Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by impaired glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Since erythropoietin-producing hepatoma (Eph)-ephrin bidirectional signalling fine-tunes GSIS from pancreatic beta cells, we investigated Eph receptor tyrosine kinases (RTK) as potential drug targets for selectively increasing GSIS. METHODS: Insulin secretion assays were carried out using mouse and human pancreatic islets as well as mouse insulinoma (MIN6) cells in the presence or absence of two Eph RTK inhibitors. Furthermore, the most potent inhibitor was injected into mice to evaluate its effects on glucose tolerance and plasma insulin levels. RESULTS: We showed that the Eph RTK inhibitors selectively increased GSIS from MIN6 cells as well as mouse and human islets. Our results also showed that the insulin secretory effects of these compounds required Eph-ephrin signalling. Finally, pharmacological inhibition of Eph receptor signalling improved glucose tolerance in mice. CONCLUSIONS/INTERPRETATION: We showed for the first time that Eph RTKs represent targets for small molecules to selectively increase GSIS and improve glucose tolerance.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors.

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics.

Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

c-Jun N-terminal kinase promotes stem cell phenotype in triple-negative breast cancer through upregulation of Notch1 via activation of c-Jun.

c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an adenosine triphosphate-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24- CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.

Combinatorial libraries and biological discovery.

Combinatorial chemistry has become a popular tool for the preparation of collections of compounds that can be used to find inhibitors and substrates for different protein targets. It has evolved to provide small molecule libraries, which, with the concomittant use of affinity chromatography, gene expression profiling and complementation, can be used to identify compounds and their protein targets in biological systems, including the neurological system.

ALK inhibitor resistance in ALK(F1174L)-driven neuroblastoma is associated with AXL activation and induction of EMT.

The crizotinib-resistant ALK(F1174L) mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALK(F1174L). Here we demonstrate acquired resistance to TAE684 and LDK378 in ALK(F1174L)-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALK(F1174L)-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance.

Discovery of a BTK/MNK dual inhibitor for lymphoma and leukemia.

Bruton's tyrosine kinase (BTK) kinase is a member of the TEC kinase family and is a key regulator of the B-cell receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as mantle cell lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and activated B-cell-diffuse large B-cell lymphoma. MNK kinase is one of the key downstream regulators in the RAF-MEK-ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has been observed to moderately inhibit the proliferation of AML cells. Through a structure-based drug-design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and noncovalent binding to MNK. Compared with the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 enhanced the antiproliferative efficacies in vitro against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells, which respond moderately to BTK inhibitor in vitro. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0-G1 stage and can induce strong apoptotic cell death. These primary results demonstrate that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances.

Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors.

BACKGROUND: Purines constitute a structural class of protein ligands involved in mediating an astonishing array of metabolic processes and signal pathways in all living organisms. Synthesis of purine derivatives targeting specific purine-binding proteins in vivo could lead to versatile lead compounds for use as biological probes or drug candidates. RESULTS: We synthesized several libraries of 2,6, 9-trisubstituted purines using both solution- and solid-phase chemistry, and screened the compounds for inhibition of cyclin-dependent kinase (CDK) activity and human leukemic cell growth. Lead compounds were optimized by iterative synthesis based on structure-activity relationships (SARs), as well as analysis of several CDK-inhibitor cocrystal structures, to afford several interesting compounds including one of the most potent CDK inhibitors known to date. Unexpectedly, some compounds with similar CDK inhibitory activity arrested cellular proliferation at distinctly different phases of the cell cycle and another inhibitor directly induced apoptosis, bypassing cell-cycle arrest. Some of these compounds selectively inhibited growth of cells derived from specific tumors. CONCLUSIONS: 2,6,9-Trisubstituted purines have various and potent biological activities, despite high concentrations of competing endogenous purine ligands in living cells. Purine libraries constitute a versatile source of small molecules that affect distinct biochemical pathways mediating different cellular functions.

Ligand-associated ERBB2/3 activation confers acquired resistance to FGFR inhibition in FGFR3-dependent cancer cells.

