Relevant Receptor Tyrosine Kinases Research Articles

One-pot two-step catalytic synthesis of 6-amino-2-pyridone-3,5-dicarbonitriles enabling anti-cancer bioactivity

We report a one-pot two-step synthesis of a bioactive 6-amino-2-pyridone-3,5-dicarbonitrile derivative using natural product catalysts betaine and guanidine carbonate. Anti-cancer bioactivity was observed in specific molecules within the library of 16 derivatives. Out of the compounds, 5o had the most potent anti-cancer activity against glioblastoma cells and was selected for further study. Compound 5o showed anti-cancer properties against liver, breast, lung cancers as well as primary patient-derived glioblastoma cell lines. Furthermore, 5o in combination with specific clinically relevant small molecule inhibitors induced enhanced cytotoxicity in glioblastoma cells. Through our current work, we establish a promising 6-amino-2-pyridone-3,5-dicarbonitrile based lead compound with anti-cancer activity either on its own or in combination with specific clinically relevant small molecule kinase and proteasome inhibitors.

Comprehensive genomic profiling (CGP) to assess mutational load in gastric and esophageal adenocarcinomas: Implications for immunotherapies.

66 Background: Gastroesophageal cancer (GEC) present a management challenge, particularly when ERBB2 is not amplified and cytotoxic therapy has failed. Tumor mutational load is linked to predicted benefit from immune checkpoint inhibitors in advanced cancers. Using CGP we assessed the relationship between mutational burden and clinically relevant genomic alterations in GEC samples in the course of routine clinical care. Methods: DNA was extracted from 40 microns of FFPE sections from patients with GEC. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of greater than 500x for 236 or 315 cancer-related genes. Mutational load was characterized as the number of somatic base substitutions and short indels per megabase. Samples were stratified by histologic subtype, and presence or absence of therapeutically relevant receptor tyrosine kinase (RTK) alterations. The majority of samples were from patients with advanced disease. Results: The genomic profiles from a total of 736 gastric and 862 esophageal carcinomas were assessed. Median patient age was 57 for gastric and 60 for esophageal cases. There was no significant difference in mutational load between gastric and esophageal samples. 10th, 25th, median, 75th, and 90th percentiles of mutation load were 1.3, 2.7, 6.3, 11.3, and 20.0 for gastric cancers and 1.8, 3.6, 6.4, 11.3, and 17.5 for esophageal cancers. 27 (3.7%) gastric and 20 (2.3%) esophageal cases had alterations in mismatch repair genes MLH1, MSH2, MSH6 which was associated with ~2.5 fold increased mutational load. Mutational load was not significantly associated with the presence of RTK alterations, which occurred in 155 (21%) gastric and 355 (41%) esophageal cases. Conclusions: CGP in the course of clinical care can be used to assess mutational load in GEC. Alterations in RTKs were not exclusive of the high mutation subgroup, contrary to some previous reports. Mutations in DNA mismatch repair genes were associated with higher mutation burden as expected. Incorporation of CGP into ongoing prospective immunotherapy trials and clinical practice is needed to refine these relationships.

Analysis of Receptor Tyrosine Kinase and G-Protein Coupled Receptor Signaling Dynamics on Micro-Structured Surfaces

Membrane-localized proteins are essential to transmit signals into the cell. An important issue is the interaction of these proteins with cytosolic proteins. To quantify such often short-lived interactions we have introduced a method based on the combination of micro-structured surfaces and TIRF microscopy.(1,2) We have used the assay to validate the efficacy of medically relevant receptor tyrosine kinase (RTK) modulators.(3) Bait epidermal growth factor (EGF) receptor molecules were forced into microscopic domains, while monitoring co-recruitment of fluorescent intracellular prey Grb2 molecules. Pretreatment with pharmacologically active ingredients used for the treatment of human cancers significantly reduced this interaction. A similar approach was used for the quantitative analysis of the interaction between different insulin receptor substrate (IRS) proteins and the insulin/IGF-I receptor.(4) The micro-patterning technique enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, which could account for the diverse IRS functions. Finally, we studied the interaction of the GPCR s2-adrenoceptor (s2AR) with arrestin-3.(5) By measuring arrestin-3 recruitment and the stability of arrestin-3-receptor complexes in real time using FRAP analysis on micro-patterned surfaces, we could demonstrate that arrestin-3 dissociates quickly and almost completely from the β2AR. Recently, we have implemented micro-structured and functionalized multi-well plates. This development step sets a milestone in terms of throughput rates of our methodology.

