Discovery of Novel 5,6-Dihydro-4H-pyrido[2,3,4-de]quinazoline Irreversible Inhibitors Targeting Both Wild-Type and A775_G776insYVMA Mutated HER2 Kinases.

HER2 mutations were seen in 4% of non-small-cell lung cancer (NSCLC) patients. Most of these mutations (90%) occur as an insertion mutation within the exon 20 frame, leading to the downstream activation of the PI3K-AKT and RAS/MAPK pathways. However, no targeted therapies have yet been approved worldwide. Here a novel series of highly potent HER2 inhibitors with a pyrido[2,3,4-de]quinazoline core were designed and developed. The derivatives with the pyrido[2,3,4-de]quinazoline core displayed superior efficacy of antiproliferation in BaF3 cells harboring HER2insYVMA mutation compared with afatinib and neratinib. Rat studies showed that 8a and 9a with the newly developed core have good pharmacokinetic properties with an oral bioavailability of 41.7 and 42.0%, respectively. Oral administration of 4a and 10e (30 mg/kg, QD) displayed significant antitumor efficacy in an in vivo xenograft model. We proposed promising strategies for the development of HER2insYVMA mutant inhibitors in this study.

Diazoxide promotes oligodendrocyte precursor cell proliferation and myelination.

BACKGROUND: Several clinical conditions are associated with white matter injury, including periventricular white matter injury (PWMI), which is a form of brain injury sustained by preterm infants. It has been suggested that white matter injury in this condition is due to altered oligodendrocyte (OL) development or death, resulting in OL loss and hypomyelination. At present drugs are not available that stimulate OL proliferation and promote myelination. Evidence suggests that depolarizing stimuli reduces OL proliferation and differentiation, whereas agents that hyperpolarize OLs stimulate OL proliferation and differentiation. Considering that the drug diazoxide activates K(ATP) channels to hyperpolarize cells, we tested if this compound could influence OL proliferation and myelination. METHODOLOGY/FINDINGS: Studies were performed using rat oligodendrocyte precursor cell (OPC) cultures, cerebellar slice cultures, and an in vivo model of PWMI in which newborn mice were exposed to chronic sublethal hypoxia (10% O(2)). We found that K(ATP) channel components Kir 6.1 and 6.2 and SUR2 were expressed in oligodendrocytes. Additionally, diazoxide potently stimulated OPC proliferation, as did other K(ATP) activators. Diazoxide also stimulated myelination in cerebellar slice cultures. We also found that diazoxide prevented hypomyelination and ventriculomegaly following chronic sublethal hypoxia. CONCLUSIONS: These results identify KATP channel components in OLs and show that diazoxide can stimulate OL proliferation in vitro. Importantly we find that diazoxide can promote myelination in vivo and prevent hypoxia-induced PWMI.

Hypoglycemia influences oligodendrocyte development and myelin formation.

Damage to central nervous system white matter is observed following hypoglycemia, raising the possibility that hypoglycemia influences oligodendrocytes and myelination. To examine effects of hypoglycemia on oligodendrocytes and myelin formation, we studied cultured oligodendrocyte precursor cells and cerebellar slice cultures. We observed that with decreasing concentrations of glucose, oligodendrocyte precursor cell proliferation, maturation, and migration decreased. We also observed that hypoglycemia induced apoptotic cell death and activation of caspase-3 in oligodendrocyte precursor cells. Slice culture studies showed that glucose is required for myelinated fiber formation, as with reduction in the glucose concentration, the density of myelinated fibers decreased. Collectively, these data show that hypoglycemia inhibits oligodendrocyte development and myelination and that hypoglycemia triggers apoptotic cell death in oligodendrocyte precursor cells.

Cytoskeletal protein 4.1G is a binding partner of the metabotropic glutamate receptor subtype 1 alpha.

