Discovery of 2,4-bis-arylamino-1,3-pyrimidines as insulin-like growth factor-1 receptor (IGF-1R) inhibitors

The insulin-like growth factor-1 receptor (IGF-1R) plays an important role in the regulation of cell growth and differentiation, and in protection from apoptosis. IGF-1R has been shown to be an appealing target for the treatment of human cancer. Herein, we report the synthesis, structure-activity relationships (SAR), X-ray cocrystal structure and in vivo tumor study results for a series of 2,4-bis-arylamino-1,3-pyrimidines.     

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.     

Targeting of the protein interaction site between FAK and IGF-1R

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.     

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.     

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R.

Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo.

INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo.

Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.     

Pyridyl and thiazolyl bisamide CSF-1R inhibitors for the treatment of cancer

The bisamide class of kinase inhibitors was identified as being active against CSF-1R. The synthesis and SAR of pyridyl and thiazolyl bisamides are reported, along with the pharmacokinetic properties and in vivo activity of selected examples.     

Novel imidazo[1,2-a]pyridine based inhibitors of the IGF-1 receptor tyrosine kinase: Optimization of the aniline

Following the discovery of imidazopyridine 1 as a potent IGF-1R tyrosine kinase inhibitor, the aniline part has been modified with the aim to optimize the properties of this series. The structure-activity relationships against IGF-1R kinase activity as well as inhibition of the hERG ion channel are discussed.     

Lipid raft-regulated IGF-1R activation antagonizes TRAIL-induced apoptosis in gastric cancer cells

Gastric cancer cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the resistance mechanism is not fully understood. In human gastric cancer MGC803 and BGC823 cells, TRAIL induces insulin-like growth factor-1 receptor (IGF-1R) pathway activation. Treatment with IGF-1R inhibitor OSI-906 or small interfering RNAs against IGF-1R, prevents IGF-1R pathway activation and increases TRAIL-induced apoptosis. The TRAIL-induced IGF-1R pathway activation is promoted by IGF-1R translocation into lipid rafts. Moreover, the translocation of IGF-1R into lipid rafts is regulated by Casitas B-lineage lymphoma b (Cbl-b). Taken together, TRAIL-induced IGF-1R activation antagonizes TRAIL-induced apoptosis by Cbl-b-regulated distribution of IGF-1R in lipid rafts.     

Focal adhesion kinase (FAK) activates and stabilizes IGF-1 receptor

Recent studies have shown a direct association between IGF-1R and FAK, two important mediators of cell growth, survival and migration. However, the mechanism by which FAK affects IGF-1R function remains unknown. This study investigates the potential role of FAK in mediating activation and stability of IGF-1R. Autophosphorylation and phosphorylation capacities of wild type and mutant IGF-1R were studied. Surprisingly, we found that the mutant IGF-1R lacking the three core tyrosine residues in the activation-loop can be phosphorylated although it is unable to undergo autophosphorylation, suggesting that another kinase possesses the ability to phosphorylate IGF-1R. By using wild type MEFs and FAK−/− MEFs we could demonstrate that FAK mediates activation-loop independent phosphorylation, as well as Akt and ERK activation. Furthermore, the stability of IGF-1R was decreased upon FAK siRNA or inactivation. Taken together, our data suggest a role for FAK in phosphorylation, signaling and stability of the IGF-1R.     

Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered a promising therapeutic target for human cancers. We identified novel tetracyclic derivatives as potent ALK inhibitors. Among them, compound 27 showed strong cytotoxicity against KARPAS-299 with an IC₅₀ value of 21 nM and significant antitumor efficacy in ALK fusion-positive blood and solid cancer xenograft models in mice without body weight loss.     

3-amido-4-anilinocinnolines as a novel class of CSF-1R inhibitor

3-Amido-4-anilinocinnolines have been identified as potent and highly selective inhibitors of CSF-1R. The synthesis and SAR of these compounds is reported, along with some physical property, pharmacokinetic and kinase selectivity data.     

Mitigation of cardiovascular toxicity in a series of CSF-1R inhibitors,and the identification of AZD7507

The potent and selective 3-amido-4-anilinoquinoline CSF-1R inhibitor AZ683 suffered from cardiovascular liabilities, which were linked to the off-target activities of the compound and ion channel activity in particular. Less basic and less lipophilic examples from both the quinoline and cinnoline series demonstrated cleaner secondary pharmacology profiles. Cinnoline 31 retained the required potency and oral PK profile, and was progressed through the safety screening cascade to be nominated into development as AZD7507.     