Discovery of dually acting small-molecule inhibitors of cancer-resistance relevant receptor tyrosine kinases EGFR and IGF-1R

Small-molecule inhibitors of cancer-relevant receptor tyrosine kinases EGFR and IGF-1R have been discovered.

Abstract 2842: Development of a novel TrkA HTS assay using template directed self assembly (TDA)

Abstract Tropomyosin-related kinases (Trks A, B, and C) are a family of membrane-associated receptor tyrosine kinases (RTKs) activated by neurotrophins. RTKs are shown to be implicated in Alzheimer's disease, depression, pain sensation and various cancers including tumor cell growth and survival signaling. Inhibitors of Trk receptor kinases might provide targeted treatments for neurodegenerative diseases, pain and cancer. One strategy to indentify Trk isoform-specific inhibitors is to develop an assay where compound libraries can be screened in a more-native membrane associated lipid environment. Template-Directed Self Assembly (TDA) is a biomimetic technology designed to restore membrane-associated proteins to more relevant physiological conditions. This technology utilizes recombinant histidine-tagged proteins bound to the surface of nickel-chelated liposomes. Recombinant HIS-tagged proteins are engineered on the correct terminus of the protein to reflect the polarity of the enzyme with respect to the membrane. This allows the HIS-tagged proteins to bind to the liposome creating an environment much like a cellular membrane. The lipids in TDA are fully fluid within the surface of the liposome and associated proteins can rotate freely. Furthermore it promotes the formation of high-order structures such as homo- or hetro-dimers and shown to recruit accessory factors. Coupling of membrane associsted kinases to liposomes reconstitutes an environment that confers proper polarity, stereochemistry, topology and other critical properties, thereby creating a more relevant biochemical assay. To date, we have shown that several RTKs have enhanced biochemical specific activity and substrate specificity that resembles physiological conditions when coupled to TDA. The TDA approach restores TrkA activity to a more-native environment; therefore, we hypothesize that we will be able to identify compounds that are more specific and selective. To address the need of an enzyme assay near a lipid membrane environment, we successfully developed a robust TrkA assay shown to exhibit up to 20-fold increase in specific activity and a reduction in Km for ATP relative to TrkA in a conventional assay. Furthermore, TDA-associated TrkA exhibited a rapid and robust trans-activation, obviating the need for a pre-activation step. Thus, assays of TrkA coupled to TDA have the potential to identify more chemically diverse and specific inhibitors. We are currently (1) validating the TDA assay with known inhibitors (2) screening a focus library to identify novel hits (3) testing the primary hits against a small kinase selectivity panel and (4) designing and developing a TrkA specific reporter cell line to confirm the primary hits. The advantages of the TDA technology will be also discussed against other therapeutically relevant RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2842. doi:1538-7445.AM2012-2842

VEGF and c-Met Blockade Amplify Angiogenesis Inhibition in Pancreatic Islet Cancer

Angiogenesis inhibitors that block VEGF receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTK). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small-molecule inhibitors that potently block multiple RTKs, including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 with those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated approximately 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis.

Abstract LB-227: Receptor tyrosine kinase phosphorylation: Simultaneous detection of 10 kinases upon inhibitor treatment

Abstract Receptor tyrosine kinases have been shown to be major pharmaceutical targets involved in disease states such as diabetes and cancer. Target driven therapeutics like small molecules or monoclonal antibodies are already in clinical use and have tremendous influence on new cancer therapies. In most cases either ligand binding to the extracellular domain followed by receptor dimerization or ATP binding to the intracellular kinase domain is inhibited. However, it is still challenging to gain specificity of those inhibitors for certain receptor tyrosine kinase targets. Bead-based assay technologies, such as the xMAP® platform, are ideal tools for the multiplex analysis of 5-20 proteins. Low sample volumes, simple assay protocols, flexibility and robustness are characteristics of bead-based assays that make them well suited for the immunological detection of bio marker and cell signaling proteins. In this presentation we will show results for the simultaneous detection of the ten most relevant receptor tyrosine kinases. The expression profile of more than 50 cell lines as well as breast tumor samples were evaluated with this powerful multiplex tool in a very robust and convenient way. Inhibitor profiling of small molecules under physiological conditions on ten receptor tyrosine kinases will be demonstrated by the detection of their phospho-Tyrosine level. These specificity profiles will help to improve the drug development process and to obtain potentially better drugs in future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-227.