Somatic alterations of fibroblast growth factor receptors (FGFRs) have been described in a wide range of malignancies. A number of anti-FGFR therapies are currently under investigation in clinical trials for subjects with FGFR gene amplifications, mutations and translocations. Here, we develop cell line models of acquired resistance to FGFR inhibition by exposure of cell lines harboring FGFR3 gene amplification and translocation to the selective FGFR inhibitor BGJ398 and multitargeted FGFR inhibitor ponatinib. We show that the acquisition of resistance is rapid, reversible and characterized by an epithelial to mesenchymal transition and a switch from dependency on FGFR3 to ERBB family members. Acquired resistance was associated with demonstrable changes in gene expression including increased production of ERBB2/3 ligands, which were sufficient to drive resistance in the setting of FGFR3 dependency but not dependency on other FGFR family members. These data support the concept that activation of ERBB family members is sufficient to bypass dependency on FGFR3 and suggest that concurrent inhibition of these two pathways may be desirable when targeting FGFR3-dependent cancers.

Synthesis and biological evaluation of myoseverin derivatives: microtubule assembly inhibitors.

Myoseverin, a trisubstituted purine, inhibits microtubule assembly in vitro, interferes with normal mitotic spindle assembly, and arrests the cell cycle in mitosis in U937 cells. We synthesized a variety of myoseverin derivatives and screened them for inhibition of spindle assembly in Xenopus egg extracts and for microtubule disassembly in vitro. Selected compounds were tested against 60 cancer cell lines at the National Cancer Institute as possible anticancer drug candidates.

Abolition of latent inhibition by a single 5 mg dose of d-amphetamine in man.

The performance of healthy volunteer subjects on an auditory latent inhibition (LI) paradigm was assessed following administration of a single oral dose of d-amphetamine or placebo. It was predicted that a low (5 mg), but not a high (10 mg), dose of d-amphetamine would disrupt LI. The prediction was supported with left ear presentation of the preexposed stimulus only. When the preexposed stimulus was presented to the right ear the predicted pattern of findings was not obtained. It is concluded that the dopaminergic system is involved in the mediation of LI in man and it is speculated that the interaction between amphetamine dose and ear of presentation of the preexposed stimulus may reflect normally occurring dopaminergic hemisphere asymmetry.

Effects of nicotine and amphetamine on latent inhibition in human subjects.

Latent inhibition (LI) is a phenomenon in which repeated non-reinforced exposure to a stimulus retards subsequent conditioning to that stimulus; it reflects a process whereby irrelevant stimuli become ignored, and has been the subject of study concerning attentional abnormalities in schizophrenia. Low doses of the indirect dopamine (DA) agonists, amphetamine and nicotine, disrupt LI in the rat. These drugs are believed to disrupt LI via DA release in the nucleus accumbens; LI in amphetamine- and nicotine-treated rats is reinstated by administration of the DA antagonist haloperidol. In human subjects, low doses of amphetamine abolish LI, and more recently haloperidol has been shown to potentiate LI. The present study investigated the effects of nicotine on LI in human subjects, and also attempted to replicate the abolition of LI by amphetamine. Nicotine failed to affect LI when administered either subcutaneously or by cigarette smoking. LI was, however, abolished in a group of subjects given 5 mg amphetamine 90 min before testing. Supplementary analyses of the data pooled from all three experiments showed that, in contrast to an earlier report, LI was no weaker in smokers than in nonsmokers.

Latent inhibition in drug naive schizophrenics: relationship to duration of illness and dopamine D2 binding using SPET.

The dual aims of the study were (1) to examine the effect of neuroleptic medication on the expression of latent inhibition (LI) by studying LI in drug naive schizophrenic patients, and (2) to investigate the relationship between LI and dopamine D2 receptor binding in the basal ganglia using single photon emission tomography (SPET). Subjects constituted a sub-set of patients investigated in a major study of in vivo D2 receptor binding in schizophrenia (Pilowsky et al., 1993). Striatal D2 receptor binding was assessed in 15 neuroleptic naive schizophrenic patients and 13 healthy volunteers. The performance of subjects on a within-subject auditory latent inhibition paradigm was also assessed. There was found to be no significant difference in LI between schizophrenic patients and normal controls, both groups showing a strong within-subject LI effect. There was also found to be no association between LI and dopamine D2 receptor binding in either the left or the right basal ganglia. This lack of association indicates that LI is not directly related to post-synaptic D2 receptor levels in the striatum. LI was, however, found to be correlated with duration of illness in the schizophrenic group. Patients with a relatively short duration of illness (< 12 months) tended to show reversed, or absent, LI whereas patients with a longer illness duration (> 12 months) showed intact LI. The effect on LI of duration of illness is consistent with previous findings that LI is disrupted specifically in acute, but not chronic, schizophrenia. Previous studies have assumed that this pattern of results is due to the stabilising effect of long-term neuroleptic medication. The present findings in a sample of neuroleptic naive schizophrenic patients indicate that this is unlikely to be the case. Rather, it appears that the reinstatement of LI in schizophrenic patients over time is due to a factor(s) intrinsic to the evolution of the schizophrenic illness.

