Browsing by Subject "Receptor tyrosine kinase"

Now showing 1 - 5 of 5
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    Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone–receptor activation
    (National Academy of Sciences, 2021) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark A.; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, M. Dodson; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Ligand-dependent conformational switches are ubiquitous in biological macromolecules, from allosteric proteins to RNA riboswitches. Molecular design of artificial switches provides a general strategy to test relationships between macromolecular structure and function. The present study exploited recent structures of complexes between an ancestral signaling protein (insulin) and the ectodomain of its cellular receptor to insert a metabolite-regulated switch into the hormone. Whereas binding of ligands often stabilizes structure, this design envisioned metabolite-dependent “opening” of a closed, inactive insulin conformation. Assessment of hormone-directed receptor autophosphorylation and a downstream signaling cascade in liver-derived cells demonstrated that binding of metabolite (a monosaccharide) enabled hormonal signaling. These results suggest a mechanism-based strategy to design “smart” glucose-responsive analogs to more safely treat insulin-dependent diabetes mellitus.
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    Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas
    (John Wiley & Sons, Inc., 2016-02) Ferguson, Michael J.; Rhodes, Steven D.; Jiang, Li; Li, Xiaohong; Yuan, Jin; Yang, Xianlin; Zhang, Shaobo; Vakili, Saeed T.; Territo, Paul; Hutchins, Gary; Yang, Feng-Chun; Ingram, David A.; Clapp, D. Wade; Chen, Shi; Department of Pediatrics, Indiana University School of Medicine
    Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1flox/− pNF murine model. Experimental Design Proliferation, β-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1+/− mast cells and fibroblasts, respectively. Krox20;Nf1flox/− mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [18F]DG-PET/CT imaging. Results Sunitinib suppressed multiple in vitro gain-in-functions of Nf1+/− mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1flox/− mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. Conclusions Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1flox/− mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.
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    Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding
    (American Society for Biochemistry and Molecular Biology, 2018-01-05) Glidden, Michael D.; Yang, Yanwu; Smith, Nicholas A.; Phillips, Nelson B.; Carr, Kelley; Wickramasinghe, Nalinda P.; Ismail-Beigi, Faramarz; Lawrence, Michael C.; Smith, Brian J.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.
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    The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1
    (Elsevier, 2023) Fukuda, Seiji; Matsuda, Nozomi; Shoji, Tsukimi; Onishi, Chie; Hirade, Tomohiro; Taketani, Takeshi; Pelus, Louis M.; Microbiology and Immunology, School of Medicine
    CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.
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    An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein
    (American Society for Biochemistry and Molecular Biology, 2018-01-05) Glidden, Michael D.; Aldabbagh, Khadijah; Phillips, Nelson B.; Carr, Kelley; Chen, Yen-Shan; Whittaker, Jonathan; Phillips, Manijeh; Wickramasinghe, Nalinda P.; Rege, Nischay; Swain, Mamuni; Peng, Yi; Yang, Yanwu; Lawrence, Michael C.; Yee, Vivien C.; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.