the developing mammalian palate allows consideration of cAMP-dPK as a key regulatory enzyme capable of transducing hormonally elevated intracellular levels of cAMP into metabolic responses during orofacial ontogenesis.