Synthesis and in vitro evaluation of [18F]BMS-754807: A potential PET ligand for IGF-1R

Radiosynthesis and in vitro evaluation of [¹⁸F](S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide ([¹⁸F]BMS-754807 or [¹⁸F]1) a specific IGF-1R inhibitor was performed. [¹⁸F]1 demonstrated specific binding in vitro to human cancer tissues. Synthesis of reference standard 1 and corresponding bromo derivative (1a), the precursor for radiolabeling were achieved from 2,4-dichloropyrrolo[2,1-f][1,2,4]triazine (4) in three steps with 50% overall yield. The radioproduct was obtained in 8% yield by reacting 1a with [¹⁸F]TBAF in DMSO at 170 °C at high radiochemical purity and specific activity (1–2 Ci/μmol, N = 10). The proof of concept of IGF-IR imaging with [¹⁸F]1 was demonstrated by in vitro autoradiography studies using pathologically identified surgically removed grade IV glioblastoma, breast cancer and pancreatic tumor tissues. These studies indicate that [¹⁸F]1 can be a potential PET tracer for monitoring IGF-1R.     

IGF受体1分子上IGF-1结合位点的探讨

为了探讨胰岛素样生长因子受体1(IGF-R1)分子上的胰岛素样生长因子1(IGF-1)结合位点,建立了研究IGF-1与IGF-R1相互作用的酵母双杂交模型;利用基因体外定点突变的方法,构建了7种IGF-R1突变体;然后通过报告基因活性的定量分析,在酵母双杂交模型中检测了IGF-1与各种IGF-R1突变体之间相互作用的大小,初步确定了IGF-R1分子中IGF-1的结合位点,并且IGF-R1分子上N237、T238在与IGF-1结合中起着重要作用.     

Inhibitors of insulin-like growth factor-1 receptor tyrosine kinase are preferentially cytotoxic to nutrient-deprived pancreatic cancer cells

Chronic deprivation of nutrients is rare in normal tissues, however large areas of tumor are nutrient-starved and hypoxic due to a disorganized vascular system. Some cancers show an inherent ability to tolerate severe growth conditions. Therefore, we screened chemical compounds to identify cytotoxic agents that function preferentially in nutrient-deprived conditions. We found that AG1024, a specific inhibitor of insulin-like growth factor-1 receptor tyrosine kinase (IGF-1R), showed preferential cytotoxicity to human pancreatic cancer cells in nutrient-deprived conditions relative to cells in nutrient-sufficient conditions. The cytotoxicity of I-OMe-AG538 (another specific inhibitor of IGF-1R kinase) was also enhanced in nutrient-deprived cells. In addition, AG1024 and I-OMe-AG538 potently inhibited IGF-1R activation to nutrient-deprived cells. In contrast, conventional chemotherapeutic drugs, as well as inhibitors of PDGFR and EGFR kinases, elicited weak cytotoxicity. These data indicate that nutrient-deprived human pancreatic cancer cells have increased sensitivity to inhibition of IGF-1R activation. IGF-1R inhibitors offer a promising strategy for anticancer therapeutic approaches that are oriented toward tumor microenvironment.     

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

Hrd1 induces cardiomyocyte apoptosis via regulating the degradation of IGF-1R by sema3a

Objective

To explore the underlying mechanisms of Hrd1/sema3a/IGF-1R on cardiomyocyte apoptosis.

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC₅₀ = 160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC₅₀ = 47 nM) was a highly specific JNK inhibitor.     

Insulin activates EGFR by stimulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells

The expression of epidermal growth factor receptor (EGFR) is an important diagnostic marker for triple-negative breast cancer (TNBC) cells, which lack three hormonal receptors: estrogen and progesterone receptors as well as epidermal growth factor receptor 2. EGFR transactivation can cause drug resistance in many cancers including TNBC, but the mechanism underlying this phenomenon is poorly defined. Here, we demonstrate that insulin treatment induces EGFR activation by stimulating the interaction of EGFR with insulin-like growth factor receptor 1 (IGF-1R) in the MDA-MB-436 TNBC cell line. These cells express low levels of EGFR, while exhibiting high levels of IGF-1R expression and phosphorylation. Low-EGFRexpressing MDA-MB-436 cells show high sensitivity to insulinstimulated cell growth. Therefore, unexpectedly, insulin stimulation induced EGFR transactivation by regulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells. [BMB Reports 2015; 48(6): 342-347]