Solid-phase synthesis of 2,3,5-trisubstituted indoles.

2,3,5-Trisubstituted indoles are synthesized in three steps starting from resin-bound aniline 2. R1 is introduced by a palladium-mediated coupling of the aryl iodide with terminal alkynes followed by intramolecular cyclization to form the indole core. Acylation at C-3 with an acid chloride in the presence of AlCl(3) catalyst introduces R2. The indole C-5 position is then diversified either by Sonagashira or Suzuki couplings with the aryl bromide. Finally, indole N-1 can be modified by post-cleavage methylation. [reaction: see text]

Insulin-like growth factor 1 and growth hormone binding protein in depression: a preliminary communication.

This study was undertaken in order to advance our understanding of the distal growth hormone axis in depression. Insulin-like growth factor 1 (IGF-1) and growth hormone binding protein (GHBP) were measured in a group of 19 depressed women and a group of 16 healthy women. Using a generalized linear model, IGF-1 levels were negatively correlated with age (p = 0.0001), influenced by menstrual phase (p = 0.016), and significantly increased in the depressed group (p = 0.02). Using the same type of analysis, GHBP was significantly related to menstrual phase (p = 0.0001) and body mass index (p = 0.0001), but was not significantly different in patients and controls. IGF-1 and GHBP were positively correlated among healthy subjects (r = 0.46, p = 0.08), but not among depressed patients (r = -0.16, p = 0.51), although these correlation coefficients were not statistically significantly different from each other. These findings confirm the importance of several physiological factors in the regulation of IGF-1 and GHBP, and suggest that depression further influences this regulation.

Case study: an infection-triggered, autoimmune subtype of anorexia nervosa.

OBJECTIVE: Certain cases of anorexia nervosa (AN) may be similar to the recently described subtype of childhood-onset obsessive-compulsive disorder hypothesized to be one of the pediatric infection-triggered autoimmune neuropsychiatric disorders (PITANDs). METHOD: Three clinical cases are reported. The first patient is a 12-year-old boy whose AN worsened acutely after a group A beta-hemolytic streptococcal (GABHS) infection. His symptoms were alleviated after antibiotic treatment. Two other patients with possible PITANDs-related AN are described. RESULTS: An infection-triggered process may contribute to the pathogenesis of a subtype of AN. CONCLUSIONS: Future research is needed to explore the nature of PITANDs and their relationship with AN.

Kamin blocking is not disrupted by amphetamine in human subjects.

The effect of oral amphetamine administration on the Kamin-blocking effect in healthy volunteer subjects was investigated. Against predictions, Kamin blocking was not disrupted by either a high or low oral dose of D-amphetamine under conditions which have, in previous studies, led to disruption of a related learning phenomenon (latent inhibition). This lack of effect of amphetamine administration upon Kamin blocking weakens hypotheses that this cognitive process is mediated by the same changes in dopaminergic activity which affect latent inhibition. Currently, the only data which show strong comparative associations between Kamin blocking and latent inhibition are when they are applied to schizophrenic populations. These results may suggest that Kamin blocking and latent inhibition may be measuring different aspects of schizophrenic cognitive dysfunction.

Effects of d-amphetamine and haloperidol on latent inhibition in healthy male volunteers.

Latent inhibition (LI) refers to a retardation of learning about the consequences of a stimulus when that stimulus has been passively presented a number of times without reinforcement. Acute positive-symptom schizophrenics, normal volunteers who score high on questionnaire measures of schizotypy and non-patients or animals treated with dopamine agonists show reduced LI. Neuroleptic drugs, such as haloperidol, administered at low doses, potentiate LI and effectively reverse disruption of LI induced by dopamine agonists in animals. However, a high dose of haloperidol, administered on its own, has been found to reduce LI. We examined the effects on LI of acute oral administration of an indirect dopamine-agonist, d-amphetamine (5 mg), and a nonselective dopamine receptor antagonist, haloperidol (5 mg), in normal male volunteers, using an associative learning task. Replicating previous reports, we found that d-amphetamine reduced LI; haloperidol also reduced LI, but only in subjects who scored low on the Psychoticism scale of the Eysenck Personality Questionnaire. In a subsequent study, no effect was found of 2 mg oral haloperidol administration on LI. The effect of 5 mg haloperidol on LI is interpreted as similar to that observed with a high dose of haloperidol in rats.