Recent evidence suggests that cytoskeletal proteins play important roles in the clustering and anchoring of glutamate receptors to the cell surface membrane. To examine further this issue, we tested for direct interactions between the metabotropic glutamate receptor subtype 1alpha (mGlu1alpha) and 4.1G, which is a member of the erythrocyte membrane, cytoskeletal protein 4.1 family. First, co-localization of 4.1G and mGlu1alpha was observed in cultured hippocampal neurons. Second, in transiently transfected HEK 293 cells and in whole rat brain tissue, direct interactions between mGlu1alpha and 4.1G were observed. Third, we were able to identify the C-terminal tail of mGlu1alpha as an essential region for mGlu1alpha-4.1G interactions. Fourth, 4.1 G influences mGlu1alpha-mediated cAMP accumulation. Finally, we found that 4.1G increases the ligand-binding ability of mGlu1alpha and alters its cellular distribution. These observations identify 4.1G as a novel binding partner of mGlu1alpha that can regulate the action of mGlu1alpha.

Cytoskeletal protein 4.1G binds to the third intracellular loop of the A1 adenosine receptor and inhibits receptor action.

To identify binding partners of the A1AR (A1 adenosine receptor), yeast two-hybrid screening of a rat embryonic cDNA library was performed. This procedure led to the identification of erythrocyte membrane cytoskeletal protein (represented as 4.1G) as an A1AR-binding partner. Truncation studies revealed that the C-terminal domain of 4.1G was essential for binding to A1ARs and that the C-terminal domain of 4.1G and the third intracellular loop of A1ARs interacted. A1AR-4.1G interaction was also confirmed in studies using brain tissue. Studies in HEK-293 (human embryonic kidney 293) cells and Chinese-hamster ovary cells showed that 4.1G interfered with A1AR signal transduction, as 4.1G reduced A1AR-mediated inhibition of cAMP accumulation and intracellular calcium release. 4.1G also altered cell-surface A1AR expression. These observations identify 4.1G as a novel A1AR-binding partner that can regulate adenosine action.

A1 adenosine receptors mediate hypoxia-induced ventriculomegaly.

Periventricular leukomalacia is characterized by a reduction in brain matter and secondary ventriculomegaly and is a major cause of developmental delay and cerebral palsy in prematurely born infants. Currently, our understanding of the pathogenesis of this condition is limited. In animal models, features of periventricular leukomalacia can be induced by hypoxia and activation of A1 adenosine receptors (A1ARs). Using mice that are deficient in the A1AR gene (A1AR-/-), we show that A1ARs play a prominent role in the development of hypoxia-induced ventriculomegaly in neonates. Supporting a role for adenosine in the pathogenesis of developmental brain injury, ventriculomegaly was also observed in mice lacking the enzyme adenosine deaminase, which degrades adenosine. Thus, adenosine acting on A1ARs appears to mediate hypoxia-induced brain injury ventriculomegaly during early postnatal development.

Lysophosphatidic acid regulates the proliferation and migration of olfactory ensheathing cells in vitro.

Olfactory ensheathing cells (OECs) are a unique type of macroglia with axonal growth-promoting properties. However, our understanding of the factors that regulate OECs is at early stages. Lysophosphatidic acid (LPA) is a lipid that influences diverse functions in the nervous system. Recent studies suggest that glial cells, including astrocytes and Schwann cells, are important targets of LPA. However, the influences of LPA on OECs are not known. To address if LPA can influence OECs, we examined effects of LPA on the proliferation and migration of OECs and intracellular effector events. Initially, we observed that OECs expressed the genes for LPA1, LPA2, and LPA3 receptors. When OECs were treated with LPA, we observed stimulated proliferation and migration of OECs. Treatment of OECs with LPA also induced actin cytoskeleton reorganization and focal adhesion assembly. These effects of LPA were blocked by treatment with C3 exoenzyme or Y-27632, which inhibit Rho-GTPase and Rho-associated kinase, respectively. Effects of LPA on OEC proliferation were blocked by the MEK inhibitors PD098059 and U0126 and by the phosphotidylinositol 3-kinase (PI 3-K) inhibitors LY0294002 and wortmannin. These results show that LPA acts via Rho-GTPase, MAPK, and PI 3-K signaling cascades to influence OEC proliferation, migration, and cytoskeleton assembly.

