Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.     

Role of IGF-1 receptor in radiation response

Insulin-like growth factor 1 receptor (IGF-1R) is a transmembrane receptor tyrosine kinase involved in the development and progression of cancer whose activation strongly promotes cell growth and survival. IGF-1R exerts its main actions through the activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. In addition to their traditional roles, IGF-1R activation has been associated with increased radioresistance both in vitro and in vivo, although the molecular mechanisms behind this process are still unclear. Recently, IGF-1R has been associated to new partners as major vault proteins, BCL-2, BAX, or Ku70/80, related to radiochemotherapy resistance, regulation of apoptosis, and nonhomologous end-joining DNA repair. Here, we review these novel associations of IGF-1R trying to explain the resistance to radiotherapy mediated by IGF-1R. Finally, we revised the role of new therapies leading to block the receptor to enhance the efficacy of radiation.     

A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Insulin-like growth factor type-1 receptor transactivation in vasoactive peptide and oxidant-induced signaling pathways in vascular smooth muscle cells

Transactivation of epidermal growth factor receptor (EGFR) is a well-documented mechanism by which vasoactive peptides and H2O2 elicit their cellular responses. However, a role for the insulin-like growth factor type-1 receptor (IGF-1R) transactivation in mediating the effects of angiotensin II (Ang II) and H2O2 in vascular smooth muscle cells from different artery types have also been recently recognized. By using a series of pharmacological inhibitors of various growth factor receptor tyrosine kinases and a direct analysis of the phosphorylation status of the beta-subunit of IGF-1R, a requirement of this growth factor receptor in Ang II and H2O2 response has been demonstrated. This review discusses some of the studies that highlight the importance of IGF-1R transactivation in mediating Ang II- and H2O2-induced mitogen-activated protein kinase and protein kinase B signaling pathways.     

Biochemical and structural characterization of a novel class of inhibitors of the type 1 insulin-like growth factor and insulin receptor kinases

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.     

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.     

A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage-induced p53 is therefore reduced in Igf-1r(-/-) mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5' untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.     

The inhibition of angiogenesis and tumor growth by denbinobin is associated with the blocking of insulin-like growth factor-1 receptor signaling

Denbinobin, which is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla, has been demonstrated to display antitumor activity. Recent reports suggest that the enhanced activity of insulin-like growth factor-1 receptor (IGF-1R) is closely associated with tumor angiogenesis and growth. This study aims at investigating the roles of denbinobin in suppressing these effects and at further elucidating the underlying molecular mechanisms. In the present study, we used an in vivo xenograft model antitumor and the Matrigel implant assays to show that denbinobin suppresses lung adenocarcinoma A549 growth and microvessel formation. Additionally, crystal violet and capillary-like tube formation assays indicated that denbinobin selectively inhibits insulin-like growth factor-1 (IGF-1)-induced proliferation (GI50=1.3×10⁻⁸ M) and tube formation of human umbilical vascular endothelial cells (HUVECs) without influencing the effect of epidermal growth factor; vascular endothelial growth factor and basic fibroblast growth factor. Furthermore, denbinobin inhibited the IGF-1-induced migration of HUVECs in a concentration-dependent fashion. Western blotting and immunoprecipitation demonstrated that denbinobin causes more efficient inhibition of IGF-1-induced activation of IGF-1R and its downstream signaling targets, including , extracellular signal-regulated kinase, Akt, mTOR, p70S6K, 4EBP and cyclin D1. All of our results provide evidences that denbinobin suppresses the activation of IGF-1R and its downstream signaling pathway, which leads to the inhibition of angiogenesis. Our findings suggest that denbinobin may be a novel IGF-1R kinase inhibitor and has potential therapeutic abilities for angiogenesis-related diseases such as cancer.     

Discovery of novel dual inhibitors of receptor tyrosine kinases EGFR and IGF-1R

Novel 4-benzylamino benzo-anellated pyrrolo[2,3-b]pyridines have been synthesized with varied substitution patterns both at the molecular scaffold of the benzo-anellated ring and at the 4-benzylamino residue. With a structural similarity to substituted thieno[2,3-d]pyrimidines as epidermal growth factor receptor (EGFR) inhibitors, we characterized the inhibition of EGFR for our novel compounds. As receptor heterodimerization gained certain interest as mechanism of cancer cells to become resistant against novel protein kinase inhibitors, we additionally measured the inhibition of insulin-like growth factor receptor IGF-1R which is a prominent receptor for such heterodimerizations with EGFR. Structure–activity relationships are discussed for both kinase inhibitions depending on the varied substitution patterns. We discovered novel dual inhibitors of both receptor tyrosine kinases with interest for further studies to reduce inhibitor resistance developments in cancer treatment.     

Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kγ in the MDA-MB-231 metastatic breast cancer cell line

Background

Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R) and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3Kγ-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE) and identified the proteins by mass spectrometry.

Results

These experiments identified eukaryotic elongation factor 2 (eEF2) as a novel downstream target of PI3Kγ after activation of the IGF-1R-CXCR4 heterodimer by IGF-I. Further analysis demonstrated that eEF2 is phosphorylated in MDA-MB-231 cells in response to IGF-I and that this is dependent on PI3Kγ activity.

Conclusions

Our data imply a novel role for PI3Kγ in facilitating cell migration by regulating phosphorylation of eEF2.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.     

The insulin-like growth factor type 1 and insulin-like growth factor type 2/mannose-6-phosphate receptors independently regulate ERK1/2 activity in HEK293 cells

Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinase-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.     

Discovery and selectivity-profiling of 4-benzylamino 1-aza-9-oxafluorene derivatives as lead structures for IGF-1R inhibitors

Recently the insuline-like growth factor receptor (IGF-1R) emerged as a promising target structure for the development of novel anti-cancer agents. IGF-1R plays a central role in both tumour progression and resistance development against anti-cancer drugs. We discovered 1-aza-9-oxafluorene derivatives as novel lead structures with submicromolar activities against IGF-1R. Structure-activity relationships (SARs) on a series of related receptor tyrosine kinases (RTKs) are discussed in the context of available crystal structures. A preliminary selectivity-profiling is demonstrated for the first compound series. Antiproliferative tumour cell line screening studies yielded one candidate as a promising cytostatic agent without significant toxic effects.     

Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells

Background

Type I insulin-like growth factor receptor (IGF-1R) and insulin receptor (INSR) are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R). The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium.

Methodology/Principle Findings

IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi) cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways.

Conclusion/Significance

In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic pathways. The development of novel therapeutic strategies designed to target the IGF-1/IGF-1R pathway must take into account the modulatory roles IGF-1R/INSR play in the epithelial cell nucleus.     

Activation of the Insulin-like Growth Factor Type 1 Receptor by Deletion of Amino Acids 870–905

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Δ870–905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Δ870–905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both α and β-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Δ870–905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Δ870–905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870–905 exert a negative effect on the tyrosine kinase domain of the β-subunit of the IGF-1 R.