Oligodendrocytes express functional A1 adenosine receptors that stimulate cellular migration.

A1 adenosine receptors (A1ARs) exert important effects in the central nervous system. However, the expression and function of A1ARs in oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLGs) is unclear. To address this issue, we examined A1AR expression during different stages of oligodendrocyte development. Radioreceptor studies showed that membranes prepared from OPCs and OLGs expressed high-affinity A1ARs with Kd values of 1.35 +/- 0.33 and 1.2 +/- 0.27 nM for [3H]CCPA, 1.17 +/- 0.24 and 1.4 +/- 0.34 nM for [3H]DPCPX, respectively. Bmax values were 64.31 +/- 6.14 and 75 +/- 6 fmol/mg protein for [3H]CCPA, and 153 +/- 12 and 205 +/- 17.8 fmol/mg protein for [3H]DPCPX, respectively. Activation of A1ARs using N6-cyclopentyladenosine (CPA) reduced both forskolin- and N-ethylcarboxyamidoadenosine (NECA)-stimulated cAMP accumulation, but did not affect basal cAMP levels. Activation of A1ARs by CPA stimulated OPC migration, but did not affect cell viability, proliferation, or differentiation. These results show that OPCs and OLGs express functional A1ARs that can stimulate the migration of OPCs.

Hepatocyte growth factor stimulates the proliferation and migration of oligodendrocyte precursor cells.

Hepatocyte growth factor (HGF) was initially identified as a potent mitogen for mature hepatocytes and has since been found to affect a variety of cells. Evidence suggests that HGF may also influence the nervous system, in that HGF stimulates the proliferation of myelin-forming Schwann cells and olfactory ensheathing cells. However, it is not known whether HGF influences oligodendrocytes. To address this issue, oligodendrocyte precursors were obtained from neonatal rat cerebra and cultured. Immunostaining and Western blotting revealed expression of both HGF and the HGF receptor (c-Met) by cultured oligodendrocytes. When the ability of HGF to stimulate oligodendrocyte division and migration was examined, we observed that treatment with HGF (10-50 ng/ml) elicited twofold increases in oligodendrocyte precursor proliferation. HGF also enhanced oligodendrocyte precursor migration, with 2.5-fold increases in rates of migration seen after treatment for 8 hr. HGF also influenced inducing the oligodendrocyte cytoskeleton by altering patterns of F-actin and beta-tubulin distribution and enhanced the expression of actin and beta-tubulin. These observations show that a functional HGF/c-Met system is present in oligodendrocytes, which can influence the growth, development, and cytoskeletal organization of oligodendrocytes.

A1 adenosine receptor activation induces ventriculomegaly and white matter loss.

A1 adenosine receptors (A1ARs) are widely expressed in the brain during development. To examine whether A1AR activation can alter postnatal brain formation, neonatal rats from postnatal days 3 to 14 were treated with the A1AR agonist N6-cyclopentyladenosine (CPA) in the presence or absence of the peripheral A1AR antagonist 8-(p-sulfophenyl)-theophylline (8SPT). CPA or CPA + 8SPT treatment resulted in reductions in white matter volume, ventriculomegaly, and neuronal loss. Quantitative electron microscopy revealed reductions in total axon volume following A1AR agonist treatment. We also observed reduced expression of myelin basic protein in treated animals. Showing that functional A1ARs were present over the ranges of ages studies, high levels of specific [3H]CCPA binding were observed at PD 4, 7 and 14, and receptor-G protein coupling was present at each age. These observations show that activation of A1ARs with doses of CPA that mimic the effects of high adenosine levels results in damage to the developing brain.